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In vitro embryo production through modification of time and gonadotropin hormone during oocytes maturation Purwantara, B; Diwyanto, K; Triwulaninngsih, Endang; Toelihere, M.R; Rutledge, J.J; Yusuf, T.L
Indonesian Journal of Animal and Veterinary Sciences Vol 6, No 3 (2001)
Publisher : Indonesian Animal Sciences Society

This research has been conducted at the laboratory of in vitro fertilization of the University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. There is transportation constraint in importing oocytes from USA. It takes more than 24 hours to bring it to Indonesia. In fact, oocytes maturation and ready to be fertilized normally requires only 24 hours in 5% CO2 incubator at 38.5Â°C. Therefore, this research is needed to study the effect of gonadotropin hormone and time for oocyte maturity and ready to be fertilized at a period more than 24 hours. If this problem could be solved then the importation of oocytes could be cheaper and easier than importation of life animals or embryos. Ovaries were collected from slaughterhouse in Wisconsin. Oocytes were matured in TCM-199 medium in 5% CO2 incubator and at 30Â°C enriched with FSH 10 Î¼l/ml, oestradiol 17 Î² 1Î¼l/ml and 10 % FCS as control of gonadotropin hormone treatment (A); with FSH 10 Î¼l/ml (B); with oestradiol 17 Î² 1Î¼l/ml (C) and without gonadotropin hormone (D) for 24 hours, 30 hours and 36 hours as time of maturation treatment I, II and III respectively. The oocytes were fertilized in vitro with motile sperm selection by Percoll gradient and incubation between sperm and oocytes in fertilization media (TALP) for 20 hours. All zygotes were cultured in modification of KSOM medium up to blastocyst and were fed serum 5 Î¼l/50 Î¼l medium on day 6. Data were analyzed by SAS program. Percentage of cleavage between time of maturation were significant (p<0.01); between gonadotropin hormone treatment A vs B and A vs C and B vs C and B vs D and between A vs D were significant (p<0.01), but between treatment C vs D were not significant (p>0.05). Percentage of blastocyst between time of maturation were not significant (p>0.05), but between gonadotropin hormone treatment A vs B and A vs C and B vs D and C vs D were significant (p<0.01), but between treatment A vs D and B vs C were not significant (p>0.05). Percentage of cleavage, morula, blastocyst, expanded blastocyst and unertilized Â ova on this study are 66.73%, 22.43%, 40.33%, 0.81% and 32.51 for 24 hours incubation (I); 61.55%, 25.69%, 32.69%, Â 0.54% and 27.61% for 30 hours incubation (II); 72.43%, 32.06%, 37.97%, 0.0% and 25.31% for 36 hours incubation (III) respectively. According to this study, in vitro production of embryo could be conducted at 30Â°C and incubation on maturation media for more than 24 hours. Â Key words: Oocytes, cleavage, embryos, blastocyst

Using CR1aa versus KSOM as the culture medium for in vitro embryo production of cattle Triwulaninngsih, Endang; Toelihere, M.R; Rutledge, J.J; Yusuf, T.L; Purwantara, B; Diwyanto, K
Indonesian Journal of Animal and Veterinary Sciences Vol 7, No 1 (2002)
Publisher : Indonesian Animal Sciences Society

This research has been conducted at the laboratory of in vitro fertilization in the Department of Animal Science University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. Oocytes were matured in TCM- 199 medium (in 5% CO2 incubator and at 390C) enriched with follicle stimulating hormone (FSH) 10 Î¼l/ml, oestradiol 17 Î² 1Î¼l/ml and 10% Fetal Calf Serum (FCS). The oocytes were fertilized in vitro with motile sperm and incubation between sperm and oocytes in fertilization medium Tyroide Albumin Lactate Pyruvate (TALP) for 20 hours. All zygotes were cultured in CR1aa (n=1549) medium versus modification of protein-free pottasium simplex optimized medium (KSOM) (n=675) up to blastocyst stage and were fed FCS 5 Î¼l/50 Î¼l medium on day 6, as treatment A and B respectively. Data were analyzed by completely randomized design with SAS program. Percentages of cleavage, morula, blastocyst, expanded blastocyst, unfertilized and degenerated ova in this study were 91.4% vs 75.6 %; 75.6% vs 58.9%; 61.5% vs 38.5%; 31.2% vs 5.1%, 8.6% vs 24.4%, 15.7% vs 8% which were significantly different (P<0.01) for treatment CR1aa and KSOM respectively. Based on this study, CR1aa medium is better culture medium than KSOM for efficient in vitro production (IVP) of bovine embryos. Â Key words: Oocytes, in vitro fertilization, embryo, blastocyst, culture medium

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

In vitro embryo production through modification of time and gonadotropin hormone during oocytes maturation Endang Triwulaninngsih; M.R Toelihere; J.J Rutledge; T.L Yusuf; B Purwantara; K Diwyanto
Jurnal Ilmu Ternak dan Veteriner Vol 6, No 3 (2001): SEPTEMBER 2001
Publisher : Indonesian Center for Animal Research and Development (ICARD)

This research has been conducted at the laboratory of in vitro fertilization of the University of Wisconsin, USA. These embryos can be used for improving genetic value of Indonesian cattle. There is transportation constraint in importing oocytes from USA. It takes more than 24 hours to bring it to Indonesia. In fact, oocytes maturation and ready to be fertilized normally requires only 24 hours in 5% CO2 incubator at 38.5°C. Therefore, this research is needed to study the effect of gonadotropin hormone and time for oocyte maturity and ready to be fertilized at a period more than 24 hours. If this problem could be solved then the importation of oocytes could be cheaper and easier than importation of life animals or embryos. Ovaries were collected from slaughterhouse in Wisconsin. Oocytes were matured in TCM-199 medium in 5% CO2 incubator and at 30°C enriched with FSH 10 μl/ml, oestradiol 17 β 1μl/ml and 10 % FCS as control of gonadotropin hormone treatment (A); with FSH 10 μl/ml (B); with oestradiol 17 β 1μl/ml (C) and without gonadotropin hormone (D) for 24 hours, 30 hours and 36 hours as time of maturation treatment I, II and III respectively. The oocytes were fertilized in vitro with motile sperm selection by Percoll gradient and incubation between sperm and oocytes in fertilization media (TALP) for 20 hours. All zygotes were cultured in modification of KSOM medium up to blastocyst and were fed serum 5 μl/50 μl medium on day 6. Data were analyzed by SAS program. Percentage of cleavage between time of maturation were significant (p<0.01); between gonadotropin hormone treatment A vs B and A vs C and B vs C and B vs D and between A vs D were significant (p<0.01), but between treatment C vs D were not significant (p>0.05). Percentage of blastocyst between time of maturation were not significant (p>0.05), but between gonadotropin hormone treatment A vs B and A vs C and B vs D and C vs D were significant (p<0.01), but between treatment A vs D and B vs C were not significant (p>0.05). Percentage of cleavage, morula, blastocyst, expanded blastocyst and unertilized ova on this study are 66.73%, 22.43%, 40.33%, 0.81% and 32.51 for 24 hours incubation (I); 61.55%, 25.69%, 32.69%, 0.54% and 27.61% for 30 hours incubation (II); 72.43%, 32.06%, 37.97%, 0.0% and 25.31% for 36 hours incubation (III) respectively. According to this study, in vitro production of embryo could be conducted at 30°C and incubation on maturation media for more than 24 hours. Key words: Oocytes, cleavage, embryos, blastocyst

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

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