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Effect of exogenous glutathione in medium fertilization to improve blastocyst rates of bovine embryos Triwulaninngsih, E; Situmorang, P; Putu, I-G; Sugiarti, T; Lubis, A.M; Kusumaningrum, D.A; Caroline, W; Sianturi, R.G
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 2 (2002)
Publisher : Indonesian Animal Sciences Society

Glutathione (C10H17N3O6S) is a tripeptide (Î³-Glu-Cys-Gly) widespread in living organism. Glutathione (GSH) at the 5 mM concentration increased the motility and fertility of frozen-thawed sperm. Intracellulair glutathione improved the cleavage rate and embryo development to the blastocyst rate. Research on in vitro embryos production through the implementation of GSH during IVF (in vitro fertilization) on embryo development has been conducted at the Laboratorium Reproductive of Physiology, Research Institute for Animal Production. Ovaries of beef cattle as source of oocytes were collected from the slaughterhouse in a thermo flask with 350C PBS as medium and transported to the laboratory. The oocytes were fertilized in vitro with selected motile sperm using Percoll gradient (90 and 45%). Ten COCs (cumulus oocytes complexes) were transfered to 44 Î¼l of fertilization medium (mTALP) was performed with either 0; 0.25; 0.50; 0.75 and 1.00 mM of glutathione as treatment A, B, C, D and E respectively, and inseminated with 2 Î¼l of capacitated sperm and added 2 Î¼l of heparin and 2 Î¼l of PHE (consisting of 20 Î¼M penicillamine, 10 Î¼M hypotaurine, 1 Î¼M epinephrine). Incubation between sperm and oocytes in the 5% CO2 incubator and RH 90% in fertilization media (TALP) for 20 hours. All zygotes were cultured in modification of CR1aa medium up to blastocyst and were fed serum 5 Î¼l/50Î¼l medium on day 6. Results of this experiment showed that the effect of concentration of glutathione (0, 0.25; 0.50; 0.75 and 1.00 mM) on fertilization medium to the percentage of cleavage rates were 69.35 + 29.40; 79.07 + 13.17; 67.88 + 10.65; 98.10 + 3.30 and 82.62 + 24.19% not significant different (P>0.05) among treatments A, B, C, D dan E respectively.The precentages of morula and blastocyst for treatment D were 50.45 + 42.64% and 31.28 + 24.28%; while 36.55 + 24.13% and 17.74 + 17.86% for treatment E respectively. Â Key words: Glutathione, in vitro fertilization, oocytes, cleavage

The effects of inclusion of exogenous phospholipid in Tris diluent with different level of egg yolk on the viability of bull spermatozoa Situmorang, P
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 3 (2002)
Publisher : Indonesian Animal Sciences Society

This study has been conducted to evaluate the effect of inclusion of phospholipid in Tris-diluent containing different level of egg yolk on the viability of bull spermatozoa after chilling and freezing. Semen was collected by means of artificial vagina, diluted in Tris-diluent to get a final concentration 100 x 106 spermatozoa/ml. Diluted semen was cooled to 50C for 45-60 minute, equiliberated in those temperature for 4 hours and frozen by placing the straw 5 cm above surface of liquid nitrogen for 10 minutes. The experiments was factorial designed with two level of phospholipid (0 and 0.5 mm) and 4 concentration of egg yolk (0, 5, 10 and 20% v/v). The viability of spermatozoa was assessed by evaluating the percentage of motile, live and condition of apical ridge after the temperature reduced to 50C, stored at those temperature for 3 and 7 days and after thawing. Inclusion of phospholipid and level of egg yolk in Tris diluent significantly increased the viability of spermatozoa for both chilling and deepfreezing. The mean percentage of motile, live and intact apical ridge for 3 and 7 days of storage times at 50C, were significantly higher (P<0.05) in diluent containing phospholipid than without phospholipid. After thawing, the mean percentage motile, live and intact apical ridges were significantly higher (P<0.05) in a diluent containing phospholipid (49.9; 60.2 and 60.0) than those without phospholipid (39.1; 54.1 and 51.5). The effects of interaction between phospholipid and level of egg yolk on the viability of spermatozoa was not significant for both chilling and freezing. Level of egg yolk significantly (P<0.05) affected the viability of spermatozoa where the optimal level of egg yolk was 10% v/v for chilled semen and the higher level (20% v/v) was needed for frozen semen. In conclusion, Tris diluent containing 0,5 mM phosphatidyl coline with 10 or 20% egg yolk gave a best protection for chilled and frozen semen respectively. Â Key words: Spermatozoa, viability, phospholipid, egg yolk

Quality of Garut ram frozen semen in various glycerol concentrations Rizal, Muhammad; Toelihere, M.R; Yusuf, T.L; Purwantara, B; Situmorang, P
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 3 (2002)
Publisher : Indonesian Animal Sciences Society

Semen was collected once a week using artificial vagina from four mature Garut rams. Immediately after initial evaluation, semen was divided into three parts and diluted with tris extender containing 3% (G3), 5% (G5), and 7% (G7) glycerol, respectively, each with the concentration of 100 million motile sperm 0.25 ml-1. Semen was loaded in 0.25 ml mini straws, and equilibrated at 50C for three hours, then frozen and stored in liquid nitrogen container. Results indicated that percentages of post thawing motility and live sperm for G5 (40 and 50.50%) were significantly higher than G3 (32.50 and 45.33%) (P<0.05), but not significantly different with G7 (39.17 and 47.67%) (P>0.05). Percentages of post thawing intact acrosomal and plasma membrane for G5 (42.67 and 43.17%) were significantly higher than G3 (36.17 and 38.17%) (P<0.05), but not significantly different with G7 (38 and 39.83%) (P>0.05). In conclusion, concentration of 5% glycerol is the optimal dose in maintaining frozen semen quality of Garut rams. Â Key words: Glycerol concentrations, frozen semen, Garut ram

The effect of cryoprotectant and equilibration period on quality and fertility of duck and muscovy sperm Setioko, A.R; Situmorang, P; Triwulanningsih, E; Sugiarti, T; Kusumaningrum, D.A
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 4 (2002)
Publisher : Indonesian Animal Sciences Society

The study was conducted to evaluate the effect of cryoprotectant and equilibration period on quality and fertility of duck and muscovy spermatozoa. Semen collected from Alabio and muscovy drakes was diluted using three different cryoprotectants:glycerol, DMSO and DMF, thereafter the semen was equilibrated 50C for 15; 30 and 60 minutes then frozen and stored in liquid nitrogen, designed by factorial 3 x 3. After thawing, semen sample was investigated on the motility and mortality rate. The best cryoprotectant and equilibration period was used in fertilization test. Duration of fertility was calculated from the second day after insemination until the last fertile egg, and the percent of fertility was calculated from the second day until the forth day after insemination. The use of cryoprotectant significantly affected sperm motility after freezing. The use of glycerol as a cryoprotectant was the lowest (P<0.05) compared to DMSO and DMF. Similarly, duck sperm motility after being freezed with glycerol, DMF and DMSO were 9.02; 21.75, and 32.86%, and for muscovy sperm motility were 11.78; 32.45 and 34.92% respectively. The percentage of live sperm for duck were 23.84; 40.14 and 42.20, while for muscovy were 29.26; 53.06 and 51.80 respectively after being freezed with glycerol, DMF and DMSO. Equilibration period did not affect the percentage of live sperm after freezing. Results of this study showed that duration of fertility of Alabio duck after being inseminated with fresh drake semen was longest compared to that of insemination using fresh muscovy semen, frozen drake semen and frozen muscovy semen (4.96 vs 3.5; 2.4 and 1.5 days respectively). Results from this study clearly indicated that preservation of sperm reduced the quality of spermatozoa. It is suggested that freezing technique of both duck and Muscovy sperm could be conducted using DMF or DMSO as a cryoprotectant with the equilibration period between 15 to 60 minutes. Â Key words: Sperm, cryoprotectant, fertility, AI, duck, muscovy

Effect of cryoprotectant and its level to survivability of drake semen D.A, Kusumaningrum; Situmorang, P; Setioko, A.R; Sugiarti, T; Triwulanningsih, E; Sianturi, R.G
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 4 (2002)
Publisher : Indonesian Animal Sciences Society

This study was conducted at Laboratory Reproduction of Physiology, Research Institute of Animal Production. The experiment was factorial design with two kinds of cryoprotectant (DMF and DMA) as the first factor and two levels of them (7 and 9%) as the second factor. This study was invented to determine the effect of cryoprotectant and its level to survivability of drake semen. Sperm was collected from fifteen drakes two times per week using artivicial vagina. Only the best quality of sperm was used in this study. Collected sperm was diluted in medium to get concentration of 400 x 106 per ml. Equilibrated at 50C in mini straw (0.25 ml) for 60 minutes, then kept them up 8 cm above the LN2 for 4 minutes before plunged in LN2. Parameters measured of this study were survability of drake semen after diluted, at 50C and after freezing-thawing at 350C for 30 seconds. Result of this study showed that percentage of motility and percentage of live sperm were significant different (P<0.05) between DMA (33.24 and 42.03) and DMF (25.82 and 34.30). Level of cryoprotectant (7 and 9%) were not significant different. Based on this study, using DMA as cryoprotectan with 7% in medium was better than that of DMF. Â Key words: Cryoprotectan, survivability, drake sperm

The effect of glutathione addition in sperm diluent on the quality of bovine chilled semen Triwulaninngsih, Endang; Situmorang, P; Sugiarti, T; Sianturi, R.G; Kusumaningrum, D.A
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 2 (2003)
Publisher : Indonesian Animal Sciences Society

This study has been conducted at the Laboratory of Physiology Reproduction, Research Institute for Animal Production (RIAP), Ciawi-Bogor, West Java. Sperms were collected from FH bulls with body weight 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 (4% v/v) glycerol to get spermatozoa concentration of 50 x 106 per ml. Sperm diluents were added with glutathione (GSH) with doses of 0.0; 0.5; 1.0 and 1.5 mM as treatments A, B, C and D respectively. The diluted semen was then cooled from 35 to 5Â°C using a cooling machine for 60 minutes then stored in the refrigerator (5Â°C). Recorded parameters were the survivability of spermatozoa by evaluating the percentage of motile and live, the condition of acrosome and plasma membrane. Data were analysed by completely randomised design with the general linear model (GLM) procedure. The characteristics of collected semen were normal. Viability of spermatozoa stored at 5Â°C for 0, 1, 4 and 8 days shown by intact acrosomal were 74.42; 69.27; 57.80 and 42.58% for A, B, C and D respectively. Those data were significantly different (P<0.01). Motility, live 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% for treatment A, B, C and D respectively. Additional 0.5 mM GSH gave the highest (P<0.01) motility, live and intact plasma membrane of sperm. Therefore, it is concluded that the effect of addition 0.5 mM of GSH to the sperm diluents can improve the viability of spermatozoa and possibly protect the spermatozoa from free radical damage. Â Key words: Glutathione, viability, spermatozoa

Characteristics of reproductive performance of Garut rams Rizal, Muhammad; Toelihere, M.R; Yusuf, T.L; Purwantara, B; Situmorang, P
Indonesian Journal of Animal and Veterinary Sciences Vol 8, No 2 (2003)
Publisher : Indonesian Animal Sciences Society

Basic information on reproductive potency of Garut rams is necessary in order to identify the capacity of rams in producing chilled or frozen semen. Eight Garut rams (three to five years old) were used in this study. The male sexual behaviors were observed and semen was collected once a week using artificial vagina. Semen quality was evaluated and its potency to produce frozen semen was calculated. Results of this study indicated that first, second, and third ejaculations were at the 29, 87 and 176th seconds, respectively. Fresh semen volume, sperm concentration, motility, intact acrosomal cap, and intact plasma membrane were 0.99 ml, 3224 million/ml; 76.67; 86.13 and 87.73%, respectively. Protein value, fructose, vitamin C, vitamin E, sodiu, potassium, calcium, magnesium, phosphor, chloride, and mangan in seminal plasma of fresh semen were 4140, 180, 3.2, 24, 180, 117, 9, 6.12, 60, 104, and 5 mg/ml, respectively. Measurement of head length, width, and length of sperm tail were 6.59, 3.99, and 42.65 Î¼m, respectively. Length and width measurement of right and left testes, and scrotal circumference were 12.71, 6.5, and 32.36 cm, respectively. Capacity of each Garut rams to produce frozen semen from three consecutive ejaculations are 35.88 mini straw with the cencentration of 200 million motile sperm per 0.25 ml. Â Key words: Reproductive characteristics, Garut rams Â

The influence of isobuthyl methylxhantine (IMX) and separation time on viability of spermatozoa and effectiveness of separation using egg albumin column Sianturi, R.G; Situmorang, P; Triwulanningsih, Elsa; Sugiarti, T; Kusumaningrum, D.A
Indonesian Journal of Animal and Veterinary Sciences Vol 9, No 4 (2004)
Publisher : Indonesian Animal Sciences Society

Supplementation of 3-isobuthyl-1-1-methylxanthine (IMX), as a cAMP inhibitor phosphodiesterase and could raise sperm motility, is expected to optimize the X and Y sperm separation. The purpose of this research was to observe the effect of IMX supplementation and separation time on the quality of separated sperm and the effectiveness of the method of sperm separation. Completely randomized design with 2 x 2 factorial was used in this research. The first factor was IMX (0.0 and 0.5 mM) while the second factor was separation time (10 and 30 minutes). The parameters observed were sperm concentration, the percentages of sperm motility, live sperm, sperm with intact apical ridge and the ratio of spermatozoa X and Y which measured by morfometric of head sperm square. IMX supplementation did not affect sperm concentration both on 10 or 30 minutes. The 30-minute separation time significantly reduced sperm motility in upper fraction while the addition of IMX significantly reduced sperm motility in lower fraction. There were no significant differences on the percentage of live sperm and sperm with intact apical ridge in every treatment even in upper or lower fraction. The albumin column sperm separation in this research changed the ratio of X and Y spermatozoa from 49.7% : 50.3% (fresh semen) to 65.1-84.0% : 16.0-34.9% in upper fraction; and to 24.0- 30.0% : 70.0-75.9% in lower fraction. The addition of IMX increased significantly X spermatozoa percentage (65.1 to 84.0%) and reduced significant Y-spermatozoa percentage (34.9% to 16.0%) in upper fraction. There was no significant differences on the ratio of X and Y spermatozoa between 10 and 30-minute of separation time treatment. In conclusion, the albumin column separation technique can be used to separate X and Y spermatozoa with the duration of 10 to 30 minutes separation time and did not severely affect the quality of separated sperm. The presence of IMX in separation media has no effect on the sperm separation effectiveness. Â Key words: Sperm separation, isobuthyl methylxanthine, X and Y spermatozoa, albumin column

Optimizing artificial insemination on swamp buffalo (Bubalus bubalis) through synchronization of estrus and ovulation Sianturi, Riasari Gail; Purwantara, B; Supriatna, I; ., Amrozi; Situmorang, P
Indonesian Journal of Animal and Veterinary Sciences Vol 17, No 2 (2012)
Publisher : Indonesian Animal Sciences Society

Artificial insemination (AI) program in swamp buffalo will be more efficient by implementing synchronization of estrus and ovulation. By synchronizing of ovulation, AI can be done at a fixed time schedule without concerning to estrus detection. Gonadotropin Releasing Hormone (GnRH) and human chorionic gonadotropin (hCG) have been used in protocols of estrus synchronization to induce ovulation. A study of AI in swamp buffalo was conducted on 83 buffaloes to evaluate the impact of protocol of estrus synchronization on reproductive efficiency of swamp buffalo. The three protocols used were Ovsynch (GnRH-PGF2Î±-GnRH-AI), convensional (PGF2a-PGF2a-AI) and Select-Synch (GnRH-PGF2a-AI). Inducing of ovulation were done by administration of GnRH or hCG after prostaglandin (PGF2Î±) injection. AI was done at 18 and 24 hour after the second GnRH injection (66 hours and 72 hours after PGF2Î± injection) for Ovsynch method and 72 hours after the last PGF2Î± injection for convensional and Select-Synch methods. Parameters observed were percentage of estrus and pregnancy from the three estrus synchronization protocols and the differences were analysed by statistics. All of buffaloes (100%) in the three synchronization protocols showed estrus behavior prior to AI. The percentage of pregnancy was 64.71; 77.14 and 83.87% for the Ovsynch, convensional and Select-synch respectively and there was no significantly different (P > 0.05) among the three protocols. hCG administration after the last PGF2Î± also did not affect pregnancy rate, ie: 76.47 vs 77.78% (with hCG vs without hCG) for the convensional and 88.24 vs 78.57% for the Select-Synch. It is concluded that the synchronization of estrus protocols in this study can synchronize the estrous and ovulation and AI can be done in a fixed-timed and could reach better pregnancy rate of swamp buffalo. Key Words: Swamp Buffalo, Synchronization, Estrus, Ovulation, AI

Effect of glutathione and bovine seminal plasma in lactose extender on viability of swamp buffalo frozen semen Sianturi, Riasari Gail; Purwantara, B; Supriatna, I; ., Amrozi; Situmorang, P
Indonesian Journal of Animal and Veterinary Sciences Vol 17, No 3 (2012)
Publisher : Indonesian Animal Sciences Society

The aim of the study was to investigate the effect on viability of frozen swamp buffalo semen of glutathione and bovine seminal plasma in lactose extender. Semen from two swamp buffalo bulls was collected twice weekly using an artificial vagina. Pooled, good-quality fresh semen was divided into three parts and centrifuged at 3,000 rpm for 15 minutes in preparation for three treatments-substitution of buffalo seminal plasma with zero, 50 or 100% bovine seminal plasma (BS0, BS50 and BS100, respectively). Each semen aliquot was then divided in two parts, on which was diluted with lactose extender containing 1 mM glutathione (GSH) and the other diluted with lactose extender without GSH (0 mM GSH). Extended semen from all six treatments was cooled to 5oC and then frozen in 0.25 ml straws.Â Mean motility percentages 0 and 30 minutes post thaw (PTM 0â² and 30â²) with GSH were 38.33 and 34.29%, significantly higher (P < 0.05) than treatments without GSH (31.67 and 25.95%). PTM 0â² and 30â² were also higher (P < 0.05) with no substitution of bovine seminal plasma (BS0) than when buffalo seminal plasma was replaced with bovine seminal plasma at either 50 or 100%. Averages were 40.00 vs 34.46 and 30.54% (BS0 vs BS50 and BS100) at thawing and 36.96 vs 28.36 and 25.36% 30 minutes post-thaw. Mean percentages of live sperm (LD), intact plasma membrane (MPU) and intact acrosomal membrane (TAU) at thawing were not significantly different with or without addition of GSH. However,at 30â² post thawing, TAU and MPU were significantly higher in GSH treatments than inthose without GSH:Â 61.50 vs. 58.19% (MPU) and 59.81 vs. 57.38% (TAU). Mean percentages of LD, TAU and MPU 30â²post thawing were higher with no substitution ofbuffalo seminal plasma (BS0) (P < 0.05) than to BS50 and BS100 treatments. In conclusion, the addition of glutathione (GSH) improved the quality of frozen swamp buffalo semen, but the partial substitution of buffalo seminal plasma with bovine seminal plasmaprovided no beneficial effects. Key Words: Swamp buffalo, Semen, Antioxidant, Glutathione, Seminal plasma

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