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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

Effect of Collection Technique on Yield of Bovine Oocytes and the Development Potential of Oocytes from Different Grades of Oocytes Sianturi, R.G; Thein, M; Wahid, H; Rosnina, Yono C
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 3 (2002)
Publisher : Indonesian Animal Sciences Society

Oocyte collection technique is important to obtain a maximum number of oocytes to be employed on in vitro production of embryos. In this study, immature bovine oocytes were collected from slaughterhouse ovaries by two techniques: aspiration of 2- to 6-mm follicles and slicing. Following collection, oocyte qualities were classified into four categories (A, B, C, and D) on the basis of cumulus attachment. Oocytes of each category were matured in vitro in CO2 incubator for 22-24 hours and cumulus expansion and maturation rates were observed. The total number of oocytes (group A+B+C+D) and yield of good quality oocytes (only group A and B) recovered per ovary by aspiration were 12.02 and 8.21, and by slicing were 29.38 and 19.65 (P<0.01), respectively. The total cumulus cells expansion rates of A, B, C and D oocytes were 97.1%, 88.3%, 6.0% and 20.6% respectively. Maturation rates for A, B and C categories of oocytes were 91.4%, 82.3% and 35.0% respectively while no matured oocyte was observed for group D oocytes. Maturation rates were significantly different between group A and C and also between B and C but not between A and B (P<0.05). In conclusion, slicing technique recovered more oocytes per ovary (2.4 times) than that of aspiration and the best maturation rate was observed from category A oocytes which surrounded by more than 3 layers of cumulus cells. However oocytes of category A and B can be considered as good quality oocytes. Â Key words: Collection technique, aspiration, slicing, oocyte quality, maturation

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

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

Effect of Collection Technique on Yield of Bovine Oocytes and the Development Potential of Oocytes from Different Grades of Oocytes R.G Sianturi; M Thein; H Wahid; Yono C Rosnina
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 3 (2002): SEPTEMBER 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Oocyte collection technique is important to obtain a maximum number of oocytes to be employed on in vitro production of embryos. In this study, immature bovine oocytes were collected from slaughterhouse ovaries by two techniques: aspiration of 2- to 6-mm follicles and slicing. Following collection, oocyte qualities were classified into four categories (A, B, C, and D) on the basis of cumulus attachment. Oocytes of each category were matured in vitro in CO2 incubator for 22-24 hours and cumulus expansion and maturation rates were observed. The total number of oocytes (group A+B+C+D) and yield of good quality oocytes (only group A and B) recovered per ovary by aspiration were 12.02 and 8.21, and by slicing were 29.38 and 19.65 (P<0.01), respectively. The total cumulus cells expansion rates of A, B, C and D oocytes were 97.1%, 88.3%, 6.0% and 20.6% respectively. Maturation rates for A, B and C categories of oocytes were 91.4%, 82.3% and 35.0% respectively while no matured oocyte was observed for group D oocytes. Maturation rates were significantly different between group A and C and also between B and C but not between A and B (P<0.05). In conclusion, slicing technique recovered more oocytes per ovary (2.4 times) than that of aspiration and the best maturation rate was observed from category A oocytes which surrounded by more than 3 layers of cumulus cells. However oocytes of category A and B can be considered as good quality oocytes. Key words: Collection technique, aspiration, slicing, oocyte quality, maturation

Effect of cryoprotectant and its level to survivability of drake semen Kusumaningrum D.A; P Situmorang; A.R Setioko; T Sugiarti; E Triwulanningsih; R.G Sianturi
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 4 (2002): DECEMBER 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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. Key words: Cryoprotectan, survivability, drake sperm

The effect of glutathione addition in sperm diluent on the quality of bovine chilled semen Endang Triwulaninngsih; P Situmorang; T Sugiarti; R.G Sianturi; D.A Kusumaningrum
Jurnal Ilmu Ternak dan Veteriner Vol 8, No 2 (2003): JUNE 2003
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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 5°C using a cooling machine for 60 minutes then stored in the refrigerator (5°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 5°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. Key words: Glutathione, viability, spermatozoa

The influence of isobuthyl methylxhantine (IMX) and separation time on viability of spermatozoa and effectiveness of separation using egg albumin column R.G Sianturi; P Situmorang; Elsa Triwulanningsih; T Sugiarti; D.A Kusumaningrum
Jurnal Ilmu Ternak dan Veteriner Vol 9, No 4 (2004): DECEMBER 2004
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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. Key words: Sperm separation, isobuthyl methylxanthine, X and Y spermatozoa, albumin column

Effect of exogenous glutathione in medium fertilization to improve blastocyst rates of bovine embryos E Triwulanningsih; P Situmorang; I-G Putu; T Sugiarti; A.M Lubis; D.A Kusumaningrum; W Caroline; R.G Sianturi
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 2 (2002): JUNE 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Glutathione (C10H17N3O6S) is a tripeptide (γ-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 μl 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 μl of capacitated sperm and added 2 μl of heparin and 2 μl of PHE (consisting of 20 μM penicillamine, 10 μM hypotaurine, 1 μM 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 μl/50μl 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. Key words: Glutathione, in vitro fertilization, oocytes, cleavage

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