THE PRODUCTION OF FREEZE-DRIED EGG YOLK POWDER AND ITS EFFECT ON THE QUALITY OF GARUT RAM LIQUID SEMEN

This research aims to evaluate the protective potency of freeze-dried egg yolk powder on Garut ram sperm during the liquid semen preservation process. Semen with good sperm quality was divided into three groups and diluted using the following Tris diluents: fresh egg yolkTris (FEY-Tris), commercial egg yolk powder-Tris (CEY-Tris), and freeze-dried egg yolk powder-Tris (DEY-Tris). Semen that had been diluted was observed every 12 hours until sperm progressive motility was 50%. Sperm quality was tested for progressive motility percentages, viability, intact plasma membrane (IPM), and intact acrosome (IA). Fresh egg yolk had a particle size of 14460.00±330.76 nm, with polydispersity index value (PI) and zeta potential (ZP) being 1.00±0.11 and -9.22±0.30 mV, respectively. The particle size of commercial egg yolk powder was 877.90±168.86 nm with PI value 0.34±0.04 and ZP-28.7±1.24 mV. The particle size of freeze-dried egg yolk powder was 1296.00±86.73 nm, with PI value 0.70±0.04 and ZP -34.5±0.64 mV. Progressive motility percentage showed that DEY-Tris diluent managed to survive to 51.11±6.06% for 168 hours, compared to FEY-Tris (51.25±6.74%) which survived for 156 hours, although no longer than CEY-Tris diluent (53.47±6.33%) which survive for 180 hours (P<0.05). Higher sperm motility was also supported by viability percentage, IPM, and IA, which were all higher (P<0.05). In conclusion, freeze dried egg yolk powder mixed with Tris buffer was able to maintain sperm quality during the liquid semen preservation process. ____________________________________________________________________________________________________________________


INTRODUCTION
Egg yolk-Tris diluent is a semen diluent that is widely used in some countries, including Indonesia. Freeze-dried egg yolk powder has been used to dilute the semen of sheep, goats, and cows (Ansari et al., 2010;Alcay et al., 2015;Alcay et al., 2016;García et al., 2018). Egg yolk is a cryoprotective agent with a collection of fat and proteins with a size of 0.8-10 µm (Stádník et al., 2015;Villarreal et al., 2018). The components of egg yolk that are responsible for the cryoprotectant effect are lecithin, phospholipid, and lipoprotein fraction (Vishwanath and Shannon, 2000;Aboagla and Terada, 2004). Egg yolk as a semen diluent functions as an energy source, a buffer for osmotic pressure, and an extracellular cryoprotectant which can cover the plasma membrane to protect sperm from cold stress and maintain plasma membrane stability during the semen preservation process (Moussa et al., 2002;Amirat et al., 2004;Khalifa and El-Saidy, 2006;Ricker et al., 2006).
Fresh egg yolk is processed into powdered egg yolk by freeze-drying it or by lyophilization, which is a method of drying biological products that involves freezing and dehydration (Alcay et al., 2016). The advantages of this method are that it is able to maintain the quality of the drying results for a long time, is more practical so that it is ready to use without having to manually crack the fresh eggs for the yolk, is available at all times, and is very suitable for protein biomolecules, like the egg yolk (Alcay et al., 2015). This method provides a solution that produces a dry egg yolk product that is stable both in quality and structure, so that it does not change the aroma, color, and other organoleptic elements. It also does not shrink or change shape in the structure of the material, and has high rehydration power with an excellent drying result that is very hollow and is also lyophile so it can return to almost the same physiological, organoleptic, and physical properties before drying (Alcay et al., 2016;García et al., 2018).
Preservation of liquid semen that is stored for long periods of time can result in physical and functional changes of spermatozoa which can reduce motility, viability, plasma membrane integrity, and acrosome integrity (Riha et al., 2006;Chelucci et al., 2015). Using egg yolk powder can be an alternative to fresh egg yolk as a semen diluent in the liquid semen preservation process. In Indonesia, the information regarding the production and use of freeze-dried egg yolk powder as a semen diluent is not yet known. Therefore, this study was conducted with the aim of making, proving, and comparing the potential of egg yolk powder with fresh egg yolk and commercial egg yolk powder on the preservation of liquid Garut ram semen. The benefit of this research is to find an alternative to fresh egg yolk as a semen diluent for Garut ram semen by utilizing freezedried egg yolk powder.

Experimental Animals
The animals used as a source of semen were six male rams in the age group of 3-6 years old with a body weight range of 35-55 kg. Males with good semen quality were identified by the following criteria: motility of more than 75%, a concentration of more than 2500x10 6 cells per mL, and abnormalities less than 15% (Yodmingkwan et al., 2016). The feed given was fresh elephant grass at 20% of body weight and concentrated in the form of pellets containing 16% protein and bran containing 12.5% protein each as much as 2% of body weight. The rams were given this feed twice a day and were watered ad libitum. The use of experimental animals and all handling procedures in the research had received approval from the Commission for the Supervision of Welfare and Use of Animal Research, Bogor Agricultural University, Number 118-2018 IPB.

Research Procedure
The production of freeze-dried egg yolk powder and its effect on the quality of liquid semen consisted of several procedures: making freeze-dried egg yolk powder, analyzing particle size, polydispersity index (PI), and zeta potential (ZP) of fresh egg yolk (FEY), freeze-dried egg yolk powder (DEY powder), and commercial egg yolk powder (CEY powder), measuring zeta potential (ZP) of Garut ram sperm cells, CEY powder and CEY powder morphology, preparing diluent media, collecting semen and evaluating fresh semen, and preserving the liquid semen.

Producing Freeze-Dried Egg Yolk Powder
The egg yolk used for the freeze-dried egg yolk was egg yolk from Lohmann Brown chicken eggs with an age range of 54-84 weeks (Global Buwana Farm, Ciampea, Bogor). The criteria for the eggs used are eggs from brood stock that are not inseminated by males; that are also fresh, oval, dark brown, thick, not cracked or dirty, and with little to no red spots on the eggshells. When the shell was broken down the yolk had to be still intact and the yolk light in color (not pale).
The yolk was separated from the egg white by placing it on filter paper and discarding the egg white. The yolk membrane was then broken down, and placed in a 500 mL measuring cup. The yolk was then frozen at 20° C for 24 hours. The yolk drying process was carried out using a freeze dryer (Christ®, Gamma 1-20) at a temperature of -20° C and a pressure of 1.030-0.630 millibars for 144 hours, and then crushed using a mortar before being filtered with a stainlesssteel sieve to separate the fine and coarse egg yolk powder. Soft egg yolk samples were stored at -20° C.

Analysis of Particle Size, Polydispersity Index and Zeta Potential of Fresh Egg Yolk, Freeze Dried Egg Yolk Powder, and Commercial Egg Yolk Powder
Each 1.04 g of DEY powder and CEY powder were dissolved in 10 mL of aquadest. FEY, CEY powder, and DEY powder were taken at upwards of 45 µL each and mixed with 20 mL of aquadest, before being put into each disposable sizing cuvette for particle measurement and PI value. For ZP measurement, the samples were put into clear disposable zeta cell. Measurements were made using the Malvern Zetasizer Nano Series Nano-ZS (Malvern, UK) in dynamic light scattering (DLS) mode for measurement of particles and PI values, and in electrophoretic light scatting (ELS) mode for ZP measurements (Bhattacharjee, 2016). Each measurement corresponded to twelve autocorrelation functions which were repeated three times.

Zeta Potential Measurement of Garut Ram Sperm Cells
The sperm cell surfaces of the Garut rams were measured using the Malvern Zetasizer Nano Series Nano-ZS (Malvern, UK). The semen diluent medium (1:100) (sperm cells: deionized water) was put into a 1 mL cuvette, and then placed into a zetasizer for measurements (Magdanz et al., 2019). Measurements were made three times and yielded a 100% mean zeta potential of -42.5±7.26 mV (Figure 1). DEY powder and CEY powder was sprinkled on the surface of the double-type carbon that coated the specimen holder, and then they were coated with gold with a sputter current of 20 mA for 60 seconds using a Quorum Q150R ES sputter coater (Quorum Technologies Ltd, UK). DEY powder and CEY powder that had been coated on the specimen holder were installed in during the stage. They were each inserted in the chamber with the imaging using SEM ZEISS Type EVOMA10 (Carls ZEISS, USA). SEM was operated with standard operating parameters using a secondary electron detector, and a working distance of 10.0 mm and an electron high tension (EHT) of 16.00 kV.

Preparation of Diluent Media
The diluent medium used was a Tris buffer diluent containing 2.98 g of Tris hydroxymethyl aminomethane, 1.65 g of citric acid monohydrate, 2 g of fructose, and 100 mL of milli-Q water (Ariantie et al., 2013). The Tris buffer diluent was added with FEY, CEY powder, and DEY powder each up to as much as 20% (Guerrero 2002;Alcay et al., 2016;Anand et al., 2017) as presented in Table 1. FEY-Tris, CEY-Tris, and DEY-Tris diluent were stirred with a speed of 500 rpm. The solution was centrifuged for 30 minutes at a speed of 3000 rpm, and each diluent was added with penicillin and streptomycin antibiotics, each with a dose of 1000 IU/mL and 1 mg/mL, respectively.

Semen Collection and Fresh Semen Evaluation
The semen was collected by using an artificial vagina once a week with six reservoirs, each consisting of two ejaculates (two reservoirs in one holding period). The collected semen was immediately stored in a water bath (32° C) during evaluation and immediately processed into liquid semen. Fresh semen evaluation was carried out both macroscopically and microscopically. The macroscopic evaluation of the semen includes volume, pH, consistency, and color. The volume was determined by observing the measuring pipette. The degree of acidity (pH) was assessed using pH indicator paper 6.4-7.2. Consistency was assessed by tilting the tube with viscous and moderate assessment criteria. The color was visually assessed and differentiated into cream and milky white. Meanwhile, microscopic evaluation of spermatozoa quality included mass movement, progressive motility, individual movement, concentration, viability, abnormal morphology, intact plasma membrane (IPM) and intact acrosomal (IA).
The mass movement was carried out by putting 5 µL of fresh semen on a microscope slide and observing it using a microscope with 100x magnification. The assessment was carried out by looking at the thickness of the mass of the spermatozoa and the speed of the spermatozoa waves migrating (David et al., 2015).
The motility of the spermatozoa and individual movements were observed under a microscope at 400x magnification with 10 µL of semen being added to 80 µL of saline solution. The percentage of spermatozoa motility was estimated from 5 fields of view by comparing the number of spermatozoa moving forward with the movements of other spermatozoa (dead spermatozoa, moving in place, turning, and moving backward) (David et al., 2015).
The concentration of total spermatozoa was counted using Neubauer's counting chamber (Marienfeld, Lauda-Konigshofen, Germany) (O'Brien et al., 2019). The semen was diluted using a formolsaline solution in a ratio of 1:500 (2 µL semen : 998 µL formolsaline) and homogenized in a 1.5 mL microtube. The composition of the formolsaline solution was 6.19 g of disodium hydrogen phosphate dihydrate, 3.54 g of potassium dihydrogen phosphate, 5.41 g of sodium chloride, 125 mL of 37% formaldehyde solution, and 875 mL of Milli-Q water (Nagorsen and Peterson, 1980;Munson, 2000). The total concentration of spermatozoa was observed under a microscope at 400x magnification.
The viability and abnormality of spermatozoa was tested using eosin-nigrosin staining (Barth and Oko, 1989). 5 µL of semen was added with 50 µL of eosin  The composition of the eosin-nigrosin staining was 20 g nigrosin, 1.5 g sodium citrate, 3.3 g eosin yellow, and was dissolved in 300 mL milli-Q water.
The calculations were carried out in 10 fields of view or with a minimum number of spermatozoa with 200 sperm cells using a light microscope with a magnification of 400x. The viability assessment was carried out by looking at the dead spermatozoa that absorbed the red color, and the living ones that did not absorb the color (transparent). Meanwhile, abnormalities were assessed by looking at primary, secondary, and tertiary abnormalities.
IPM spermatozoa was carried out by dissolving 10 µL of semen in 1000 µL of hypoosmotic swelling test (HOS-test) solution before being incubated at 37° C for 30 minutes. The composition of the HOS solution followed the method by Jeyendran et al. (1984), with 7.5 g sodium citrate and 13.52 g fructose dissolved in 1000 mL milli-Q water. The osmolarity of the HOS solution was 150 mOsm. The spermatozoa were calculated in 10 fields of view or with a minimum number of spermatozoa with 200 sperm cells at a microscope magnification of 400x. The IPM was characterized by a coiled spermatozoa tail, while the damaged one was characterized by a straight tail.
IA spermatozoa was done by diluting the semen using formolsaline solution ,which functions to fix the spermatozoa, and was made into a peripheral smear and dried on a bunsen and stained with 5% (v/v) aniline blue and 0.5% (v/v) crystal violet for 15 seconds (Martecikova et al., 2010). Spermatozoa that still have intact acrosomal were marked with 1/2 to 2/3 of the anterior part of the head darker than the posterior part. Meanwhile, incomplete spermatozoa were seen without any boundaries between the anterior and the posterior parts (Petkov et al., 2007). Spermatozoa were calculated in 10 fields of view or with a minimum number of spermatozoa with 200 spermatozoa cells using a CKX41 phase-contrast microscope (Olympus, Tokyo, Japan) at 400x magnification.

Liquid Semen Preservation
Fresh semen from the two ejaculates was mixed into one, then divided into three parts and diluted with FEY-Tris, CEY-Tris, and DEY-Tris diluents ( Table 1). The diluted semen was inserted into a 0.2 mL centrifuge tube with a dilution dose of 50x10 6 spermatozoa per mL. The diluted semen was packed in a tube with a water jacket cooling system, and then stored in a refrigerator (3-5° C) and observed every 12 hours until the motility of spermatozoa was 50% (Ariantie et al., 2014). The quality of the spermatozoa tested was measured by the percentage of progressive motility, viability, IPM and IA.

Data Analysis
The data was analyzed using comparative descriptive analysis and analysis of variance (ANOVA). If a significant difference was found between treatments, the Duncan test was then performed.

Particle Measurement
The results of the FEY particle size measurement were 14460.00±330.76 nm as presented in Table 2. Table 2 shows that the three groups of FEY particle size distribution were not readable on the particle measuring instrument because the accurate size range read on the particle meter ranged from 10-10000 nm. FEY which went through a freeze-drying process for 144 hours at a temperature of -20° C and a pressure of 1.030-0.630 millibars was dried until FEY became a DEY powder. DEY powder was measured using a particle measuring instrument resulting in a size of 1296.00±86.73 nm ( Table 2). Based on Table 2, there are three groups of DEY powder particle sizes, 74.3% DEY powder particles with a size of 749.2 nm and a concentration of 74.3% and the smaller size (191.9 nm) with a concentration of 19.4%. In addition, CEY powder particle size measurements were also carried out and resulted in a size of 877.90±168.86 nm, of which 82.6% of CEY powder particles were 446.7 nm in size and 12.3% had a size of 126.4 nm ( Table 2). The uniformity of particles assessed based on the PI value showed that the FEY particles, DEY powder and CEY powder were 1.00±0.11, 0.70±0.04, and 0.34±0.04, respectively ( Table 2).
The level of particle stability was indicated by the ZP value and the ZP values of the FEY particles, CEY powder and DEY powder were -9.22±0.30 mV, -28.7±1.24 mV and -34.5±0.64 mV, respectively ( Figure 2).
The morphology of all CEY powder particles and DEY powder particles were spherical and polymorphic, respectively ( Figure 3). CEY powder is uniformly spherical, which is also evidenced by the PI value of 0.34±0.04 and ZP -28.7±1.24 mV, which means that the CEY powder has a fairly good uniformity (medium monodipersion) and has a stable charge. DEY powder is in the form of polymorph, proven by the PI value of 0.70±0.04, which means that the DEY powder particles are high in polydispersion, but have a particle charge of -34.5±0.64 mV which means that they are very stable. If the particle morphology of CEY powder and DEY powder were related to particle size using a particle measuring instrument, the size in the particle morphology image was greater than the size on the particle measuring instrument. This is because when taking the image, the area that was taken was an area that had a large particle size. Fresh egg yolk with a size of 14460.00±330.76 nm ( Table 2) was processed into freeze-dried egg yolk powder. The fresh egg yolk freeze-drying process lasted for 144 hours at a temperature of -20° C and a pressure of 1.030-0.630 millibars. In addition to produce freeze-dried egg yolk, the process could reduce the particle size of fresh egg yolk with a size of 1296.00±86.73 nm, with a ZP value of -34.5±0.64 mV which means it is very stable. The freeze-dried egg yolk powder will be dispersed after diluted with distilled water. This monodisperse dilution will induce more free movement of the freeze-dried egg yolk. The size of the freeze-dried egg yolk could not be reduced further by commercial milling equipment because of the hygroscopic nature of the egg yolk. Because egg yolk absorbs water molecules, during the process of reducing the size of the egg yolk powder it had a tendency to stick to the commercial milling tool ( Figure  4). However, the freeze-dried egg yolk powder can be used as a semen diluent and was tested in comparison with fresh egg yolk and commercial egg yolk powder. The particle size of commercial egg yolk powder was measured and had a size of 877.90±168.86 nm.
The shrinkage of freeze-dried egg yolk powder is estimated to only last as long as the yolk becomes freeze-dried egg yolk powder, and the size of the frozen egg yolk powder is estimated to return to its initial size  before the yolk is processed into egg yolk powder. This is based on the statement from Alcay et al. (2016) and García et al. (2018) which states that the freeze-drying process can cause shrinkage of the size of a particle but the freeze-drying product has a high rehydration element with a very hollow and lyophile drying result. This is so that it can return to almost the same physiological, organoleptic and physical properties before drying.

Fresh Semen and Liquid Semen Quality
Macroscopic and microscopic examination results of fresh semen from Garut ram in this study was semen that had a volume of 1.50±0.18 mL, pH 6.4, with a cream-color with moderate to thick consistency. It also tended to have thick mass waves and rapidly changed places, with a percentage of progressive motility of 79.31±1.93% with fast and very fast individual movement.
The average difference between progressive motility and viability of spermatozoa was 11.17%, and the difference between the percentage of IPM and IA and the percentage of progressive motility of spermatozoa up to 50% at the same hour was 11.17%, 9.28%, and 8.35% respectively ( Table 3). The difference in the mean percentage of viability, IPM, and IA was higher than the percentage of motility because of the number of living spermatozoa, having intact plasma membranes and acrosome, but not necessarily all progressive motile.
The concentration of FEY, DEY, and CEY mixed with Tris diluent was 20% each (Guerrero, 2002;Alcay et al., 2016;Anand et al., 2017). The quality of liquid semen using CEY-Tris diluent was longer in maintaining the progressive motility of spermatozoa with a percentage of 53.47±6.33% for 180 hours compared to DEY-Tris and FEY-Tris diluents with the percentage of progressive motility of 51.11±6.06% and  Figure 5). The percentage of progressive motility of spermatozoa in the older CEY-Tris diluent was also supported by the higher percentage of viability, IPM and IA compared to the DEY-Tris and FEY-Tris diluents (Figure 6-8).
The percentage of spermatozoa quality using CEY-Tris diluent was higher because the commercial powder egg yolk added to the Tris diluent was smaller (877.90±168.86 nm) compared to freeze-dried egg yolk powder (1296.00±86.73 nm) and fresh egg yolk (14460.00±330.76 nm) ( Table 2). The smaller size is thought to have less movement resistance, which makes it easier for spermatozoa to move. In addition, small particle sizes have large surface areas (Khan et al., 2019). Ober and Gupta (2011) further explained that the surface area and the percentage of molecules for particle sizes of 100 nm, 1000 nm, 10000 nm are 1.26 x 105 nm2 and 2.97%; 1.26 x 107 nm2 and 0.30%; and 1.26 x 109 and 0.03%. The particle surface area of commercial egg yolk powder that is larger than fresh egg yolk and freeze-dried powder is thought to be more optimal in protecting spermatozoa cell membrane during the preservation process of liquid semen.
Freeze-dried egg yolk powder and commercial egg yolk powder had a particle size distribution marked with a PI value of 0.70±0.04 and 0.34±0.04, respectively, which means that the two powdered egg yolks have moderate levels of polydispersion (Ponnuraj et al., 2015). The PI value of fresh egg yolk was 1.00±0.11, which means that it has a very high level of polydispersion (Ponnuraj et al., 2015). Ponnuraj et al. (2015) further described that the range of PI values is divided into 4: 1) 0-0.05 (high monodispersion), 2) 0.05-0.08 (moderate monodispersion), 3) 0.08-0.7 (moderate polydispersion), and 4) >0.7 (high polydispersion). Based on the PI value, it shows that freeze-dried egg yolk powder and commercial egg yolk powder have a slightly better uniformity levels than fresh egg yolk, although based on the literature it has a moderate polydispersion level.
Freeze-dried egg yolk powder had a particle charge marked with a ZP value of -34.5±0.64 mV which means it is very stable, and commercial egg yolk powder had a charge value of -28.7±1.24 mV which means that it is also quite stable, while fresh egg yolk had a charge value of -9.22±0.30 mV which means it is very unstable (Bhattacharjee, 2016). Further explained by Bhattacharjee (2016), the ZP value is classified into 4 parts: 1) very unstable (0-10 mV), 2) quite stable (10-20 mV), 3) stable (20-30 mV) ), 4) very stable (˃30 mV). ZP values greater than +30 mV or less than -30  mV will produce a stable particle. If the particles have a large negative or positive ZP value, the particles will repel each other and there will be no aggregation. On the other hand, if the particles have a small ZP value there is no force to prevent the particles from attracting each other and aggregation will occur (Yedurkar et al., 2016). The overall surface of the Garut ram spermatozoa cells was -42.5±7.26 mV. Meanwhile, the surface of bovine spermatozoa cells has a ZP value of -13.00 to -27.7 mV (Magdanz et al., 2019). Magdanz et al. 2019 explained that even though the overall cell load of spermatozoa is negative, it is possible that some areas are positively charged on the surface of the spermatozoa cell membrane due to the loss of integration of negatively charged glycoproteins. Spermatozoa cells from the epididymis and ejaculation will cause a dynamic change in the surface charge of the spermatozoa from negative to positive (Simon et al., 2016). Simon et al. (2016) explained that changes in the surface charge of spermatozoa occur due to the accumulation of components in the form of proteins and glycoproteins from the epididymis which gives the sperm a negative charge. The glycosylation of sialic acid residues forms a negative layer of 20-60 nm thick around the sperm which gives a negative charge of -16 to -20 mV (Yudin et al., 2005;Chan et al., 2006). Spermatozoa become positively charged when they lose glycoproteins attached to the plasma membrane (Simon et al., 2016). Loss of glycoproteins occurs during apoptosis when the integrity of the membrane is lost due to translocation of phosphatidylserine from the inside to the outside of the plasma membrane and affects the fluidity of the plasma membrane (Mourdjeva et al., 2005).
Egg yolk components that are involved in protecting spermatozoa during the preservation process of liquid semen include phospholipids, low-density lipoproteins (LDL), and cholesterol (Moussa et al., 2002;Aboagla and Terada, 2004;Bergeron and Manjunath, 2006;Mocé et al., 2010). These components are believed to be able to associate with the plasma membrane to form a protective layer on the surface and are able to replace plasma membrane phospholipids and maintain plasma membrane integrity (Kampschmidt et al., 1953;Quinn et al., 1980;Graham and Foote, 1987;Moussa et al., 2002;Hu et al., 2011). The use of fresh egg yolk can be replaced with freezedried egg yolk powder and commercial egg yolk powder because the two egg yolk powders have been shown to maintain the quality of spermatozoa during the liquid semen preservation process.

CONCLUSION
Freeze-dried egg yolk powder-Tris has the potential to maintain spermatozoa quality (up to a percentage of spermatozoa motility of 50%) longer (168 hours) than fresh egg yolk-Tris (156 hours). Commercial egg yolk powder-Tris can maintain the quality of liquid semen for 180 hours longer than freeze-dried egg yolk powder-Tris and fresh egg yolk-Tris.