INTRODUCTION

The cryopreservation of sperm is preservation process of sperm cell performed at very low temperature (commonly at -196°C by using N₂ liquid). Temperature decreasing process causes changing media environment (osmolatiry increase and ice crystal formation) which damaging the cell.

During cryopreservation process, sperm undergo stress due to temperature decrease. Temperature stress will be decrease the function and fertility of sperm. Some sperm cryopreservation  problem were decreasing of viability of sperm more than 50%, induction of acrosome reaction, decrease of motility and problem of fertility (Chavero et al. 2006; Wongtawan et al. 2006). Sperm damage occurred through two mechanisms: due to mechanic process through ice crystal formation and due to ostmotic effect and solute concentration known as solute effect (Wowk 2007). Addition of cryoprotectant agent was conducted to decrease the lethal effect of the ice crystal formation and solute effect.

According to ability to penetrete cell membrane, cryoprotectant was divided as intracellular and extracellular cryoprotectant. Intracellular cryoprotectant can penetrate membrane cell, such glycerol, methanol, dimethyl sulfoxide (DMSO), 1.2 propanediol, butanediol, acetamide, propylene glycol. Extracellular cryprotectant have high molecular weight, can’t penetrate into the cell and works outside the cell. Lactose, raffinose, mannase, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), trehalose and proteins such as lipoprotein of egg yolk ware extracelullar cryoprotectant. (Wowk 2007; Lemma 2011).

Glycerol (C3H8O3), alcoholic sugar with BM 92.09, high solubility in water, freezing point at -38°C, and low toxicity (Fuller 2004). Glycerol is the most popular cryoprotectant for crypreservation of bovine sperm. The aplication of glycerol concentration dependent on species, in livestock animal 2-10% were applied. In bovine, 5-10% was applied but optimum concentration was 6-8% (Miller & Van Demark 1953). Glycerol applicating in one step was reported slightly toxic for sperm, then the glycerol was added in extender and given stepwise during the cooling process in a hour.

Action mechanism of intracellular cryoprotectant in maintaining cell during cryoproservation process was by replacing and filling the room left by water cell. By the low frozen point, glycerol was able to reduce cell damage caused by solution injury and ice crystallization (Fuller 2004; Andrabi 2007; Wowk 2007; Lemma 2011).

Intracellular cryoprotectant toxicity was highly depended on species response, concentration, temperature, and delivery technique (Wowk 2007). Glycerol were not toxic, but some research indicated toxicity glycerol in any species. The combination of intracelullar cryoprotectan glycerol with any extracelullar cryoprotectant can reduce the toxicity.

External cryoprotectant only worked on outside the cell with the same mechanism with the intracellular cryoprotectant. The extracellular cryoprotectant commonly is a carbohydrate or protein polimer. Alginate as encapsulation media component of sperm microencapsulation is expected may be act as extracellular cryoprotectant. Alginate is carbohydrate polimer with high molecule weight, not toxic, and can not penetrate spermatozoa cell membrane. There were no study of alginate action as extrecelullar cryoprotectant but carbohydrate polimer methyle cellulose was reported could be combined with glycerol to reduce glycerol negative effect against chicken’s sperm fertility (Phillips et al. 1996). Alginate has also been applied in cryopreservation of prebiotic and fish.(Venugopal 2011; Pop et al. 2015).

This studys were conducted to determine the cryopropreservation technique of sperm microencapsulated using glycerol, to observe function of  alginate "micro-encapsulation component " as  extracellular cryoprotectant agent, whether process of microcapsulation  affects sperm viability and to determine glycerol concentration at the cryopreservation of sperm microencapsulation. The final goal of this study was to increase artificial insemination efficiency.

MATERIAL AND METHODS

Sperm cryopreservation was performed in two experiment. First experiment was to determine alginate and microencapsulation effect against sperm viability and second experiment was to determine glycerol concentration and equilibration time against sperm viability.

Time and location

This study was conducted on June 2013-April 2014 at Laboratory of Reproduction Physiology, Indonesian Research Institute for Animal Production.

Semen sample and semen quality evaluation

Three FH bulls (3-4 years old) were used and semen was collected twice a week using artificial vagina. Fresh semen quality observation was performed microscopically and macroscopically including color, consistentce, mass movement, live sperm percentage (%L), and intact acrosome ridge (%IAR).

Alive spermatozoa (%L) were observed using smear preparate by eosin-negrosin dye. Observation was performed using light microscope with 100 x 10 magnifications. Dead spermatozoa would absorb the dye represent red color and the live were trasparant. Two hundred live and dead sperm were counted in 1 object glass and the live sperm were counted by following formulation:

Live sperm (%L) =    Number live sperm        x 100%

Total sperm observed

Observation of %IAR was performed by observation sperm in NaCl formaline solution (0.01% w/v). One hundred sperm were observed under contrast phase microscope with 100 x 10 magnification using emersy oil. Percentage of IAR was calculated using the following formula:

% IAR = The number black hood sperm x 100%

Total sperm observed

Semen from 2 ejaculates (minimum mass movement was ++, %L was >80%, and concentration was more than 800 x 106/ml) were mixed proportionally to be used as sample. The pool semen was evaluated to obtain fresh semen quality data and used as repetition.

Microcapsulation Sperm Preparation

Twenty five ml encapsulation media (Table 1) was mixed with semen sample to get  total concentration  sperm 100 million/ml. Microgel formation was performed using microencapsulator Buchi B 390 (750 Hz, 550 volt). Droplets of media were collected into baker tube consisting 1.5% CaCl₂ and stirred using magnetic stirrer.

Table 1 .  Composition of Tris Citrate yolk-Alginat (TCEY-A) encapsulation media

Chemical materials	Composition
Tris Hidroxymethil Aminomethan (g)	3.027
Citric Acid (g)	1.675
Fructose (g)	1.25
Streptomycin (mg)	100
Penicylin (IU)	100000
Glutation (mM)	1
Aquabides (ml)	80
Yolk (ml)	20
Alginate (g)	1.5

Microgel formed was washed using physiological NaCl (3x) and weighed as 4 g as sample. Microcapsule membrane formation was performed using 1% chitosan (pH 5.5) for 15 minutes. The microcapsule was rinsed using physiological NaCl (3x) and liquid core performing was performed using 2% Na Citrate for 10 minutes. Evaluation of sperm viliability was conducted as soon as after microencapsulation process finished. The evaluation of sperm viability do using freezing media A (Table 3). It was used as initial data of sperm viability. Spermatozoa viliability variables observed were motility percentage (%M), %L, and %IAR.

Percentage of motility (%M) in the microencapsulation was observed by damaging microcapsule membrane using coverslip. Motility was observed under light microscope (100x10 magnification). The %L and %IAR were observed after microencapsule membrane were damaged using repetitive pipetting method (Kusumaningrum 2016).

Research 1

Research 1 was performed to determine the effect of alginate and microencapsulation process to viliability of sperm at each stage of freezing. Completely randomized design was used with 3 treatments and 6 repetitions. The treatments were T-I (semen sample in Tris citrate Egg Yolk_TCEY as control), T-II (semen sample in TCEY-A) and T-III (semen sample in TCEY-A followed by microcapsulation forming).

Semen sample was diluted using 3 diluent treatments to reach the final sperm cocentration 100 x 10⁶/ml. Evaluation of sperm viliability both of T-I, T-II, and T-III (%M, %L and %IAR) was performed after microencapsulation process (T-III) finished. Those data were used as initialy sperm viability data.

Feezing process both of T-I, T-II, and T-III was carried out by freezing standard of bovine’s sperm using Tris citric extender consisting of 20% yolk and 7% glycerol (Table 3) with equilibration time was 3 hours. Addition of freezing media (media A and B) was performed gradually started from room temperatur decrease until 5°C by using cooling machine.

Four ml sample semen (TI and TII) and four grams of microencapsule were add with 4 ml media A and B (Table 2) as following:

• Addition of media A was performed after microcapsulation finished (2 ml)
• Additional of media B was performed at 15 and 10^oC (2 ml)

The final concentration of sperm after that process was 50 x 10⁶/ml with glycerol concentration 7%. After temperature was at 5°C, evaluation of sperm viability (%M, %L and %IARAU) was carried out. Equlibration was carried out at 5^oC for 3 hours. In first 1 hour equilibration, semen was packed into medium straw (0.5 ml, 25x10⁶/straw). Equlibration was continued in the straw until 3 hours equilibration.

Straw were freezed on 8 cm upper N₂ liquid for 10 minuts, then plung in N₂ liquid. Frozen sperm evaluation was carried out after 1 week storage. The straw were thawed using warm water (37°C) for 30 seconds, and sperm vialbility (%M, %L, and %IAR) was evaluated. The datas were record as post-thawing data. Viliability data (initialy, 5°C, and post-thawing data) was analyzed by ANOVA and mean difference was tested using Duncan (SAS 9.1).

Research 2

This study was conducted by 2 x 2 factorial Completely Ramdomized Design with 6 repetitions. As first factor was glycerol concentration by 5 and 7% and the second factor was equilibration time by 3 and 4 hours.

2 ml of media A (Table 4) were added in 4 gram microcapsule, in room temperature. Temperature of suspension of semen was decreased using cooling mechine, temperature will decrease gradually until 5°C (±60 minutes). Two ml freezing media B (Table 4) was added stepwise at 15°C and 10°C to obtain sperm concentration by 50 x 10⁶/ml with glycerol concentration by 5 and 7%.

Equilibration was carried out in the cool top (5°C) for 3 and 4 hours. Packaging of sperm microencapsulated were performed using medium straw (0.5 ml, 25 x 10⁶/straw) at 1 hour after equilibration. Straws were seal using poliviline powder and equilibration was continued until 3 and 4 hours.

The freezing of straw were perfomed using the same methode before. The evaluation of post thawing were done 1 week after storage in N₂ liquid using same methode.

Sperm viability was analyzed using ANOVA. Difference between treatment was tested using Duncan and LSD if there was significant interaction between glycerol concentration and equilibration time (SAS 9.1).

RESULT AND DISCUSSION

Quality of fresh semen

Fresh semen used in this study was feasible to be used in sperma cryopreservation process. The semen has sufficient volume and concentration, average volume 5 ml, concentration 1160 x 10⁹, average percentage of live sperm 82.6% and 87.7% IAR.

Research 1

Alginate and sperm microencapsulation process was affacting sperm vialbility at initial of cryopreservation process.

Sperm motility (%M) in media consisting alginate (T-1II) was lower (P<0.05) than control (T-I), meanwhile microencapsulation process caused %M on encapsulated T-III (55.00%) was lower (P<0.05) than unencapsulated. (66.71 and 60.00% for T-I and T-II, respectively).

The percentage of live sperm (%L) T-III (82%) was also lower (P<0.05) than non-encapsulated (90.14 and and 86.57 for T-I and T-II, respectively).

The percentage of IAR (%IAR) was not significantly different caused by alginate and microencapsulation process (Table 5).

 

Viability of sperm at initialy of cryopreservation

Sperma vialbility (%M, %H dan %IAR) at the beginning of cryopreservation varied depending on process and media where spermatozoa stayed for a hour at the room temperature. Sperm viability inT-PI and T-II was in line with previous study conducted by Kusumaningrum et al. (2105), where %M in media TCEY-Alginate (T-II) lower (P<0.05) than TSKT (T-I), but %L and %IAR were not significant different.

TCEY-Alginate (T-II) lower (P<0.05) than TSKT (T-I), but %L and %IAR were not significant different. Microencapulation process (T-III) significant lower (P<0.05) than non-microencapsulation (T-I and T-II) at initial quality (%M and %H).

Microencapsulation process consisting of 3 stage and every stage is physically and chemicaly give stress to the sperm. At the first stage, formation of microgel: interaction sperm with media TCEY consisting 1.5% alginate which high osmilarity and viscosity decrese the sperm motility (Kusumaningrum et al. 2015). A contact between microencapsulation media with gel former media (1.5% CaCl₂) causing the sperm in immobilization phase. That contact also change sperm membrane condition, where Ca would stimulate change of distribution of lipid and composition pospolipid membrane as occurred on capacitation and acrosome reaction (Landim-Alvarenga et al. 2004).

In the second stage (alginate-chitosan membrane forming), sperm linked with chitosan solution with pH 5.5 and followed by washing process. Low pH might cause lost of spermatozoa membrane integrity (Bohloodi et al. 2012).

In the third stage, reaction of microcapsule-sodium citric solution causing sperm condition change from immotile into motile sperm inside the encapsulation