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The most common therapy on men who suffered fertility decline due to aging was called “T Therapy”, but that’s therapy has long-term risks of sexual dysfunction, metabolic syndrome, prostate, and cardiovascular system. Stem cells are an alternative therapy can be used for ameliorate testicular fuction because of their ability to differentiate into various cell types. The aim of this study was to evaluate the injection of hWJ-MSC in physiologic aging male rats on testicular function. This study was used 3 young male rats (8-12 weeks) and 6 physiological aging male rats (22-24 months) which divided into 3 groups, (i) the young rats, (ii) physiological aging male rats, and (iii) physiological aging male rats that injected with hWJ-MSCs. The young rat group did not give any treatment, physiological aging male rats received NaCl (0.9%) 0.4 mL, and the treatment group received 1x106 cells/kg BW of hWJ-MSCs. The observations were performed on the macroscopical and histological analysis. The result indicates that the younger group had the lowest body weight (154.6 g) and the percentage of the testis weight on the body weight was highest (2.2%) compared to the other groups (P>0.05). The physiological aging rats group had the smallest tubule (9726.9 ìm2) with a largest interstitial area (1117.1 ìm2) compared the other groups (P>0.05). After injection of hWJ-MSC, the tubule area became wider followed by narrowing of the interstitial area (P>0.05). The difference in the body weights is due to the different age of the rat. Improvement of tubule area and interstitial area due to the ability of hWJ-MSCs to improve spermatogenic cells within the tubule. Injection of hWJ-MSCs has been shown to increase fertility in aging rats.

Oocyte vitrification is the important part of gamete preservation for further purpose. The objective of this study was to evaluate the response or development of vitrified-mice oocyte following activation using various concentrations of Strontium Chloride (Sr Cl2). Oocytes were collected from superovulation-induced female mice. Oocytes vitrification was then performed using a gradual equilibration of 2 M Ethylene Glycol in 0.25 M sucrose and 7 M Ethylene Glycol on 0.5 sucrose. Subsequently, the vitrified oocytes were thawed and activated using various Strontium Chloride concentration in each group. Control 1 is unvitrified oocyte and without Sr Cl2. Control 2 is unvitrified oocyte then activated by 20 mM Sr Cl2. Zero (0) mM Sr Cl2 is vitrified oocyte without Sr Cl2. Group Ten (10) mM is vitrified oocyte then activated by 10 mM Sr Cl2. Group Twenty (20) mM is vitrified oocyte then activated by 20 mM Sr Cl2. The viability of vitrified-thawed oocytes was observed based on ooplasm integrity. Whereas the oocytes respond to artificial activation was observed based on pronucleus formation after 10 hours of activation. The result showed that 39% of oocyte degenerated following vitrification. The respond of vitrified-thawed oocytes following artificial activation using Strontium Chloride was significantly lower compared to fresh oocytes (p

The use of cryoprotectants for the cryopreservation of human spermatozoa, oocytes, zygote, earlycleavage stage of embryos and blastocyst is an integral part of almost every human In Vitro Fertilizationprogram. Moreover, the cryopreservation of these types of cells by direct plunging into liquid nitrogen (-196°C) usually requires a high concentration of cryoprotectant with a consequent of cytotoxic effect. Theaim of this study was to observe the effect of ethylene glycol concentration on the spermatozoa recoveryrate following vitrification process. Earle’s balanced salt solution + 0.25 M sukrosa + 1 % human albumineserum as basic solution supplemented with some different concentrations of etylene glycol (ie: 36.25%;18.25%; 9.12%; 4.56%; 1.14% and 0.57%) were used to evaluate the motility and viability of spermatozoafollowing vitrification. Human’s spermatozoa from ejaculates with progressive motility and viability above50% were used as samples. Samples were mixed with vitrification solution and then loaded into 0.25 mLstraws, equilibrated for 10 minutes at room temperature before plunged into liquid nitrogen. Spermatozoathawing was done at 24 hours after the vitrification. The results showed that, the decrease of spermatozoamotility and viability were observed at the highest (100%, 96.70%, respectively) in the samples that wereadded with vitrification medium contained 36.25% of ethylene glycol. On the other hand, the decrease ofthe spermatozoa motility and viability were found at the lowest (14.11%, 43.81 %, respectively) in thesamples without ethylene glycol supplementation. It can be concluded that the highest spermatozoa recoveryrate was obtained from the vitrification using a low concentration of ethylene glycol.

Ovarian dynamics in the garut ewes had never been studied continuously by using ultrasonography.The aim of this study was to observe development of the follicles and corpus luteums in the estrous cyclein the garut ewes. Garut ewes (n=6) with body weight 30.00±4.05 kg which had normal estrous cycle wereused in this study. All ewes were synchronized by using CIDR-G implantation for 14 days. Ovulation of thedominant follicle, development of the follicle waves and corpus luteum were observed continuously everyday during the estrous cycle after CIDR-G removal. The number of small (2-3 mm in diameter), medium (4-5 mm in diameter) and large (>5 mm in diameter) follicles were aligned during the estrous cycle. Follicleand corpus luteum diameters were measured by using built in caliper in the ultrasound. The resultsshowed 1) the average length of estrous cycle was 19,2±0,8 days; 2) ovarian follicle growth occurred inthree waves during the estrous cycle; 3) the number of preovulatory follicles were 1-2 follicles; 4) theaverage maximum diameters of preovulatory follicle was 7.5±0.5 mm; 5) the average maximum diametersof corpus luteum was 7.3±0.4 mm. In conclusion, the estrous cycle in garut ewes was 18-20 days with 3follicular waves.

The purpose of this study was to verify the effectiveness of cumulus cells as a feeder layer in supportingthe growth of mouse embryonic stem cells. Embryos at blastocyst stage were exposed in pronase solution,and then incubated in rabbit anti-mouse antibody and guinea pig complement to lyse and separate thetrophoblast cells from the inner cell mass. Inner cell mass subsequently cultured in a petri dish containinga cumulus feeder layer, mouse embryonic fibroblasts, or without a feeder layer, in stem cells medium. Theresulting stem cells colony passaged in trypsin solution, pipetted repeatedly to produce subcolonies orsingle cells, and cultured as before in some new petri dishes. Characterization of stem cells was identifiedby using alkaline phosphatase staining. The results showed the effectiveness of cumulus cells as feederlayer for culture of mouse embryonic stem cells was comparable with mouse embryonic fibroblasts, andboth of them were better than without a feeder layer method. The number of primary colony, cell lines, andcolony growth rate increased 41.30, 8.70, and 54.20%, respectively, while doubling time was shorter 10:21hours compared to the growth without feeder layer method. These results prove that the cumulus feederlayer effectively supports the growth of mouse embryonic stem cells.

Cloned embryo and parthenogenetic embryo are a potential source of stem cells for regenerativemedicine. Stem cells derived from those embryos are expected to overcome the ethical issues to the use offertilization embryos for therapeutic purposes. The pre-implantation development is a critical step fordeveloping embryos reach the blastocyst stage. The objectives in vivo of this research are to produce mousecloned embryo, parthenogenetic embryo, and fertilized embryo and to study stages of in vitro pre-implantation development culture. In vivo fertilized embryos, mouse oocytes, and cumulus cells were usedin this study. Treatment was performed on female mice superovulated with PMSG and hCG injections.Two-cell stage of in vivo fertilized embryos were collected on the second day post hCG injection. Clonedembryos were produced through Somatic Cell Nuclear Transfer (SCNT), which included enucleation, nucleartransfer and artificial activation. Parthenogenetic embryos were produced with artificial activationtechnique. The result of the research indicated that SCNT application was able to produce cloned embryos which could develop to blastocyst stage (3,2%). In addition, artificial activation of oocytes could produceparthenogenetic embryos which were able to develop up to the blastocyst stage (8,6%). In conclusion,efficiency level of parthenogenetic embryos that is able to reach the blastocyst stage was higher than in thecloned embryos. Fertilized embryos shows a better development and more efficient compared to in vitrocloned embryos and parthenogenetic embryos cultures.

The aim of the study was to investigate the role of zona pelucida (ZP) as a barrier of embryo againstE.coli K99 contamination. The complete randomized design was used in this study. The embryos weregiven treatment as a follow : 1) embryos without ZP were contaminated with E.coli K99; 2) embryo withintact ZP were contaminated with E.coli K99; and 3) embryos with intact ZP were not contaminated withE.coli K99, as a control. In each treatment there was 15 replication and in each replication there was oneembryo. The embryos were incubated in incubator at 37°C and 5% CO2 atmosphere. The embryos wereobserved every six hours in 24 hours using inverted microscope. The result showed that embryos withintact ZP could develop in culture contaminated with E.coli K99, while embryos without ZP becomedegenerated. The viability of intact embryos was 75% and the embryos without ZP were 65%. Embryosculture in contaminated medium could develop from eight cells embryo into morulla stage of embryo,compact morulla, and blastocyst. E.coli K99 contamination could inhibit embryo development. In conclusion,ZP could protect embryo against E.coli K99 contamination.

One of the factors that inhibit the formation of male pronuclei following injection of freeze-dried ramspermatozoa was the absence of artificial activation during oocyte incubation after the injection. Therefore,in this experiment the ability of strontium chloride (SrCl2) to improve oocyte activation followingintracytoplasmic sperm injection (ICSI) was evaluated. Aceto lacmoid staining was used to assessdecondensation and pronucleus formation following ICSI. Results of this experiment revealed that freezedriedspermatozoa had the ability to decondense and to form 1PN following injection into oocytes evenwithout artificial activation, but failed to form 2PN. However, 40% of 2PN oocytes were obtained when theinjected oocytes was first incubated for 20 minutes in medium containing 50 mM strontium chloride thensubsequently incubated for 10 hours in medium without strontium. On the contrary, the 2PN oocytes werenot observed either in injected oocyte neither without artificial activation nor in non-injected oocytes withartificial activation. In conclusion, freeze-dried ram spermatozoa were able to decondense and to support2PN formation following ICSI and artificial activation using strontium.

The purpose of this study was to evaluate the effect of cooling and freezing of ewes ovarian tissue ontheir follicles morphology. The study was carried out in two experiments. Experiment I, ovaries weremaintained in Phosphate Buffered Saline (PBS) at -20oC and room temperature (RT) for 24 h, and at 5oCfor 24 h and 72 h, respectively. After storage, follicles were histologically evaluated. Experiment II, theovarian cortex was isolated and tissue slices (±1 mm3) were prepared. Following this tissues were loadedinto hemistraw then transferred to equilibration solution (PBS+20% FBS+7,5% EG+7,5 % DMSO) atroom temperature and held for 10, 20, 30 minutes, respectively. Afterward tissues were tranferred tovitrification solution (PBS+20%FBS +15%EG+15%DMSO ) for 3 minutes, then the hemistraw was placeddirectly into liquid nitrogen. After thawing, the tissues were prepared for histological examination. All ofthe follicles were deteriorated after 24 h storage at RT. The percentage of morphologically normal follicleswas significantly reduced when ovarian tissues were stored at -20oC for 24 h and at 5oC for 24 h and 72 h.However, it seemed to have a minor deterioration effect when the tissues were kept at 5oC for 24 h(P0.05). It can be concluded that exposingtissues to equilibration solution for 10 minutes prior to freezing would kept the ovarian follicles morphologyin good condition.

The aim of the study was to assess the viability of vitrified embryo using cryoloop as a carrier ofembryo. The blastocyst stage embryos were collected from superovulated mice. Embryos were frozenusing vitrification method and vitrified embryos were loaded on copper filament cryoloop before dipped inliquid nitrogen. The viability of vitrified embryos was assess in vitro by medium cultered and in vivo bytransfered them to recipient mice. The result shows the viability of vitrified embryos was 85,7% after 24hours cultured and the embryos were born from two pregnant recipient mice out of nine (22%) or fouroffspring out of 63 trasfered embryos (6%). In conclusion, vitrified blatocyst stage embryos using cryoloopas a carrier could keep the viability of the embryos and they could be transfered to the recipient mice andwere born normally.

ABSTRAK Telah dilakukan penelitian untuk mengetahui viabilitas oosit domba yang dikoleksi dari ovarium domba pascatransplantasi intrauterus pada kelinci pseudopregnant. Ovarium domba ditransplantasikan dalam uterus kelinci pseudopregnant pada hari pertama bunting semu dan kemudian diambil kembali setelah lima (P5) atau tujuh (P7) hari transplantasi. Sebagai kontrol digunakan ovarium segar. Oosit dikoleksi dari ovarium pascatransplantasi dengan metode slicing (pencacahan) di dalam media phosphate buffer saline (PBS) yang disuplementasi dengan 5% fetal bovine serum (FBS) dan 100 IU penicillin-streptomisin/ml. Oosit hasil koleksi selanjutnya dimaturasi dalam Tissue Culture Medium (TCM)-199 yang disuplementasi dengan 10% FBS, 10 IU follicle stimulating hormone (FSH)/ml dan 100 IU penicillin-streptomisin/ml. Oosit selanjutnya diinkubasi dalam inkubator CO2, dengan kandungan CO2 5% dan suhu 38ºC, selama 24 jam. Setelah dimaturasi, oosit diwarnai dengan aceto-orcein 2% untuk menentukan status inti oosit. Hasilnya memperlihatkan bahwa oosit yang mampu mencapai tahapan perkembangan dengan status inti M-II setelah maturasi pada P5 (35,05%) dan P7 (35,24%) nyata lebih sedikit (p

ABSTRAK Tujuan penelitian ini adalah untuk mengevaluasi dinamika folikel pada ovarium domba pascatransplantasi secara intrauterin pada kelinci pseudopregnansi. Transplantasi dilakukan pada kelinci pseudopregnant hari ke 1 atau ke 7. Ovarium kembali diambil pada hari ke 5, 7, atau 9 setelah transplantasi. Untuk menentukan dinamika folikel pada ovarium domba pascatransplantasi dan menghitung jumlah folikel pada berbagai tahap perkembangan, ovarium domba pascatransplantasi dijadikan preparat histologis dengan metode parafin dan pewarnaan HE. Hasilnya masih ditemukan semua tahapan perkembangan folikel (folikel primordial, primer, preantral, dan antral) pada semua kelompok perlakuan. Jumlah folikel pada 5, 7 atau 9 hari pascatransplantasi menurun nyata (p

Low productivity of in vitro embryo production at blastosis level appears to be as a result of imperfectoocyte maturation which causes imperfect oocyte growth, and in turn will affect the embryo growth. Inaddition to hormonal factor, growth factor plays significant role in maturation process of oocytes. Asgrowth factor might has a significant role during maturation process, a study was conducted to determinewhether transforming growth factor ? (TGF ?) isolated from oocytes of cumulus complex is required for coculture and in vitro embryo production.. Oocytes were collected from follicles with the diameter of 3-5 mmand > 5 mm. Then the oocytes were cultured for 22 hours at 38.5o with 5% CO2 atmosphere in tissueculture medium (TCM) 199 supplemented with 5 ?g/mg luteonizing hormone (LH), 3% bovine serumalbumin (BSA) 50 ?g/ml gentamycin sulfat and three different levels of TGF ? (12,85 pg/ml, 25,7 pg/mland 38,55 pg/ml). The oocyte maturation and number of apoptosis cells were examined. The result showedthat oocyte maturation in medium supplemented TGF ? at the dose of 38,55 pg/ml was better than in thatat dose of 12,85pg/ml or 25,7 pg/ml. The best maturation was observed at metaphase II stage of oocytedevelopment. No apoptosis was observed during maturation of oocytes. Supplementation of TGF ? at thedose of 38,55 pg/ml in culture medium increased the oocyte maturation without causing a significantapoptosis in vitro.

Several methods i.e. cooling, freezing, and freeze-drying have been widely used to preserve spermatozoa with various degree of success. Freeze-drying appears to provide a method to preserve spermatozoa in a dry state without requiring liquid nitrogen for storing frozen spermatozoa. Freeze-drying procedures can have a detrimental effect on plasma membrane and acrosomal cap of the spermatozoa. In this experiment study, the viability and subcellular changes of freeze-dried ram spermatozoa were evaluated using staining method and scanning electron microscopy. The results revealed that all freeze-dried spermatozoa were dead following evaluation using eosin staining and Hoechst-propidium iodide staining methods. Morover, plasma membrane and acrosomal cap of freeze-dried ram spermatozoa was disrupted observed using scanning electron microscope.

Cryopreservation is an ultra rapid freezing process to preserve tissue or organ. The studywas conducted to identify the effectiveness of cryoloop vitrification method and the viability ofembryos following vitrification. Embryos at blastocyst stage were vitrified by placing them inequilibration medium containing 10% ethylene glycol (EG) and phosphate buffer saline (PBS) wichsupplemented with 20% new born calf serum for 8-10 minutes. The blastocysts were then removedand put in vitrification medium (15% dimethyl sulfoxide, 15% EG, and 0.5M sucrose), and theprocess in the vitrifivcation medium not longer than 25-30 seconds. The blastocysts were immediatelytransferred to the vitrification medium film in the cryoloop and plunged into 100 ml liquid nitrogen.The warming process was done by immersing the cryoloop which carried the vitrified blastocstsinto PBS supplemented with 20% serum and 0.5M sucrose for 1 minute, and then removed to samesolution supplemented with 0.25M sucrose and 0.1M sucrose for 2 minutes respectively. Theblastocysts were washed 4 times in kalium simplex optimized medium (KSOM) and cultured indrops of KSOM in 5% CO2 incubator at 370C. The observations were done every 6 hours for 48hours using inverted microscope ( Olympus IX70 Japan). The viability of embryos was assessed onthe basis of the intact morphology, reexpansion of the blastosul, and the development of embryosinto advance stage. The results showed that 85.71% of vitrified embryos, developed into advancestages and 19% of them hatched. In conclusion the cryoloop can be used to vitrify the embryos.

Preparation of a good quality of sperm is required in order to increase the efficacy of in vitro fertilization.One method of a good sperm preparation is the use of Percoll density gradient centrifugation. This studywas aimed to determine the optimal combination of speed and time of centrifugation for the spermpreparation with the lowest mortality rate. Fresh semen collected from Garut sheep with the concentrationof 2 million cells per ml was used in this study. The experimental design used was a completely randomizedfactorial design with one control group and sixteen combination of speed and time of centrifugation. Theywere 1),(1) 200 xg for 5 min; (2) 200 xg for 10 min; (3) 200 xg for 15 min; (4) 200 xg for 20 min; (5) 300 xg for5 min; (6) 300 xg for 10 min; (7) 300 xg for 15 min; (8) 300 xg for 20 min; (9) 400 xg for 5 min; (10) 400 xg for10 min; (11) 400 xg for 15 min; (12) 400 xg for 20 min; (13) 500 xg for 5 min; (14) 500 xg for 10 min; (15) 500xg for 15 min; and (16) 500 xg for 20 min. All of control and treatments combination were replicated forthree times. Each control and combined treatment were replicated for three times. The variables observedwere the concentration of sperm, the percentage of live sperms, the percentage of progressively motilesperms and the percentage of normal sperm. The data obtained were analyzed by two way analysis ofvariance (ANOVA) and the differences between treatment groups were subjected for Duncan’s MultipleRange Test (DMRT). The best result was obtained using 400 xg for 15 minutes which showed the spermconcentration of 66.66 ± 12.22 millions per ml, the living sperms of 74.82± 1.53%, the percentage ofprogressively motile sperm of 57.56±8.42% and the percentage of normal sperm of 86±1.73%).

This study was designed to optimalize the use of epididymal or ejaculate sperm and plasma for in vitro fertilization, that sperm agglutination was found at preparation. The rate of sperm agglutination was calculated the head-to-head sperm agglutination that were incubated in Krebs Ringer-(N-(2hydroxyethyl)piperazine-N’-(2-ethenesulfonic acid) or KR-HEPES medium in 38.50C with 5% CO2 at 1, 3, 5 and 7 hours culture in vitro. The rate of head-to-head sperm agglutination were decreased with time treatments. The cauda of sperm agglutination was lower than that caput, corpus epididymal and ejaculate sperm with statistically significant (P

The purpose of the present study was to investigate the viability of mice and hamster embryos developed in Kalium Simplex Optimized Medium amino acid (KSOMaa) and Hamster Embryo Culture Medium-6 (HECM-6) medium. Female DDY mice were superovulated by injection i.p. of 5 IU Pregnant Mare Serum Gonadotropine (PMSG) and 5 IU Human Chorionic Gonadotropine (hCG) in 48 h interval, hamster (Phodopus campbelli) injected by 2.5 IU PMSG and 2.5 IU hCG 48 h later. Then females were mated with fertile males. Eight-cell embryos were recovered at day 3 after natural mating. The mice embryos were cultured in KSOMaa+5% NBCS (New Born Calf Serum) (T1) and HECM-6+5% NBCS (T2), the hamster embryos were cultured in KSOMaa+5% NBCS (T3) and HECM-6 + 5% NBCS (T4) for further development at 37oC in a humidified atmosphere of 5% CO2 in air for 48 h. The examinations were replicated five times. The T1 embryos developed to compact morulla and early blastocyst 100% (140/140), 92.1% (129/140) to blastocyst and expanded blastocyst, and 22.9% (32/140) became hatching/hatched. The T3 reached 100% (60/60) to compact morulla and early blastocyst, 85.0% (51/60) blastocyst, and 48.3% (29/60) expanded blastocyst, no embryo observed hatching/hatced. The T2 embryos had more expanded blastocyst than T3 (P

The objective of this study was to evaluate the development of ewe follicles and oocytes viabilityfollowing an intrauterine transplantation of the ovaries into pseudopregnant rabbit. The experiment wasconducted in day 1 of pseudopregnant rabbit. Following transplantation for 5, 7, and 9 days, the ovarieswere recollected. The development of follicles determined by counting the number of follicles in paraffinembedded ovaries after staining with haematoxylin-eosin (HE). The viability of oocytes was determined byslicing the ovaries. Oocytes were incubated in CO2 incubator with 5% CO2, 38°C for 24 hours. After maturation,the oocytes were stained with 2% aceto-orcein to determine the nuclear oocytes status. The result showedthat follicles were detected in all stages of their development (primordial, primary, preantral, and antralfollicle stages), but their number decreased significantly (P

The objective of the research is determining the ICM isolation method to produce ESC. Blastocyst stage of DDy mice embryos were used in this study. Zona pellucida of blastocysts were removed by 0.25% pronase, the ICM isolation were done by enzimatic or immunosurgery method, and then they were cultured in DMEM-high glucose supplemented with mercaptoethanol, gentamycin, fetal bovine serum, and cumulus cells as feeder layer. The result of the research indicated that immunosurgery method yielding attachment rate and number ESC colony 93.85% and 43.08%, respectively, higher (P0.05) that are 93.59% in enzymatic method and 98.56% in immunosurgery method. This research concluded that immunosurgery more effective method for isolation of ICM and ESC production than enzymatic method.

Pada penelitian ini dikaji kemampuan spermatozoa domba hasil pengeringbekuan untuk melakukan dekondensasi dan membentuk pronukleus setelah diinjeksikan ke dalam oosit dengan menggunakan teknik intracytoplasmic sperm injection (ICSI). Metode pewarnaan aceto lacmoid digunakan untuk mengevaluasi kejadian dekondensasi dan pembentukan pronukleus pada oosit setelah ICSI. Hasil penelitian menunjukkan bahwa spermatozoa hasil pengeringbekuan dapat melakukan dekondensasi (2%) dan mendukung pembentukan 1PN (34%) tetapi belum mampu mendukung pembentukan 2PN setelah diinjeksikan ke dalam oosit. Sedangkan injeksi dengan menggunakan spermatozoa segar mampu mendukung pembentukan 2PN (30%) dan 1PN (40%). Sebagai kesimpulan dapat dikemukakan bahwa spermatozoa hasil pengeringbekuan mampu melakukan dekondensasi dan mendukung pembentukan 1PN setelah ICSI

Penelitian untuk mengetahui tingkat aglutinasi antarkepala spermatozoa dan sebaran antiaglutinin dalam plasma asal epididimi dan ejakulat telah dilakukan untuk mengoptimalkan pemanfaatannya dalam fertilisasi in vitro. Tingkat aglutinasi dan aktivitas antiaglutinin plasma epididimis dan ejakulat domba dihitung dengan cara menghitung jumlah aglutinasi antarkepala spermatozoa setelah diinkubasikan selama 1, 3, 5, dan 7 jam in vitro dalam media Krebs Ringer- HEPES.