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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 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

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

Ovulation rates and twinning birth following Follicle Stimulating Hormone (FSH) treatment at differents stages of estrus cycle Situmorang, Polmer; Kusumaningrum, D.A; Sianturi, R
Indonesian Journal of Animal and Veterinary Sciences Vol 17, No 1 (2012)
Publisher : Indonesian Animal Sciences Society

Twinning rate could be increased through genetically approach or through hormonal induction approach. The aim of this study was to observe the effect of first injection of FSH initiated on the ovulation rate. The experiment design was completely randomized with 3 different time of FSH was initiated as treatment using 12 lactating dairy cattle. Injection of FSH was initiated at day 2 (Treatment I), day 10 (Treatment II) and at day 18 (Treatment III) of estrus cycle. A total of 6 ml Folltropin (Equivalent 120 mg FSH) was intramussculary injected in decreasing dossis method twice a day with 12 hours interval for 4 days. Blood was collected on day 12 of estrus cycle for progesterone level. Data recorded were the diameter of ovary (DO), total corpus luteum (TCL), concentration of progesterone (P), percentage of pregnancy and number of birth. The means DO, TCL and P were significantly (P < 0.05) higher after injection (2.0 cm; 2.1 and 1.6 ng/ml) than those before injection (1.4 cm, 1.0 dan 0.6 ng/ml). The time of first injection of FSH was initiated, significantly affect the ovulation rate. The mean DO, TCL and P were significantly higher (P < 0.01) in treatment II (2.6 cm; 4.0 and 2.9 ng/ml) than those in treatments I (1.9 cm; 1.3 and 0.9 ng/ml) or in treatment III (1.6 cm; 1.3 and 0.9 ng/ml). There was no significant diference between treatment I and III. The percentage of pregnant were 25.0; 75.0 and 25.0 for treatments I; II and III respectively. One twin birth and 1 single birth were obtained in treatments II but only one single birth for each treatment I and III. Number of CL were positively correlated with the concentration of progesterone but were not fully useful for prediction number of birth. In conclusion, the dairy cattle gave a better response to exogenous gonadotropin hormone when the first injection was initiated at day 10 of estrus cycle. Key Words: Hormone, FSH, Estrus Cycle, Ovulation, Twin Birth

The effect of cryoprotectant and equilibration period on quality and fertility of duck and muscovy sperm A.R Setioko; P Situmorang; E Triwulanningsih; T Sugiarti; D.A Kusumaningrum
Jurnal Ilmu Ternak dan Veteriner Vol 7, No 4 (2002): DECEMBER 2002
Publisher : Indonesian Center for Animal Research and Development (ICARD)

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. Key words: Sperm, cryoprotectant, fertility, AI, duck, muscovy

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

Ovulation rates and twinning birth following Follicle Stimulating Hormone (FSH) treatment at differents stages of estrus cycle Polmer Situmorang; D.A Kusumaningrum; R Sianturi
Jurnal Ilmu Ternak dan Veteriner Vol 17, No 1 (2012): MARCH 2012
Publisher : Indonesian Center for Animal Research and Development (ICARD)

Twinning rate could be increased through genetically approach or through hormonal induction approach. The aim of this study was to observe the effect of first injection of FSH initiated on the ovulation rate. The experiment design was completely randomized with 3 different time of FSH was initiated as treatment using 12 lactating dairy cattle. Injection of FSH was initiated at day 2 (Treatment I), day 10 (Treatment II) and at day 18 (Treatment III) of estrus cycle. A total of 6 ml Folltropin (Equivalent 120 mg FSH) was intramussculary injected in decreasing dossis method twice a day with 12 hours interval for 4 days. Blood was collected on day 12 of estrus cycle for progesterone level. Data recorded were the diameter of ovary (DO), total corpus luteum (TCL), concentration of progesterone (P), percentage of pregnancy and number of birth. The means DO, TCL and P were significantly (P < 0.05) higher after injection (2.0 cm; 2.1 and 1.6 ng/ml) than those before injection (1.4 cm, 1.0 dan 0.6 ng/ml). The time of first injection of FSH was initiated, significantly affect the ovulation rate. The mean DO, TCL and P were significantly higher (P < 0.01) in treatment II (2.6 cm; 4.0 and 2.9 ng/ml) than those in treatments I (1.9 cm; 1.3 and 0.9 ng/ml) or in treatment III (1.6 cm; 1.3 and 0.9 ng/ml). There was no significant diference between treatment I and III. The percentage of pregnant were 25.0; 75.0 and 25.0 for treatments I; II and III respectively. One twin birth and 1 single birth were obtained in treatments II but only one single birth for each treatment I and III. Number of CL were positively correlated with the concentration of progesterone but were not fully useful for prediction number of birth. In conclusion, the dairy cattle gave a better response to exogenous gonadotropin hormone when the first injection was initiated at day 10 of estrus cycle. Key Words: Hormone, FSH, Estrus Cycle, Ovulation, Twin Birth

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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