THE EFFECT OF GLUTATHIONE ADDITION IN SPERM DILUENTS ON THE QUALITY OF BOVINE CHILLED SEMEN 1)

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1 64 Indonesian Journal of Agriculture 1(1), 2008: E. Triwulanningsih et al. THE EFFECT OF GLUTATHIONE ADDITION IN SPERM DILUENTS ON THE QUALITY OF BOVINE CHILLED SEMEN 1) E. Triwulanningsih, P. Situmorang, T. Sugiarti, R.G. Sianturi, and D.A. Kusumaningrum Indonesian Research Institute for Animal Production Jalan Raya Tapos, Ciawi, PO Box 221, Bogor 16002, Indonesia ABSTRACT. This study was conducted at the laboratory of physiology reproduction of the Indonesian Research Institute for Animal Production, Ciawi-Bogor, West Java. Sperms were collected from FH bulls with body weight of 613 kg (FH-1) and 480 kg (FH-2) twice a week. Briefly after quality evaluation, semen was diluted in Tris-citrate buffer medium containing egg yolk (20% v/v) and glycerol (4% v/v) to get spermatozoa concentration of 50 x 10 6 per ml. Sperm diluents were then added with glutathione with doses of 0.0, 0.5, 1.0, and 1.5 mm as treatment A, B, C, and D, respectively. The diluted semen was then cooled from 35 o C to 5 C using a cooling machine for 60 minutes then stored in the refrigerator (5 C). Parameters observed were the survivability of spermatozoa by evaluating the percentage of motile and live, and the condition of acrosome and plasma membrane. The experiment was arranged in randomized split block design and the data were analyzed with general linear model procedure. The results showed that the characteristics of collected semen were normal. Viability of spermatozoa stored at 5 C for 0, 1, 4 and 8 days shown by intact acrosomal were 74.42, 69.27, 57.80, and 42.58%, respectively, and for A, B, C, and D treatments were 60.82, 62.75, and 58.73%, respectively. Those data were significantly different (P<0.01). Motility, live, and intact plasma membranes were 46.72, 52.34, and 51.09%; 63.59, 69.11, and 66.89%; and 66.01, 69.75, and 68.44% for treatment A, B, C and D, respectively. Addition of 0.5 mm glutathione gave the highest (P<0.01) motility, live and intact plasma membranes of sperm. Therefore, it is concluded that addition of 0.5 mm glutathione to the sperm diluents can improve the viability of spermatozoa and possibly protect the spermatozoa from free radical damage. [Keywords: Glutathione, viability, spermatozoa] INTRODUCTION Artificial insemination technology has widely been implemented, particularly on dairy cattle. However, the success of pregnancy level still very much depends on 1) Article in bahasa Indonesia has been published in Jurnal Ilmu Ternak dan Veteriner Vol. 8 No. 2, 2003, p the technology in preserving spermatozoa in addition to the human factor (farmers and inseminators). Spermatozoa chilling process by decreasing the temperature to -196 C in liquid nitrogen causes around 30% spermatozoa died (Goldman et al. 1991; Park and Graham 1992). In the semen chilling process, the problem commonly encountered is the damage of spermatozoa plasma membranes due to the formation of lipid peroxidation. This condition occurs as the spermatozoa membranes contain a lot of unsaturated fatty acids which very susceptible to peroxidation damage (Maxwell and Watson 1996). Furthermore, Sikka (1996) stated that mammal spermatozoa are rich of unsaturated fatty acids and very easily subjected to reactive oxygen species (ROS) that can decrease spermatozoa mortality and increase the morphological damage influencing the sperm capacity and acrosome reaction. Peroxidation of lipid on plasma membranes is the key mechanism in ROS causing the damage of spermatozoa and infertility apart from the fact that peroxidation can damage DNA and protein. Glutathione (C 10 H 17 N 3 O 6 S) and its derivates that are tripeptide (γ-glu-cys-gly) can influence a lot of metabolism aspects (De Matos and Furnus 2000), such as assist detoxification and transportation of γ-glutamilamino acid. Wijaya (1996) reporeted that glutathione is a primary antioxidant working by preventing the formation of new free radicals. This antioxidant alters the available free radicals to molecules that have less negative impacts. According to Karyadi (1997), the free radicals are very unstable atoms or molecules as they possess one or more unpaired electrons, thus to obtain paired electrons, this compound reacts with other atoms or molecules such as unsaturated fatty acids, proteins, nucleic acids or lipopolyssacharides that result in abnormal compounds. Among the negative effects of lipid peroxide on somatic cells are the inhibition of the oxidative and glycolisis metabolisms, lyses of erythrocytes, oxidation on sulfyhydril and the inhibition of the work of SH enzyme, modification of proteins and amino acids, membrane damages, inactivation of membrane bonding enzymes, and

2 The effect of glutathione addition in sperm diluents denaturation of DNA (White 1993; Sikka 1996). The addition of glutathione to spermatozoa diluents is expected to decrease or prevent the emergence of free radicals that will ruin the plasma membranes, thus the fertility of liquid semen increases and eventually it will improve the conception (C/R) and pregnancy percentage. Other inhibiting factors are the availability of liquid nitrogen and expensive liquid nitrogen tube in regions. Therefore, the use of chilled semen is an alternative that can be implemented to overcome those problems. In chilled semen, spermatozoa concentration is lower compared to that in frozen semen, thus one ejaculation can be used to inseminate more estrus female animals. However, the problem faced is the slight decrease of fertility due to the increase of storage time of the chilled semen. In the previous research, Situmorang et al. (2001a) reported that the percentage of pregnancy decreased after the chilled semen was stored more than 4 days. One of the causes is the presence of free radicals capable of damaging spermatozoa cells. Parris (1998) stated that glutathione is a crucial antioxidant that can protect mitochondria due to the presence of free radicals. Glutathione can control homeostatic both inside and outside the cells. Glutathione is a sulfhydril antioxidant (-SH), an antitoxin and enzyme cofactor. As on the antioxidant characteristic that can neutralize free radical, the addition of glutathione as a primary antioxidant is expected to reduce the damage of plasma membranes. A research was conducted to find out the preserving technology for chilled semen by the addition of glutathione to the sperm diluents. MATERIALS AND METHODS This research was conducted at the laboratory of physiology reproduction of the Indonesian Research Institute for Animal Production, Ciawi-Bogor, West Java, using two bulls ages of around 3.5 years with life weights of 613 kg (FH-1) and 480 kg (FH-2). As an elicitor a FH cow was used. The feed given were elephant grass, 5 kg of tofu meal, and 6 kg of commercial concentrate. Semen was collected twice weekly by using artificial vagina. Semen evaluation was conducted macroscopically and microscopically. The macroscopic evaluation comprises smell, color, consistency, and volume and it was conducted immediately after the semen was collected. Meanwhile, the microscopic evaluation consists of concentration, mobility, life percentage, whole acrosom cover, and percentage of whole plasma membranes. The treatments used were the addition of glutathione to the diluents as much as 0 mm (control), 0.5, 1.0 and 1.5 mm each as treatment A, B, C and D, respectively. Dilution was conducted by using Tris-citrate buffer at 35 C by Table 1. The composition of tris-citrate buffer solutions. Materials Solution A Solution B Tris (hydroxymethyl) amino methane (g) Citric acid (g) Fructose (g) Streptomycin (mg/ml) Benzyl penicillin (IU/ml) Glycerol (% v/v) Egg yolk (% v/v) Glutathione (mm) 0; 0.5; 1.0; 1.5 0; 0.5; 1.0; 1.5 adding diluents medium A containing 20% (v/v) egg yolk and 2.4% (v/v) glycerol so that a spermatozoa concentration of 100 x 10 6 per ml was obtained (Table 1). The solution containing the semen was then cooled from 35 to 5 C using cooling machine for 60 minutes. During the temperature lowering, solution B containing Tris-citrate buffer with 20% (v/v) egg yolk and 5.6% (v/v) glycerol was added when the temperature reached 15, 10 and 5 C thus the concentration of the spermatozoa became 50 x 10 6 per ml. Semen solution in line with the treatments was examined for its viability at 5 C by counting the percentages of life motility, whole acrosome cover, and whole plasma membranes at day 0, 1, 4, and 8. The experiment was arranged in completely randomized design with four factors of treatments (four doses of glutathione and four levels of storage time), with total semen collecting of eight times as replicates (Snedecor and Cochran 1974; Steel and Torrie 1991). Data collected were analyzed by general linier (SAS 1984). RESULTS AND DISCUSSION The average quality of FH bull semen (Table 2) used in this experiment was within the normal range and suitable to be diluted and stored as chilled semen at 5 C. The average volume of fresh semen from both bulls ranged from 3.70 to 6.18 ml for FH-1 and from 3.02 to 6.22 ml for FH-2. These results were within the normal ranges. The semen volume of each bull was influenced by life weight, feed, libido, individual, collecting frequency, and species (Toelihere 1993). The average spermatozoa concentration used is in line with that reported by Toelihere (1993), that the concentration of fresh semen ranged between 1000 and 2000 millions or more spermatozoa per ml, having viscous consistency and creamy color. The semen concentration is very much influenced by age, individual variation, feed, testes size, and sexual development (Salisbury and Van Demark 1985).

3 66 E. Triwulanningsih et al. The percentages of motility and life spermatozoa in line with the glutathione doses and storing periods at 5 C is presented in Table 2. Factually, the longer the storage period, the lower the motility is. This is in line with that reported by Situmorang et al. (2000b) who used vitamin E (α-tocoferol) 0.1% and phospholipids as the antioxidants. However, it is apparent that the effect of glutathione as the antioxidant could increase the motility (P < 0.01) by %; % and %, for glutathione doses of 0.5, 1.0, and 1.5 mm, respectively, compared to those without glutathione ( %). However, among the glutathione treatments, it is insignificantly different (P > 0.05), hence the addition of 0.5 mm Table 2. The average quality of fresh semen from two FH bulls. Semen assessment FH-1 Bull FH-2 Bull Macroscopic Volume (ml) Smell normal normal Consistency medium-viscous medium-viscous Color creamy creamy Microscopic Concentration (10 6 per ml) Mass movement Percentage of life spermatozoa Percentage of whole acrosome cover Percentage of whole plasma membrane +++ = very good mass movement. glutathione to the diluents is sufficiently effective to protect the plasma membranes and maintain the percentage of spermatozoa motility stored at 5 C. This results is in line with that reported by Sinha et al. (1996) that the addition of 5 mm glutathione to goat semen diluents increased the motility (55.7% versus 47.81%) accompanied by the lowness of acrosome abnormality (6.22% versus 10,10%) compared to that without glutathione. The percentage of pregnancy also increased from 49.23% to 59.18% after insemination with chilled semen that had been thawed. Furthermore, Foote et al. (2002) stated that the addition of 0.5 mm glutathione, with or without superoxide dismutase (SOD), can improve the percentage of the bull spermatozoa motility. The results obtained in this experiment showed that addition of 0.5 mm glutathione as an antioxidant can prevent the damage of membrane cells due to the presence of free radicals as stated by Parris (1998). The spermatozoa motility percentages using 0.1% vitamin E as the antioxidant at 5 C were 51.4, 40.0, and 25.0% for storing period of 0, 3, and 7 days, respectively (Situmorang et al. 2001b). When compared with the results of this experiment, the use of glutathione is slightly better as at day 0 and day 4 the motilities obtained were and 45.62%, respectively. As stated before, mammal spermatozoa are extremely susceptible to lipid peroxidation that can cause structural damage of spermatozoa and motility (Sinha et al. 1996). In this experiment, 20% (v/v) egg yolk was used that might cause different results compared to those in the previous experiments. Situmorang (2002) reported that for chilled semen (5 C) it is sufficient to use 10% (v/v) egg yolk and 20% (v/v) for frozen semen, in which the percentages of motilities obtained were 42 and 30% on day 3 and 7, respectively. When the egg yolk Table 3. The average percentages of motility and life spermatozoa based on glutathione doses and chilled semen storing period at 5 C. Storing period (day) Glutathione dose (mm) % Average Motility (%) a b c d Average b a a a Life (%) a b c d Average c a ab b Numbers followed by different letters in the same lines and columns are very significantly different (P < 0.01).

4 The effect of glutathione addition in sperm diluents was increased to 20%, the motility obtained was 40.5% (day 3) and 30.5% (day 7). In this experiment, the motilities obtained on day 4 were 41.25% (control), 48.75% (0.5 mm), 48.12% (1 mm) and 44.37% (1.5 mm). These results are slightly better compared to those from the previous experiment in which the average motility on day 4 was higher (45.62%) compared to that on day 3 of 40.5% as reported by Situmorang (2002). In Table 3 it can be seen that the average percentages of life spermatozoa significantly (P < 0.1) decreased in line with the length of storing period, i.e , 72.00, 66.42, and 54.11% for semen stored on day 0, 1, 4 and 8, respectively. The glutathione addition to diluents obviously improved the spermatozoa condition (P < 0.01). The average percentages of life spermatozoa were 63.59, 69.11, 68.64, and 66.89% for treatments with 0.0, 0.5, 1.0, and 1.5 mm glutathione, respectively. However, treatment B and C and between treatment C and D were insignificantly different (P > 0.05), while treatment B and D were significantly different (P < 0.01). From the results obtained it can be seen that the use of 0.5 mm glutathione is more effective compared to other treatments. This shows that glutathione can suppress the presence of free radicals capable of decreasing the percentage of life spermatozoa stored at 5 C. In the experiment using 0.1% vitamin E as the antioxidant, the life percentages obtained were 70.1, 49.7 and 41.7% for storing on day 0, 3 and 7, respectively (Situmorang et al. 2001b). In this experiment, it is apparent that the addition of glutathione is more effective compared to that of 0.1% vitamin E, as on day 4 and 8 the percentages of life spermatozoa were and 54.11%. Meanwhile, in the experiment where 0.5 mm phospholipids in the diluents of Tris citrate buffer containing 10% (v/v) egg yolk, the life percentages obtained were 55 and 46% (Situmorang 2002); and when the egg yolk was increased to 20%, the life percentages became 52 and 47.3% on day 3 and 7, respectively. The percentage of spermatozoa whole plasma membranes and whole acrosome covers in line with glutathione doses and semen storing period at 5 C is presented in Table 4. Obviously the addition of glutathione can increase the percentage of whole plasma membranes (P < 0.01), but intertreatment was not significantly different (P > 0.05). The averages of each whole plasma membranes were 66.01, 69.75, 68.38, and 68.44% for treatments of 0.0, 0.5, 1.0, and 1.5 mm glutathione, respectively. Based on the results of this experiment, the percentages of whole plasma membranes and whole acrosome covers decreased (P < 0.01) in line with the increase in storing period. It can be stated that the addition of 0.5 mm glutathione is adequately effective in maintaining plasma membranes from damages due to the presence of free radicals. Meanwhile the percentage of whole acrosome covers increased due to the addition of 0.5 mm glutathione then decreased as the increase in glutathione concentration in the diluents, but between treatments of 0.5 mm (62.75%) and 1.0 mm (61.77%) they were insignificantly different (P > 0.05). The percentages of whole acrosome covers in each treatment were 60.81, 62.75, and 58.73% for 0.0, 0.5, 1.0 and 1.5 mm, respectively. Sinha et al. (1996) reported that goat semen diluents in the form of Tris added with 2 mm glutathione and Tris added with 5 mm glutathione evidently decreased the abnormality of goat spermatozoa after being chilled and thawed again each 10.10, 8.63 and 6.98%. Meanwhile, Situmorang et al. (2002) stated that the addition of g/100 ml catalyst to the diluents can increase motility percentage, but the catalyst addition can be more effective if 0.5 mm glutathione is simultaneously Table 4. The average percentages of whole plasma membranes and whole acrosom cover of spermatozoa based on glutathione doses and semen storing period at 5 C. Storing period (day) Glutathione dose (mm) Average Whole plasma membrane (%) a b c d Average b a a a Whole acrosome cover (%) a b c d Average ab a a b Numbers followed by different letters in the same lines and columns are very significantly different (P < 0.01).

5 68 E. Triwulanningsih et al. added. Furthermore, the addition of merely 0.5 mm glutathione has the same effectiveness as the addition of catalyst and glutathione. This is caused by different preventive mechanism. Generally, the damage of acrosome membranes occurs at the last stage or at capacity time. The presence of glutathione and catalyst protects acrosome plasma membranes resulting in delayed damages or the damages have not yet occurred. Beconi et al. (1993) reported that the addition of 1 mg/ ml vitamin E to FH bull semen diluents can overcome the damage speed of plasma membranes and protect the plasma membranes against lipid peroxidation by protecting the activity of superoxide dismutase (SOD) enzyme and maintaining cell metabolism and functions. Plasma membrane damages may be caused by several factors such as: (1) the availability of nutrition for decreasing spermatozoa; (2) unsuitable environments like low temperature; (3) the formation of lactic acid from metabolism residue that can decease the ph; (4) inappropriate diluting and chilling processes; (5) the formation of lipid peroxidation reaction; (6) low quality semen as a lot of abnormal spermatozoa were found; and (7) the occurrence of cell damages. Based on the results of this experiment, the use of 0.5 mm glutathione in diluents is adequately effective in making chilled semen as it can increase the percentages of whole plasma membranes and whole acrosome covers of spermatozoa. Kim et al. (1999) reported that the addition of 1 mm glutathione in fertilization medium in the in vitro fertilization process produced different blastosis, depending on the males used. For males with low fertility by in vitro, the addition of 1 mm glutathione became effective, in which the blastosis percentage increased from 29.8% (without glutathione) to 39.4%. This is caused by the increase in ROS during in vitro culture that can cause cell damages. Sikka (1996) stated that ROS has a potential implication in biological reproduction, including superoxide (O 2- ) anion, hydrogen peroxide (H 2 O 2 ), peroxil radicals (ROO - ), reactive hydroxyl (OH - ), nitrite oxide radicals (NO - ), and peroxynitrite (ONOO - ). The effect of (NO - ) depends on the concentration and interaction with hydrogen peroxide. ROS increase causes the damages of DNA, protein and lipid, increases oxide stress including lipid peroxide, thus decreasing motility, viability, capacitating, acrosome reactions and spermatozoa functions in general resulted in the declining of fertility. CONCLUSION The addition of 0.5 mm glutathione to Tris citrate buffer diluents is sufficiently effective in improving the condition of chilled semen plasma membranes stored at 5 C, thus it can increase the percentages of motility, life, whole membranes, and whole acrosome covers. To prove the results of this experiment, a deeper experiment is necessary to be conducted. ACKNOWLEDGEMENT This acknowledgement is extended to Ir. Ahmad Arifin and all parties that have helped the smoothness of this research. REFERENCES Beconi, M.T., C.R. Francia, N.G. Mora, and M.A. Affranchine Effect of natural antioxidants on frozen bovine semen preservation. Theriogenology 40: De Matos, D.G. and C.C. Furnus The importance of having high glutathione (GSH) level after bovine in vitro maturation on embryo development. Effect of beta-mercaptoethanol, cystein and cystine. Theriogenology 53: Foote, P.A., C.C. Brocket, and M.T. Caproth Motility and fertility of bull in whole milk extender containing antioxidant. 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