Mohamad Agus Setiadi
Bagian Reproduksi dan Kebidanan Departemen Klinik Reproduksi dan Patologi Fakultas Kedokteran Hewan Institut Pertanian Bogor, Bogor

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STATUS DNA DAN KARAKTERISTIK SPERMATOZOA KAUDA EPIDIDIMIS DOMBA PASCAPENYIMPANAN PADA SUHU 4OC (DNA STATUS AND CHARACTERISTIC OF SPERM CAUDA EPIDIDYMAL RAM AFTER STORAGE AT 4OC) Masir, Ummul; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani
Jurnal Veteriner Vol 18 No 2 (2017)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (177.669 KB) | DOI: 10.19087/jveteriner.2017.18.2.167

Abstract

The aim of this study was to evaluate the deoxyribonucleic acid (DNA) status and characteristics of ram spermatozoa, maintained within epididymides stored at 4ºC. A total of 12 pairs of ram cauda epididymis were kept by means that one of the pair in a tube contain media (isotonic NaCl solution) and the other pair into a clean ziplock without medium then stored at 4ºC for three days. Following this, the motility and viability and DNA status of spermatozoa was observed using halomax sperm method, prior and post freezing process. The results showed a decreased in the percentage of motility and viability of spermatozoa from cauda epididymis which were kept in and without media, before or after cryopreservation (P <0.05). Based on the storage method, a significant difference in motility percentage occurs at day two and day three of the storage. Storage using media had a lower percentage of motility (23%; 10%) than without using media (50%; 37%) (P <0.05). The DNA status following storage at 4ºC for three days, descriptively showed impaired of spermatozoa DNA less than 1%. The percentage of impairment increased following the cryopreservation of spermatozoa due to the freezing process. This condition was especially observed on the third day of storage of cauda epididymis which were kept in media (10.16%). The characteristics of spermatozoa from cauda epididymis which were kept in the media were lower compared to without medium. The DNA status of cauda epididymis spermatozoa is not affected by the storage method. ABSTRAK Penelitian ini bertujuan untuk mengevaluasi status DNA dan karakterisik spermatozoa asal kauda epididimis domba yang disimpan pada suhu 4ºC. Sebanyak 12 pasang kauda epididimis domba disimpan dengan cara salah satu dari setiap pasang kauda epididimis dimasukan ke dalam tabung berisi media (NaCl fisiologis) dan pasangan yang lain ke dalam ziplock bersih (tanpa media). Penyimpanan dilakukan selama tiga hari pada suhu 4ºC kemudian dilihat motilitas dan viabilitasnya serta status DNA spermatozoa dengan metode sperm sus halomax, sebelum dan setelah pembekuan. Hasil penelitian menunjukkan bahwa persentase motilitas dan viabilitas spermatozoa asal kauda epididimis baik yang disimpan dengan atau tanpa media mengalami penurunan, sebelum atau setelah kriopreservasi (P<0,05). Berdasarkan metode penyimpanan, perbedaan persentase motilitas yang nyata terjadi pada hari ke dua dan ke tiga penyimpanan. Penyimpanan menggunakan media memiliki persentase motilitas yang lebih rendah (23%; 10%) dibandingkan dengan tanpa menggunakan media (50%; 37%) (P<0,05). Status DNA spermatozoa setelah dilakukan penyimpanan pada suhu 4ºC selama tiga hari, secara deskriptif memerlihatkan kerusakan DNA spermatozoa kurang dari 1%. Persentase kerusakan kemudian meningkat setelah kriopreservasi spermatozoa akibat dari pembekuan, terutama pada hari ke tiga penyimpanan kauda epididimis dengan media (10,16%). Karakteristik spermatozoa dari kauda epididimis yang disimpan dengan media lebih rendah dibandingkan tanpa media. Status DNA spermatozoa kauda epididimis tidak dipengaruhi oleh metode penyimpanan.
KEMAMPUAN FERTILISASI SPERMATOZOA SEXING DAN PERKEMBANGAN AWAL EMBRIO SECARA IN VITRO PADA SAPI Aini, Alvien Nur; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani
Jurnal Sain Veteriner Vol 34, No 2 (2016): Desember
Publisher : Fakultas Kedokteran Hewan Universitas Gadjah Mada bekerjasama dengan PB PDHI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (659.562 KB) | DOI: 10.22146/jsv.27562

Abstract

The aim of the present study was to investigate the fertilization ability of bovine oocytes and early bovine embryonic development in vitro, fertilized by frozen X and Y sperm separated by bovine serum albumin (BSA) gradient column. Oocytes were collected from slaughter house ovarian by flushing and slicing technique. Oocytes were than maturated in tissue culture medium (TCM) 199 supplemented with 10 IU/ml pregnant mare?s serum gonadotropin (PMSG), 10 IU/ml human chorionic gonadotropin (hCG) and 10% fetal bovine serum (FBS) for 24 h in 5% CO2 incubator 39oC. Oocytes then fertilized with three kind of different frozen spermatozoa (X,Y and unsexing spermatozoa as control) for 14 h with final concentration 2x106 spermatozoa/mL. Embryos were cultured insynthetic oviductal fluid (SOF) supplemented with essential and non essential amino acid and 0.3% bovine serum albumin (BSA) for 96 h. Results of the experiments revealed that there was no significant difference (P>0.05) in thefertilization ability (49.17%; 51.40%; 53.42%) for X, Y and control group, respectively. No significant difference (P>0.05) in the number of embryos development (47.77%; 48.25%; 54.43%) for X, Y and control group, respectively. Furthermore, only small number of embryos could pass development blockade (23.80%; 26.08%; 23.61%) for X, Y and control spermatozoa with statistically no significant difference (P>0.05). It is concluded that sexed spermatozoa separated by BSA gradient column had comparable fertilization ability with unsexing spermatozoa and had ability to supported early embryonic development.
EFEKTIVITAS EKSTRAK BIJI KAPAS (GOSSYPIUM HIRSUTUM L.) TERHADAP JUMLAH DAN VIABILITAS EMBRIO MENCIT (MUS MUSCULUS L.) Zayani, Nofri; Supriatna, Iman; Setiadi, Mohamad Agus
Jurnal Sain Veteriner Vol 34, No 2 (2016): Desember
Publisher : Fakultas Kedokteran Hewan Universitas Gadjah Mada bekerjasama dengan PB PDHI

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (816.406 KB) | DOI: 10.22146/jsv.27563

Abstract

The cottonseed (Gossypium hirsutum L.) contained gossypol as antifertility agent. The effect of cottonseed extract treatment could be decrease and impaired follicles development were accompanied by oocytesdamage. Damage of oocytes resulted reduction of number and viability of embryos. The objective of this study was to evaluate the effectiveness of cottonseed extract (Gossypium hirsutum L.) on the number and viability ofmice embryo (Mus musculus L.). Doses of the cottonseed extract were used consists of 0 (control), 1.5; 2.1; and 2.7 g/kg of body weight (BW) for 24 days via the oral route. This research used 24 animals healthy of femaleDDY mice 14-15 weeks old and 30-35 g BW. Embryos were collected at day 4 of pregnancy by flushing the utery cornua. The collected embryos were cultured in vitro for 48 hours in modified phosphate buffered saline (PBS) supplemented with 10% fetal bovine serum (FBS) culture medium according to the stage of its development to observe the viability of embryos. The result showed that the cottonseed extract with doses 1.5; 2.1; and 2.7 g/kg of body weight (BW) made the number of embryos which collected in D4 of pregnancy significantly lowerthan control (P<0.05). Data from embryos culture in vitro for 48 hours decreased embryos number (P<0.05) that developed in to the expanded and hatched blastocysts. At 2.7 g/kg BW, embryos only can develop to theblastocysts stage. Retardation (4-8 cells) and degeneration embryos did not develop in culture
TINGKAT MATURASI DAN FERTILISASI OOSIT DOMBA YANG DIMATURASI DALAM MEDIA DENGAN IMBUHAN B-MERCAPTOETHANOL SECARA IN VITRO (IN VITRO MATURATION AND FERTILIZATION RATE AT SHEEP OOCYTES MATURATED IN MEDIA WITH B-MERCAPTOETHANOL) Bintara, Okky Adi; Setiadi, Mohamad Agus; Karja, Ni Wayan Kurniani
Jurnal Veteriner Vol 16 No 4 (2015)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

The objectives of this study to evaluate the effect of b-mercaptoethanol addition in maturation mediumon maturation and fertilization rate of sheep oocytes in vitro. Collected oocytes were then matured inmaturation medium without (control group) or with b-mercaptoethanol in concentration of 50 ?M or 100?M. There was a significant increase in the percentage of oocytes that reached the metaphase II (MII) stageafter cultured in maturation medium with b-mercaptoethanol either in concentration of 50 ?M or 100 ?M(p&lt;0.05) (62.67%; 77.78%; 82.05% for control, 50 ?M, and 100 ?M group respectively). However no differencewas found in maturation rate of those oocytes in 50 ?M and 100 ?M groups (p&gt;0.05). When maturedoocytes were fertilized, no difference was found on fertilization rate among the groups (P&gt;0.05) (70.93%;77.50%; 69.14% for control, 50 ?M and 100 ?M group respectively). These data indicated that the additionof b-mercaptoethanol increased the percentage of sheep oocytes that reached the MII stage in vitro, but hadno effect on the fertilization rate.
RESPONS SUPEROVULASI SAPI PERANAKAN ONGOLE TERHADAP PENYUNTIKAN TUNGGAL FOLLICLE STIMULATING HORMONE KE DALAM RUANG EPIDURAL (SUPEROVULATION RESPONSES IN ONGOLE CATTLE CROSSBREED TREATED WITH A SINGLE EPIDURAL INJECTION OF FOLLICLE STIMULATING HORMONE) Imron, Muhammad; Supriatna, Iman; ., Amrozi; Setiadi, Mohamad Agus
Jurnal Veteriner Vol 17 No 1 (2016)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Super-ovulation is conventionally performed by injection of FSH twice daily for four days. Thistreatment needs frequent attention by farm-personnel and relatively increases the possibility of failuresdue to mishandling and errors in administration of the treatment. This study was conducted to evaluatethe responses of superovulation trough a single injection of FSH into epidural space in ongole cattlecrossbreed. In Experiment 1, a combination of single dose injection of FSH was applied into epidural spaceplus intramuscular (epi+i.m group) compared to the group of intramuscular injection of FSH, which wastreated twice daily for four days (intramuscular group), using equal total dose of FSH 400 mg. Superovulationresponse of epi+im group (n=4) was not significantly different compared with intramusculargroup (n=4). In experiment 2, it compared two treatmentof FSH in different concentration(280 mg versus160 mg) in a single dose applied into epidural space. Data of Epi+im group from experiment 1 was used ascontrol. Group of 280 mg FSH (n=4) resulted total collection of ova/embryo and transferable embryos(9,00+2,65 and 3,33+2,52 respectively) was significantly different compared to the 160 mg group (n=4)(which were 2,00+1,26 and 0,00) P &lt;0,05, although they werenot significantly different compared to thecontrol (9,33+5,68 and 3,67+3,21). In conclusion, injection of a single dose of FSH at 280 mg into epiduralspace result in a comparable transferable embryo which similar to the conventional method that appliedintramuscular injection of FSH twice daily for four days.
PENAMBAHAN GLUTATHIONE PADA MEDIUM FERTILISASI EFEKTIF MENDUKUNG PEMBENTUKAN PRONUKLEUS DAN PERKEMBANGAN AWAL EMBRIO SAPI (SUPPLEMENTATION OF GLUTATHIONE IN FERTILIZATION MEDIUM EFFECTIVELY SUPPORT NORMAL PRONUCLEUS FORMATION AND EARLY BOVINE EMBRYONIC DE Nugroho, Aras Prasetiyo; Supriatna, Iman; Setiadi, Mohamad Agus
Jurnal Veteriner Vol 18 No 3 (2017)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (148.616 KB) | DOI: 10.19087/jveteriner.2017.18.3.327

Abstract

The objective of this study was to determine fertilization rate effectiveness and early embryonic development competency with glutathione (GSH) supplementation in fertilization medium and culture This study consisted of two experiments comprising each of the four treatment and six repetitions with completely randomized design (CRD) using 651 oocytes. In the first experiment, a total of 317 bovine oocytes were matured in tissue culture medium (TCM) 199 at incubator 5% CO2 with temperature 39 ºC for24 h, then fertilized with sperm separated by swim up technique. Oocyte and sperms were incubated in fertilization medium supplemented with 0.25 mM, 0.50 mM, 1.00 mM GSH. In the second experiment, bovine oocytes were matured in maturation medium and fertilized with same procedure as mentioned before, then cultured in modified synthetic oviduct fluid (mSOF) with the following treatment: supplementation GSH only in fertilization medium (T1), supplementation GSH only in culture medium (T2), and supplementation GSH in both fertilization and culture medium (T3), while control not supplementation GSH (T0). Result of the first experiment showed that supplementation 1.00 mM GSH in fertilization medium can increase higher normal pronucleus (2PN) formation (86,9%) compared to other treatments, 0.50 mM (80.3%), 0.25 mM (73.8%), and control (58.9%) (P&lt;0.05). In the second experiment showed that early bovine embryonic development on 2nd day cultured which reached 5-8 cell on treatment T1 (56.0%) and T3 (53.6%) were higher (P&lt;0.05) compared to treatment T2 (26.2%) and T0 (control) (31.3%). Result of the other were showed that early bovine embryonic development on 4th day cultured which reached 9-16 cell on treatment T1 (26.2%) and T3 (27.4%) were higher (P&lt;0.05) compared to that T2 (11.9%) and T0 (control) (10.8%). In conclusion, 1.00 mM GSH supplementation in medium was more effective in supporting normal pronucleus formation and early fertilization bovine embryonic development compared to in culture medium. ABSTRAK Penelitian ini bertujuan untuk mengetahui tingkat fertilisasi dan kompetensi perkembangan awal embrio sapi dengan penambahan glutathione (GSH) pada medium fertilisasi in vitro (IVF) dan kultur in vitro (IVC). Penelitian ini terdiri atas dua penelitian yang terdiri dari masing-masing empat perlakukan dan enam kali ulangan dengan rancangan acak lengkap (RAL) menggunakan 651 oosit. Penelitian I, sebanyak 317 oosit sapi dalam tissue culture medium (TCM) 199 dimatangkan pada inkubator 5% CO2 dan suhu 39°C selama 24 jam, kemudian difertilisasi dengan spermatozoa yang telah diseleksi menggunakan teknik swim up. Oosit dan spermatozoa diinkubasi pada medium fertilisasi dengan penambahan 0,25 mM, 0,50 mM, dan 1,00 mM GSH. Penelitian II, sebanyak 334 oosit sapi dimatangkan pada medium pematangan dan difertilisasi, kemudian dikultur pada medium modified synthetic oviduct fluid (mSOF), dengan perlakuan: penambahan GSH hanya pada medium fertilisasi (T1), penambahan GSH hanya pada medium kultur (T2), dan kombinasi penambahan GSH pada medium fertilisasi dan kultur (T3). Hasil penelitian I, menunjukkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi dapat meningkatkan pembentukan pronukleus normal (2PN) yang lebih tinggi (86,9%) dibandingkan dengan perlakuan yang lain yaitu 0,50 mM (80,3%), 0,25 mM (73,8%), dan kontrol (58,9%) (P&lt;0,05). Penelitian II menujukkan bahwa perkembangan awal embrio sapi pada hari ke-2 kultur yang mencapai pembelahan 5-8 sel pada perlakukan T1 (56,0%) dan T3 (53,6%) lebih tinggi (P&lt;0,05) dibandingkan dengan perlakuan T2 (26,2%) dan T0 (kontrol) (31,3%). Hasil penelitian lain menunjukkan bahwa perkembangan awal embrio sapi pada hari ke-4 kultur yang mencapai pembelahan 9-16 sel pada perlakuan T1 (26,2%) dan T3 (27,4%) lebih tinggi dibandingkan dengan perlakukan T2 (11,9%) dan T0 (kontrol) (10,8%) (P&lt;0,05). Dapat disimpulkan bahwa penambahan 1,00 mM GSH pada medium fertilisasi lebih efektif dalam mendukung pembentukan pronukleus normal dan perkembangan awal embrio sapi dibandingkan pada medium kultur.
KEMAMPUAN MATURASI DAN FERTILISASI OOSIT SAPI YANG DISELEKSI MENGGUNAKAN TEKNIK PEWARNAAN BRILLIANT CRESYL BLUE (SELECTING MATURATION AND FERTILIZATION ABILITY OF BOVINE OOCYTES USING BRILLIANT CRESYL BLUE) Muttaqin, Zultinur; Karja, Ni Wayan Kurniani; Setiadi, Mohamad Agus
Jurnal Veteriner Vol 16 No 2 (2015)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

The objective of this study was to evaluate the use of brilliantcresyl blue (BCB) in selecting potentialbovine oocytes for maturation and fertilization in vitro. Brilliant cresyl blue (BCB) is a dye that can assessintracellular activity of glucose-6-phosphate dehydrogenase (G6PD), a synthesized enzyme in maturedoocytes. Oocytes were exposed to 26 ?M BCB diluted in modified Phosphate Buffer Saline (mPBS, PBS +10% Fetal Bovine Serum) for 90 minutes is 5% CO2 incubator at 390C. The oocytes were classified accordingto their cytoplasm coloration: oocytes with blue cytoplasm (BCB+) and unstained oocytes (BCB-). Oocytesof the control group were incubated shortly after morphological selection without being exposed to BCB.Afterwards, all groups of oocytes (BCB+, BCB-, and control) were matured and fertilized in vitro. Maturedoocytes were those oocytes that reach metaphase II after 24 hours culturing; whereas oocytes showing twoor more pronuclei at 14 hours post incubation were classified as fertilized oocytes. The nuclear maturationrate was significantly (P&lt;0.05) higher in BCB+ group (78.7% ± 0.41) than the BCB- group (33.3% ± 0.13).However, there was no significance difference (P&gt;0.05) between the BCB+ and the control group (77.1% ±0.32). The fertilization rate was significantly higher (P&lt;0.05) in the BCB+ group (30.5% ± 0.04) comparedto the BCB- (13.6%±0.03) and control group (23.6% ± 0.05). In conclusion, BCB staining of bovine oocytesprior in vitro maturation could be used in selecting potential developing oocytes.
PROFIL PROGESTERON AIR SUSU DAN TINGKAT KEBUNTINGAN SAPI PERAH PASCASINKRONISASI ESTRUS MENGGUNAKAN PROSTAGLANDIN F2ALFA ATAU PROGESTERON-CIDR (MILK PROGESTERONE PROFILE AND PREGNANCY RATE ON DAIRY CATTLE AFTER ESTROUS SYNCHRONIZATION WITH PROSTAGLANDIN F Suprihatin, Novi; Tumbelaka, Ligaya ITA; Setiadi, Mohamad Agus
Jurnal Veteriner Vol 17 No 3 (2016)
Publisher : Faculty of Veterinary Medicine, Udayana University and Published in collaboration with the Indonesia Veterinarian Association

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Abstract

Research on estrus synchronization, progesterone profiles of post-synchronization and early pregnancyhas been conducted using 16 Holstein Frisian dairy cows. Treated cows were divided into 2 groups. Cowsin group I were synchronized with double injection of prostaglandin F2á (PGF2á, Lutalyse®) 25 mg / head at11 days apart, then were inseminated twice at 72 hours and 96 hours after the second injection of PGF2á(FTAI = fixed timed insemination). Group II were synchronized with progesterone implant-CIDR® for 11days. At the time of progesterone implant withdrawal the animals were injected with PGF2á at 25 mg /head then inseminated twice at 48 hours and 72 hours. Milk samples were collected on the day before, atthe treatment day and at day 1st, 3rd, 5th, 7th following the first insemination in order to determine theprofile of progesterone after synchronization, while for early pregnancy examinations, sampling of milkwere collected at day 21th, 24th and 27th after the first insemination. The milk samples were analyzed byRadioimmunoassay (RIA) method. Rectal palpation to confirm pregnancy was undertaken at day 60thafter the first insemination. The results showed a marked decrease in milk progesterone (nmol / l) at thefirst insemination (H0) in both group PGF2á and CIDR® (0.84; 0.49 vs. 0.92; 0.32), which indicated theoccurrence of estrus. Gradually increased of milk progesterone level (0.52; 0.68; 1.17; 1.69, respectively)started from day 1st, 3rd, 5th, 7th was seen in animals PGF2á group, whereas in the CIDR® group the milkprogesterone level was found fluctuate (0.21; 0.39; 0.33; 1.61). However, at day 7th the concentration ofprogesterone in both groups was significantly increased which indicated functional activity of the corpusluteum. Meanwhile the progesterone concentrations (nmol/l) of pregnant cows at day 21st, 24th and 27th ingroup PGF2á were 3.63; 3.51; 1.58 and in CIDR® group were 2.50; 2,79; 4.35, respectively. In non-pregnantcows, the progesterone concentrations (nmol/l) were lower (0.63; 0.42; 1.41 vs. 0.20; 0.27; 1.33), than thoseof pregnant cows. The results of rectal palpation after 60 days of the first Artificial Insemination (AI)confirmed that 5 cows with higher milk progesterone concentrations at day H21, H24, H27 from the firstinsemination were pregnant, with the possibilities at 62.5% in each group. It is concluded that estroussynchronization using either PGF2á or CIDR® in lactating dairy cows will give the same response and thiscould be detected using the milk progesterone profiles. Measurement of milk progesterone concentrationsby RIA began at day 21 of the first AI was effective for early pregnancy diagnosis.
KOLEKSI SEL TELUR DENGAN TEKNIK LAPAROSKOPI UNTUK PRODUKSI EMBRIO DAN TRANSFER EMBRIO PADA DOMBA Setiadi, Mohamad Agus; Supriatna, Iman; Boediono, Arief
Jurnal Ilmu Pertanian Indonesia Vol. 12 No. 2 (2007): Jurnal Ilmu Pertanian Indonesia
Publisher : Institut Pertanian Bogor

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An experiment was carried out to analyze the application of laparoscopic technique for oocyte collection, in vitro embryo production and embryo transfer in sheep. The first experiment was conducted to observe effect of gonadotropin stimulation on follicle development and laparoscopic technique for oocytes aspiration. In the second experiment, effect of culture system on the embryo development in vitro was assessed and in the third experiment, the application of laparoscopic for embryo transfer has been conducted. The result showed that single dose of gonadotrophin was sufficient to support follicle development significantly and it could help follicle visualization. It also showed that laparoscopic ovum-pick up could be conducted weekly without any restriction The second series experiment showed CR1aa culture system was better than TCM 199 (29.90°/o vs 8.00%) and the changing of media was required to ensure better metabolism process for embryos. The third experiment revealed that embryo transfer could be conducted with an aid from laparoscope. In conclusion, single dose PMSG stimulation is sufficient to support follicle development for /aparoscopic ovum-pick up, the culture media changing affects the successful rate of in vitro embryo production (8% vs 25.66%) and the laparoscopy technique can be used safely for embryo transfer on sheep.Keyword: laparoscopic, oocyte, embryo transfer, sheep
Status of ram spermatozoa DNA after freeze-drying process Saili, Takdir; Prasetyaningtyas, wahono Esthi; Setiadi, Mohamad Agus; AgungPriyono, Srihadi; Boediono, Arief
Indonesian Journal of Animal and Veterinary Sciences Vol 11, No 3 (2006)
Publisher : Indonesian Animal Sciences Society

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (260.586 KB) | DOI: 10.14334/jitv.v11i3.528

Abstract

The process of freeze drying caused detrimental effect on plasma membrane and acrosome of the spermatozoa, even it potentially could alter the chromatin and DNA integrities. On the other hand, DNA integrity is essential for spermatozoa to participate in pronucleus formation during fertilization event. Therefore the evaluation of DNA integrity should be carried out to study the effect of freeze drying process. EDTA, EGTA, and PBS were used as dilution media of spermatozoa prior to freeze drying process to protect the DNA. Toluidine blue staining and comet assay methods were used to evaluate the alteration on chromatin and DNA integrities of spermatozoa, respectively. The results revealed that the highest compacted chromatin after 6 months storage of freeze-dried spermatozoa were observed from EGTA-3 (98%) and EGTA-1 (97%) treatments that had significant differences compared to all PBS treatments (90-92%), but not for fresh spermatozoa (100%). Whereas, the highest compacted DNA integrity of freeze-dried spermatozoa were observed from EGTA-2 (92%) and EGTA-3 (92%) but had no significant differences compared to other treatments including fresh spermatozoa (97%). These results demonstrate that EDTA and EGTA tend to be able to protect chromatin and DNA integrities of ram spermatozoa during freeze-drying and storage compared to PBS. Key Words: Freeze-Drying, Spermatozoa, DNA, Toluidine Blue, Comet Assay