EFFECT OF GLUTATHIONE ADDITION TO THE SPERM DILUENT MEDIUM ON QUALITY OF BOVINE CHILLED SEMEN 1)

Transcription

1 60 Indonesian Journal of Agriculture 3(1), 2010: Endang Triwulanningsih et al. EFFECT OF GLUTATHIONE ADDITION TO THE SPERM DILUENT MEDIUM ON QUALITY OF BOVINE CHILLED SEMEN 1) Endang Triwulanningsih, P. Situmorang, T. Sugiarti, R.G. Sianturi, and D.A. Kusumaningrum Indonesian Research Institute for Animal Production, Jalan Banjarwaru, PO Box 210, Bogor 16002, West Java Phone: (0251) , , Facs.: (0251) , ABSTRACT A study was conducted at the Laboratory of Reproductive Physiology of the Indonesian Research Institute for Animal Production, Bogor, West Java. Semen was collected from two FH bulls, each with body weight of 613 kg (FH-1) and 480 kg (FH-2), twice a week. After evaluation, the semen was diluted in a Tris-citrate buffer medium containing egg yolk (20% v/v) and glycerol (4% v/v) to obtain a sperm concentration of 50 x 10 6 per ml. Glutathione at dosages of 0.0, 0.5, 1.0, and 1.5 mm were added to the semen diluents as treatments A, B, C, and D, respectively. The diluted semen was then cooled down from 35 C to 5 C for 60 minutes using a cooling machine and stored in a refrigerator (5 C). Parameters observed were survivability of the sperms based on the percent of motility and percent of survival, as well as conditions of the acrosome and the plasma membrane. The data were analysed by the completely randomized design using the general linear model. The results showed that the characteristics of the collected semen were normal. There were significant differences (P<0.01) among the treatments. Survivabilities of the sperms stored at 5 C for 0, 1, 4 and 8 days as indicated by the percentages of intact acrosomal cap (IAC) were 74.42, 69.27, and 42.58%, respectively. Among the treatments, the sperm survivabilities were 60.82, 62.75, 61.76, and 58.73% for treatments A, B, C, and D, respectively. The sperm motility, sperm viability, and intact plasma membrane were 46.72, 52.34, 53.44, and 51.09%; 63.59, 69.11, 68.64, and 66.89%; and 66.01, 69.75, 68.38, and 68.44% in treatments A, B, C and D, respectively. Addition of 0.5 mm glutathione to the semen diluent gave the highest motility, viability, and intact plasma membrane of the sperm (P <0.01). It was concluded that addition of 0.5 mm glutathione to the semen diluent improved survivability of the sperms and probably prevented the sperms from free radical damages. [Keywords: Glutathione, semen preservation] INTRODUCTION Artificial insemination has been widely performed, especially in dairy cows, but the pregnancy is still dependent on the success of semen preservation technology, besides 1) Article in bahasa Indonesia has been published in Jurnal Ilmu Ternak dan Veteriner Vol. 8 No. 2, 2003, p on the human factors (breeders and inseminators). Semen freezing process by lowering the temperature until it reaches -196 C in liquid nitrogen causes approximately 30% death of the sperms (Goldman et al. 1991; Park and Graham 1992). In semen freezing process, problem that often occurs is damage of the sperm plasma membrane due to the lipid peroxidation. This is because many of the sperm membre contain unsaturated fatty acids that are highly susceptible to peroxidation (Maxwell and Watson 1996). Furthermore, Sikka (1996) stated that mammalian sperms are rich in unsaturated fatty acids and highly susceptible to reactive oxygen species (ROS), which lead to the decrease in sperm motility and increase in morphological damages that affect the sperm capacitation and acrosome reaction. Lipid peroxydation in the plasma membranes is the key mechanism of ROS that causes damage and infertility of sperms. Besides, fat peroxydation can damage the DNA and protein. Glutathione (C 10 H 17 N 3 O 6 S) and its derivatives, which are tripeptides (γ-glu-cys-gly) affect many aspects of metabolism (De Matos dan Furnus 2000), among others, help detoxification and transport of γ-glutamyl-amino acid. Wijaya (1996) stated that glutathione is a primary antioxidant that works by preventing the formation of new free radicals. This antioxidant changes the existing free radicals into molecules that have less negative impact. According to Karyadi (1997), free radicals are atoms or molecules that are very unstable because they have one or more electrons which are not paired. To obtain paired electrons, these compounds have to react with atoms or other molecules, such as unsaturated fatty acids, proteins, nucleic acids or lipopolysaccharides that would lead to the formation of abnormal compounds. Among the negative effects of lipid peroxides to somatic cells are inhibitation of oxidative metabolism and glycolysis, lysis of erythrocytes, oxidation of sulfyhydril, inhibitation of SH enzyme function, modification of protein and amino acids, membrane damages, inactivate membrane binding enzymes, and denaturation (White 1993; Sikka 1996). The addition of glutathione into the semen diluent medium is expected to reduce or prevent the presence of free radicals that potentially damage the plasma membrane, so that

2 Effect of glutathione addition to sperm diluent medium fertility of the liquid semen increase, and at the end improve the degree of conception (C/R) and gestation. Another inhibiting factor is the availability of liquid nitrogen and liquid nitrogen tube which are expensive in the local region. The use of liquid semen is therefore a better alternative to overcome these problems. In liquid semen, the sperm concentration is lower than that in frozen semen, so that in one ejaculate can be used to inseminate more estrus female cattle. The problem, however, is the slight decrease in fertility of the liquid semen with the increase in length of storage period. Situmorang et al. (2001a) stated that the percentage of gestation decreased after the liquid semen was stored for more than 4 days. One reason is the presence of free radicals that damage the sperms. Parris (1998) mentioned that glutathione is an important antioxidant that can protect mitochondria from damage caused by free radicals. Glutathione controls homeostatic both inside and outside the cells. Glutathione is a sulfhydril (-SH) antioxidant, antitoxin and enzyme cofactor. Based on antioxidant properties that can neutralize free radicals, addition of glutathione into the medium can reduce plasma membrane damages. A study aimed to determine liquid semen preservation technology with the addition of glutathione into the sperm diluent medium. MATERIALS AND METHODS Research was conducted at the Laboratory of Reproductive Physiology of the Indonesian Research Institute for Animal Production (IRIAP), Bogor, West Java. Two 3.5 year-old FH bulls each weighing of about 613 kg (FH-1) and 480 kg (FH-2) were used as sources of semen, whereas a female cow was used as bait. The bulls were feed with elephant grass, 5 kg tofu, and 6 kg commercial feed concentrate. Semen samples were collected twice a week using an artificial vagina. Evaluations on the semen characteristics were made both macroscopically and microscopically. The macroscopic evaluation was done immediately after the semen was collected; this includes semen odor, color, consistency, and volume. The microscopic examination included sperm concentration, sperm motility, survival percentage, percentage of intact acrosome cap (IAC), and percentage of intact plasma membrane (IPM). The treatments were addition of glutathione into the sperm diluent medium at dosages of 0 (control), 0.5, 1.0, and 1.5 mm, respectively, which were named treatments A, B, C and D. Dilutions were made using Tris-citrate buffer at 35 C by adding diluent medium A that contained 20% (v/v) egg yolk and 2.4% (v/v) glycerol to obtain a sperm concentration of 100 x 10 6 per ml (Table 1). The semen Table 1. Composition of the Tris-citrate buffer solution as bovine semen diluent medium. Material Solution A Solution B Tris (hydroxymethyl) amino methane (g) Citric acid (g) Fructose (g) Streptomycine (mg/ml) Benzylpenicillin (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 diluents were cooled from 35 C to 5 C using a refrigerator for 60 minutes. During the temperature decline, solution B that contained Tris-citrate buffer, 20% (v/v) egg yolk, and 5.6% (v/v) glycerol was added to the diluent when the temperature reached 15 C, 10 C, and 5 C, respectively, so that the sperm concentration was 50 x 10 6 per ml. The semen diluents, according to their treatments, were observed for the sperm viability at 5 C by counting the percentage of sperm motility, viability, IAC, and IPM on days 0, 1, 4, and 8. The experimental design used was factorial in a completely randomized design (four dosages of glutathione and four levels of storage day) with the eigth semen sampling as replicates (Steel and Torrie 1991). The data were analyzed with the general linear model (SAS 1984). RESULTS AND DISCUSSION Average quality of the FH bull semen used in this study was in the normal range and reasonable for dilution and use as source of chilled semen at 5 C (Table 2). The average volume of fresh semen per sampling from the two FH bulls ranged from 3.70 to 6.18 ml for the FH-1 and 3.02 to 6.22 ml for the FH-2. These results are in the normal range. The volume of semen produced by each individual bull was affected by life body weight, feed, libido, individuals, frequency of sampling, and the nation (Toilihere 1993). The average sperm concentration used in this study was in line with that reported by Toelihere (1993), which ranged from 10 x 10 8 to 20 x 10 8 or more sperms per ml with thick consistency and creamy color. The sperm concentration was much influenced by age, individual variation, diet, testes size, and the sexual development (Salisbury and van Demark 1985). Table 3 shows percentages of motility and viable sperms according to glutathione dosages and storage times at 5 C. The sperm motility declined with the longer time of storage. This is in accordance with the report of Situmorang et al. (2001b),

3 62 Endang Triwulanningsih et al. Table 2. Characteristics of fresh semen collected from FH-1 and FH-2 bulls. Semen characteristic FH-1 FH-2 Macroscopic Volume (ml) Odor Normal Normal Consistency Medium-thick Medium-thick Color Creamy Creamy Microscopic Concentration (10 6 per ml) Mass motility Viable spermatozoa (%) Intact acrosomal cap (%) Intact plasma membrane (%) = high mass motility which uses vitamin E (γ-tocoferol) 0.1% as an antioxidant and phospholipid in the semen diluents. However, the use of glutathione at 0.5, 1.0 and 1.5 mm increased the sperm motility significantly (P < 0.01) by %; %; and %, respectively, compared to that without glutathione ( %). Among treatments with different dosages of glutathione, however, there was no significant different effect on the sperm motility (P > 0.05). This means that the addition of 0.5 mm glutathione into the semen diluent medium is sufficiently effective to protect the sperm plasma membrane and minimize the percentage of sperm motility stored at 5 C. These results are in accordance with that reported by Sinha et al. (1996) that the addition of 5 mm glutathione into the goat semen diluent improved sperm motility (55.7% vs 47.81%) followed by a low level of acrosome abnormalities (6.22% vs 10.10%) than those without glutathione addition. Similarly, the percentage of gestation increased from 49.23% to 59.18% after insemination using frozen semen that has been thawed back. Meanwhile, Foote et al. (2002) reported that addition of 0.5 mm glutathione with or without superoxide dismutase (SOD) improved sperm motility of cows. Results of the study illustrate that the addition of glutathione 0.5 mm can prevent cell membrane damage caused by free radicals, such as reported by Parris (1998). Situmorang et al. (2001b) reported that the percentages of sperm motility upon storages at 5 C for 0, 3 and 7 days in a medium containing 0.1% vitamin E as an antioxidant were 51.4%, 40%, and 25%, respectively. When compared with results of this experiment, the effect of glutathione was a little better than that of vitamin E because at days 0 and 4 of storages, the sperm motilities were 73.44% and 45.62%, respectively. As has been stated, mammalian sperms are susceptible to lipid peroxidation; it damages the sperm cell structure that eventually lowered metabolism and motility of the cells (Sinha et al. 1996). The use of egg yolk in this study has probably given the different results from that of the previous researchers. Situmorang (2002) reported that addition of 10% (v/v) egg yolk to liquid semen (5 C) and 20% (v/v) egg yolk to frozen semen resulted in 42% and 30% sperm motilites after storage for 3 and 7 days, respectively. When the amount of egg yolk was increased to 20% (v/v), the sperm motilities were 40.5% and 30.5% at 3 and 7 days of storage, respectively. In this research, the sperm motility on day 4 of storage at 5 C were 41.25% (control), 48.75% (treatment with 0.5 mm glutathione), 48.12% (treatment with 1 mm glutathione), and 44.37% (treatment with 1.5 mm glutathione), respectively. The results were slightly better than that of the previous research; at day 4 of storage, the average of sperm motility was higher (45.62%) than that of the previous research (40.5%) at day 3 of storage as reported Situmorang (2002). Data in Table 3 show that the mean percentages of life sperms significantly (P < 0.01) decreased with the increase in storage period, i.e %, 72.00%, 66.42%, and 54.11% for semen stored for 0, 1, 4 and 8 days, respectively. The addition of glutathione into the sperm diluent seemed to improve the sperm condition (P < 0.01). The mean percentages of viable sperms were 63.59%, 69.11%, 68.64%, and 66.89% in treatments with 0.0, 0.5, 1.0, and 1.5 mm glutathione, respectively. The mean percentages of viable sperms in treatment B and C, as well as in treatment C and D, were not significantly different (P > 0.05), while between treatment B and D were significantly different (P < 0.01). These results showed that the use of 0.5 mm glutathione was more effective than the other treatments. Glutathione can reduce the presence of free radical, which decrease the percentage of viable sperms stored at 5 C. Situmorang et al. (2001b) reported that the use of 0.1% vitamin E as an antioxidant resulted in percentages of viable sperms of 70.1%, 49.7%, and 41.7% after storage for 0, 3, and 7 days, respectively. Results of this study indicated that the use of glutathione as an antioxidant was more effective than 0.1% vitamin E, because on the days 4 and 8 of storage, the percentage of viable sperms were 66.42% and 54.11%, respectively. Whereas in another study, Situmorang et al. (2002) reported that the addition of 0.5 mm phospholipids in Tris-citrate buffer containing 10% (v/v) egg yolk resulted in 55% and 46% sperm viability at 3 and 7 days of storage, respectively. When the egg yolk content was increased to 20%, the percentages of sperm viability were 52.0% and 47.3%. Table 4 shows the percentage of IPM and IAC of the sperms according to glutathione dosage and duration of semen storage at 5 C. The use of glutathione might increase the percentage of IPM (P < 0.01), although among the

4 Effect of glutathione addition to sperm diluent medium Table 3. Mean percentages of bovine sperm motility and sperm viability after storage at 5 C in the form of liquid semen added with different dosages of glutathione. Time of storage (day) Glutathione dosage (mm) Mean Sperm motility (%) a b c d Mean b a a a Sperm viability (%) a b c d Mean c a ab b Table 4. Average percentages of the intact acrosomal cap (IAC) and intact plasma membrane (IPM) of bovine sperms according to dosage of glutathione addition to the diluent medium and storage time at 5 C. Time of storage (day) Glutathione dosage (mm) Mean IAC (%) a b c d Mean b a a a IPM (%) a b c d Mean ab a a b treatments the values were not significantly different (P > 0.05). The mean percentages of each IPM were 66.01%, 69.75%, 68.38%, and 68.44% for control and addition of 0.5, 1.0, and 1.5 mm glutathione, respectively. Results of the study also indicated that the percentages of IPM and IAC decreased (P < 0.01) in line with the storage period. It may be concluded that the addition of 0.5 mm glutathione to the semen diluent medium was effective enough to prevent plasma membrane damage caused by free radicals. Meanwhile, the percentage of IAC increased in line with the addition of 0.5 mm glutathione, but then declined with increasing glutathione concentration in the diluent medium, although the differences among treatments of 0.5 mm (62.75%) and 1.0 mm (61.77%) were not significantly different (P > 0.05). The percentages of IAC were 60.81%, 62.75%, 61.77%, and 58.73% for the control and 0.5, 1.0 and 1.5 mm glutathione treatments, respectively. Sinha et al. (1996) reported that the effect of use of Tris as diluent of goat semen was better than that of Tris + 2 mm GSH and Tris + 5 mm GSH; it decreased goat sperm abnormality after frozen and re-thawed by 10.10%, 8.63%, and 6.98%, respectively. Meanwhile, Situmorang et al. (2002) stated that the addition of catalase g/100 ml to the diluent increased the percentage of motility. The addition of catalase, however, was more effective when added together with 0.5 mm glutathione. Furthermore, it was mentioned that the addition 0.5 mm glutathione alone gave the same result as that by addition of catalase and

5 64 Endang Triwulanningsih et al. glutathione. This was caused by different mechanisms of protection. In general, acrosome membrane damage occurred in the last stage or at the time of capacitation. The presence of glutathione and catalase can protect acrosome plasma membrane damage. Beconi et al. (1993) reported that the addition of antioxidant and vitamin E as much as 1 mg/ml into FH bull semen diluent can protect plasma membrane against lipid peroxidation by preventing activity of SOD enzyme and maintaining its metabolism as well as cell function. Plasma membrane damage may be caused by several factors, such as (1) decrease in availability of nutrients for spermatozoa; (2) non-conducive environmental conditions such as low temperatures; (3) formation of lactic acid as a residual metabolism that lower the ph; (4) dilution and cooling process that less precise; (5) formation of fat peroxidation reaction; (6) low-quality semen due to the presence of abnormal sperms, and (7) the occurrence of cell damages. Results of this study showed that the addition of 0.5 mm glutathione to the sperm diluent medium was sufficiently effective in the production of liquid semen because it increased the percentage of IPM and IAC sperms. Kim et al. (1999) reported that the addition of 1 mm glutathione into the fertilization medium during the in vitro fertilization process yielded different blastocysts depending on the stud (male) used. In a male that has a low fertilization capacity in vitro, addition of 1 mm glutathione improved its effectivenes, where the percentage of blastocysts increased from 29.8% (without glutathione) to 39.4%. This was because the increase in ROS in the medium during the in vitro culture caused cell damage. Sikka (1996) mentioned that ROS had a potential implication in reproductive biology, including the superoxyde (O 2- ) anion, hydrogen peroxyde peroxyl radicals (Roo - ), reactive hydroxyl (OH - ), nitrite oxide radical (NO - ), and peroxynitrite (ONOO - ). The effect of NO - is dependent on its concentration and interaction with H 2 O 2. The increase in ROS may cause damages to DNA, protein and fat, increase stress oxide, including lipid peroxides, thus resulting in the decrease in acrosome motility, viability and capacitation, as well as sperm function in general, thereby reducing its fertility. CONCLUSION Addition of 0.5 mm glutathione in Tris citrate buffer was effective enough to improve the condition of the plasma membrane of liquid semen stored at 5 C. It increased percentages of sperm motility, life sperms, as well as acrosomal cap and membrane integrity. A more indepth assessment is needed to prove further results of this study. ACKNOWLEDGEMENTS Thanks are conveyed to Mr. Arifin Ahmad and to all those who helped 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 cysteine. Theriogenology 53: Foote, P.A., C.C. Brocket, and M.T. Caproth Motility and fertility of bull in whole milk extender containing antioxidant. Anim. Reprod. Sci. 71: Goldman, E.E., J.E. Ellington, F.B. 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