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Oocyte competence is a determining factor that influences the embryo development. Embryos produced in vitro have a reduced developmental competence than embryos produced in vivo. Therefore, human Chorionic Gonadotropin (hCG) injection was carried out to improve the quality of the oocytes. The objective of this study was to evaluate the effect of ovarian stimulation with hCG before ovary collection on oocyte quality in the domestic cat. Oocyte donors were either 1) treated with a single dose of 200 IU hCG four days before ovary collection (hCG group), or, 2) no treatment before ovary collection (control group). The oocytes were collected by the slicing method. Immature cumulus oophorus complexes (COCs) from both groups were pooled and matured in vitro for 24-26 hours. Then mature oocytes were fertilized with epididymal sperm and cultured in vitro for seven days. The results study showed that the number of the dominant follicle (DF) and the number of COCs in the hCG group was higher than the control group in right and left ovaries (p

Objective: To determine the correlation of different serum estradiol levels on the trigger day with the clinical and laboratory outcomes of in-vitro fertilization (IVF) cycles comprising a single fresh top-quality blastocyst transfer. Methods: This was a retrospective observational study performed in Morula IVF Clinic Jakarta. Five hundred forty-two women were recruited and grouped according to their serum estradiol levels on the trigger day of follicular maturation as follows: 0.05). Nonetheless, the study demonstrated a positive correlation of the serum estradiol levels with the overall laboratory outcomes including the number of retrieved, mature, and fertilized oocytes, the total sum of derived embryos, and top-quality embryos at cleavage and blastocyst stage (P

Background: Ca2+ signaling pathway is suggested to play an essential role in mediating oocyte maturation. Aims: The aim of this study was to evaluate intracellular Ca2+ of resistant immature oocytes that failed to resume meiosis following subsequent in vitro culture reach metaphase II after calcium ionophore A23187 activation. Settings and Design: This in vitro analytical experimental study was conducted at Animal Science Laboratory of Indonesian Medical Education and Research Institute (IMERI), Human Reproductive Infertility and Family Planning of IMERI, and Electrophysiology Imaging of Terpadu Laboratory, Faculty of Medicine, University of Indonesia. Methods: A total of 308 oocytes classed as resistant immature following in vitro culture were randomly allocated to control (n = 113) and treatment groups (n = 195). The oocyte activation group was exposed to A23187 solution for 15 min and then washed extensively. Maturation was evaluated by observing the first polar body extrusion 20‒24 h after A23187 exposure. Ca2+ imaging was conducted using a confocal laser scanning microscope to identify the dynamic of Ca2+ response. Statistical Analysis: SPSS 20, Chi-square, and Mann–Whitney U-test were used in this study. Results: Activation of resistant immature oocytes with A23187 significantly increased the number of oocyte maturation compared with the control group (P < 0.001). Furthermore, fluorescent intensity measurements exhibited a significant increase in the germinal vesicle stage when activated (P = 0.005), as well as the metaphase I stage, even though differences were not significant (P = 0.146). Conclusion: Artificial activation of resistant immature oocyte using chemical A23187/calcimycin was adequate to initiate meiosis progress.

Background:Luteinizing hormone (LH) supplementation may have beneficial effect on the maturity and fertilizability of oocytes in poor ovarian reserve (POR) and may influence the progesterone level, thus increasing the pregnancy rate. However, previous studies on the effect of LH activity supplementation on poor responders have shown conflicting results. This study aimed to compare the clinical effectiveness of two different forms of gonadotropin (highly purified human menopausal gonadotropin (HP-HMG) vs. recombinant human follicle-stimulating hormone (r-hFSH)-only) in Indonesian population. Methods: Women diagnosed with poor ovarian response who received gonadotropin-releasing hormone (GnRH) antagonist protocol with either HP-HMG or r-hFSH-only were investigated. Women who underwent freeze all cycles, mini stimulation, and natural stimulation were excluded. Multiple logistic regression was performed to assess the effect of follicle-stimulating hormone (FSH) + human chorionic gonadotropin (HCG)-driven LH activity combination in HP-HMG to pregnancy event adjusting for progesterone level, demographic variables, and clinical characteristic variables. Results: A total of 101 subjects in the HP-HMG treatment group and 89 subjects in r-hFSH-only treatment group were involved in the study. There was no significant difference of clinical pregnancy rate between HP-HMG group and r-hFSH-only group (adjusted OR: 0.94, 95% CI: 0.39–2.25; p-value: 0.890). Conclusion: Compared to r-hFSH-only group, combination of FSH + HCG-driven LH activity in HP-HMG group had similar effectiveness in poor responders undergoing in vitro fertilization (IVF) using the antagonist protocol.

Objective: To assess the effect of short-term gavage of ethanolic extract of Imperata cylindrica L root on the ovarian activity and estrus behavior of female mice. Methods: Eighteen virgin female ddY mice, 8 to 10 weeks of age, weighing 22–25 g with regular estrus cycle, were divided into three groups. Group 1 received 0.5% carboxymethylcellulose, whereas Groups 2 and 3 received the ethanolic extract of Imperata cylindrica L root at 90 and 115 mg/kg body weight (b.w.) per day by gavage for 20 days, respectively. All of the groups were checked before 9 a.m. daily for vaginal cytology to determine the estrus phase. On day 21, the mice were sacrificed to collect serum samples to quantify the concentrations of reproductive hormones using enzyme-linked immunosorbent assay and to determine changes in the reproductive organs based on their reproductive organ weight, histomorphology, and histomorphometry of ovarium and uterus. Results: The reproductive organ weight in the treatment groups was similar compared with that in the control group. The 90 mg/kg b.w. treatment group showed an increase in corpus luteum number when compared with the control group, with few degenerated follicles and diminished oocytes. Moreover, the 115 mg/kg b.w. treatment group showed fewer primordial and primary follicles and an increase in corpus luteum number and a prolonged diestrus phase compared to the control and 90 mg/kg b.w. treatment groups. The histomorphology examination of the uterus showed that the thickness of myometrium and epithelium in the treated animals was similar to the control group. In addition, there was a significant decrease in follicle-stimulating hormone level in the 115 mg/kg b.w. treatment group (P

An investigation was conducted to determine the influence of two sperm selection modalities, IMSI and ICSI, on the morphokinetics, dynamic development and ploidy status of embryos derived from males with sub-optimal sperm profiles during IVF program. A total of 209 PGTA-tested top-quality blastocysts (IMSI = 129, ICSI = 80) from 84 couples (IMSI = 51, ICSI = 33) were assessed retrospectively. This study found that both IMSI and ICSI yielded comparable embryo morphokinetics, except for the T7, TEB and CC3 parameters (p 0.05). A significant lower incidence of multinucleation was observed in the IMSI group when compared to the ICSI group (48.8% vs. 71.3%, p = 0.002), while other parameters of embryo development such as direct cleavage, distorted cytoplasmic movement, reverse cleavage and vacuole(s) appearance did not differ (p 0.05). No differences were noticed in the proportion of generating chromosomally euploid embryos (44.2% vs. 51.3%, p = 0.394, respectively, for IMSI and ICSI). The implementation of IMSI or ICSI in couples with sub-optimal sperm profiles resulted in embryos with comparatively similar morphokinetics. Furthermore, the incidence of multinucleation at the two- to four-cell stage was lower following the practice of IMSI, although the method did not improve the proportion of gaining euploid embryos.

Hasanah U, Binawanto, Alimuddin A, Boediono A, Kursini E. 2020. Short Communication: Effect of cryopreservation on ultrastructure and mitochondrial function of albino Pangasius catfish spermatozoa. Biodiversitas 21: 4524-4528. Cryopreservation techniques have been carried out on many endangered species and animals with unique characteristics. Successful cryopreservation techniques vary between species depending on various factors. The study used cryopreserved spermatozoa of the albino Pangasius catfish as samples. The cryopreserved spermatozoa were analyzed by its ultrastructure, functional mitochondria, and viability. The cryopreservation was performed using a combination of 10% methanol, skim milk, and fish ringer extender. A deep freezer is used for cryopreservation at -80 °C with a storage period of 14 days. Observations were made on fresh spermatozoa, post-equilibration spermatozoa, and frozen-thaw spermatozoa. This study found there were differences in ultrastructure and morphology in the three treatments. Fresh spermatozoa and post-equilibration spermatozoa appeared intact membrane, mitochondrial, and flagellar structures. In contrast, in frozen-thaw spermatozoa, there was damage to the cell membrane. The study showed different percentages of yields on functional mitochondria of fresh spermatozoa (98 ± 2%), spermatozoa post-equilibration (57 ± 7%), frozen-thaw spermatozoa (42 ± 3.21%). Cell viability showed that there were differences in viability of fresh spermatozoa and frozen-thaw (p

Female reproductive system showing the fastest signs of aging. The ovarian aging characterized by a decrease in follicular development. Stem cells are undifferentiated cells and can form a variety of different cells as the foundation of tissues and organs. Previous studies reported that Bone Marrow Mesenchymal Stem Cells (BM-MSCs) transplantation can restore follicular development in damaged ovarian rats. This study aimed to analyze the number of follicular development in aged rats and to analyze the capability of human Umbilical Cord Mesenchymal Stem Cells (hUC-MSCs) to improving follicular development in aged rats. This study used 3 mature rats (4 months old), and 9 nine aged rats (22-24 months old), Spraque Dawley (SD) strain. They were divided into four groups. The first and the second group was mature rats and aged rats without injection. The third and the fourth group was aged rats injected hUC-MSCs dose 106 cells/kgBW and hUC-MSCs dose 107 cells/kgBW. The injection carried out 4 times at 3-month intervals. The parameters observed were follicular development and homing image of hUC-MSCs in ovarian tissue. The results showed that the number of follicular developments in aged rats 22-24 months decreased significantly compared to mature rats 4 months old. Injection of hUC-MSCs at dose 106 cells/kgBW and 107 cells/kgBW did not increase follicular development in aged rats. hUC-MSCs did not found in ovarian tissue. It could be concluded that aged rats 22-24 months old no longer productive indicated from the number of follicular developments and corpus luteum decreased. The injection of hUC-MSCs intravenously did not indicate an improvement of follicular development in aged rats 22-24 months old.

An experiment to compare age of Macaca fascicularis (Mf) as pheripheral blood (PB) mesenchymal stem cell (MSC) isolate sources and the impact of its concentration on the pheriperal blood mononucleous cells (PBMC) development has been conducted. Twelve male Mf were used in this experiment. Three different age groups (infant (A1), juvenil (A2) and adult (A3)) of the Mfs were compared as treatments. Isolate of pheriperal blood MSC were created by taking 1 ml, 5 ml or 10 ml the Mfs pheriperal blood, processed them into PBMC, counted, isolated, cultured, subcultured, pelleted, extracted for their messenger Ribonucleic Acid (mRNA). Reverse transcriptase - polymerase chain reaction (RT-PCR) were conducted to obtain complentary Deoxyribonucleic Acid (cDNA). PCR amplification were performed to look cluster differentiation (CD) of the MSC gene expression. Incomplete block design was used and the data were analysed using descriptive statistic and T-Test. The results showed that PBMC counted from infant, juvenil and adult were 6.78 – 7.28, 6.18 – 7.30, and 6.01 – 7.34 log cell, respectively. The subculture and pelleting cells were only obtained from A3 with positive 73, 90, 105 and negative 34, 45 CD markers. It is concluded that pheriperal blood of adult Mf can be utilized as MSC source.

Preservation of ovarian tissue from severely injured or dead valuable animals has the potential to preserve female germ cells of animals. The ability to mature and fertilize of oocytes from preserved ovary of endangered species will allow us to sustain genetic and global biodiversities. The aims of this study were to investigate the viability of oocytes collected from the preserved ovary and its potential utilization for the production of cat embryos followed by in vitro maturation and fertilization. Ovary was preserved immediately in phosphate buffer saline (PBS) at 4 °C for 24 or 48 hours. The quality and viability of oocytes after the maturation process were identified microscopically using aceto-orcein staining. Biological function of the oocytes was evaluated by using in vitro culture technique for the maturation and fertilization rate in CR1aa medium culture. The results showed that the percentage of oocytes collected from preserved ovary for 24 and 48 hours that remained at the stage of metaphase-II were 29.4% and 21.9% respectively. Fertilization rates produced in the IVF using oocytes collected from ovary preserved for 24 or 48 hours were significantly lower (30%) than that of unpreserved control (36.7%). In conclusion, female germ cells of cat ovary preserved at 4 °C in PBS for 2 days were still viable for in vitro fertilization and thus can be utilized for in vitro production of cat embryos. Information obtained can be used as a basis of knowledge of using a combination of physiological reagent and cold-based preservation technique in modern reproductive technology for animals.

Multiple ovulation and embryo transfer (MOET) is a reproductive technology to increase the livestock population in a short period. The success of ovary stimulation programs is influenced by an individual’s response to follicle-stimulating hormone (FSH) stimulation. In this study embryo production was carried out on local cows represented by Peranakan Ongole (PO) cows and exotic cows represented by Simmental cows. FSH stimulation was performed on 10 PO cows and 10 Simmental cows. On Day-1 Cue Mate® (progesterone) was inserted intravaginally. FSH injection (400 mg) was carried out intramuscularly from D-10 at reduced dose with 12 hours intervals (Day-10: 100 mg; Day-11: 60 mg; Day-12: 40 mg per injection). On Day-12, prostaglandin (PGF2α) was injected and Cue Mate® was removed. Artificial insemination (AI) was performed 48 hours after PGF2α injection for 3 times at 12 hours intervals. The embryo was collected 6 days after the last AI (Day-21). Superovulation response was detected on 70% of PO cows and 90% of Simmental cows. The average number of transferable embryos in Simmental cows (9.11±7.27) was higher than PO cows (7.86±7.78). This research shows that Simmental cows are more responsive to FSH stimulation, and can produce more transferable embryos than PO cows.

Interspecies embryo transfer is a seldom-used method to increase the successful conservation of endangered species. The study aimed to determine the potential development of interspecies reconstructed embryos. The present study used two animal models, Campbell hamsters (Phodopus campbelli) and mice (Mus musculus). The isolated inner cell mass (ICM) of hamster embryos were injected into the mice embryos. The embryos were transferred to the pseudopregnant mice using non-surgery embryos transfer methods. The fetuses were collected at day 13.5 of gestation for morphometric measurement and cytochrome b (Cyt b) analysis which used to determine the species of obtained fetuses. The results showed that the viability, pregnancy rate, and embryonic implantation ability of the interpecies reconstructed embryos did not differ significantly (p>0.05) compared to non-reconstructed embryos. Morphometric measurement showed that the crown-rump (CR) and the weight of fetuses in the reconstructed group were significantly higher than non-reconstructed group (p

Objective This study aimed to examine the effect of vitrification on apoptosis and survival in human preantral follicles after thawing. Methods This experimental study was conducted at an acute tertiary care hospital from March 2012 to April 2013. Ovaries were sliced into 5×5×1-mm pieces and divided into the following three groups: preantral follicle isolation, ovarian tissue vitrification-warming followed by follicle isolation, and immunohistochemistry of fresh ovarian tissue. For statistical analyses, the Student t-test, chi-square test, Kruskal-Wallis test, and Kaplan-Meier survival analysis were used. Results A total of 161 preantral follicles (70% secondary) were collected from ovarian cortex tissue of six women between 30 and 37 years of age who underwent oophorectomy due to cervical cancer or breast cancer. There were no significant differences in the follicular morphology of fresh preantral follicles and vitrified follicles after thawing. The mean Fas ligand (FasL) mRNA expression level was 0.43±0.20 (relative to β-actin) in fresh preantral follicles versus 0.51±0.20 in vitrified follicles (p=0.22). The mean caspase-3 mRNA expression level in fresh preantral follicles was 0.56±0.49 vs. 0.27±0.21 in vitrified follicles (p=0.233). One vitrified-thawed secondary follicle grew and developed to an antral follicle within 6 days of culture. Conclusion Vitrification did not affect preantral follicle morphology or mRNA expression of the apoptosis markers FasL and caspase-3. Further studies are required to establish whether vitrification affects the outcomes of in vitro culture and the maturation of preantral follicles.

Background: Dimethyl sulfoxide (Me2SO) is a common cryoprotective agent widely used in cell preservation system. Me2SO is currently known to cause epigenetic changes which are critical in stem cells development and cellular differentiation. Therefore, it is imperative to develop cryopreservation techniques that protect cellular functions and avert Me2SO adverse effect. Trehalose was able to protect organism in extreme condition such as dehydration and cold. This study aimed to verify the protective effect of trehalose preincubation procedure in cryopreservation.Methods: The study was conducted using experimental design. Thawed mesenchymal (CD271+) stem cells from YARSI biorepository were used for the experiment. Trehalose preincubation was performed for 1 hour, internalized trehalose was confirmed by FTIR-ATR measurement. Three groups consisted of (1) cryopreserved without trehalose preincubation, (2) cryopreserved with trehalose preincubation, and (3) did not undergo cryopreservation were evaluated after 24 hours in LN2 for viability in culture. The absorbance from each group was measured at 450 nm. The analysis performed using paired student t test.Results: Viability of thawed mesenchymal (CD271+) stem cells that undergo trehalose preincubation prior cryopreservation was significantly higher (p

Sperms are vulnerable to oxidative stress due to the high proportion of polyunsaturated fatty acids within their membranes. This condition could decrease sperms quality during preservation. β-carotene is among antioxidants able to neutralize reactive oxygen species, natural by-products of oxygen metabolism in sperms. This study was done to investigate the capability and protective effect of this antioxidant on motility, viability, acrosomal integrity, and plasma membrane integrity of frozen-thawed sperms of Aceh swamp buffalo. The study was conducted using completely randomized design with four treatments and five replications. Fresh semen was diluted in egg-yolk tris-based extenders with the addition of antioxidant of 0% (w/v) (control, P0), 0.625% (w/v) (P1), 1.25% (w/v) (P2), and 2.5% (w/v) (P3) prior to freezing. Variables of sperms quality examined were motility, viability, acrosomal integrity, and plasma membrane integrity of semen after dilution, equilibration, and thawing. Semen having the best post-thawing quality after β-carotene treatment was used for artificial insemination (AI) for accessing its pregnancy rate. The results showed that the addition of 0.625% (w/v) β-carotene (P1) resulted in the highest motility, viability, acrosomal integrity, and plasma membrane integrity. Pregnancy rate of buffalo inseminated with semen treated with 0.625% β-carotene (P1) was 50%. In conclusion, administration of 0.625% β-carotene was able to maintain and could protect motility, viability, acrosomal integrity, and plasma membrane integrity of frozen sperms of Aceh swamp buffalo that are good for artificial insemination.

Quality of epididymal and ejaculated sperms of spotted buffalo in three different extenders (i.e. Andromed, Andromed supplemented with 0.2% dextrose, and Andromed supplemented with 0.4% dextrose) was studied. The results showed that there was no significantly different (P > 0.05) quality of ejaculated and epididymal sperms. The motility percentage of post thawing epididymal and ejaculated sperms was 41-46% and 41-45%, respectively. And percentage of cytoplasmic membrane integrity of the post thawing epididymal and ejaculated sperms was 66-67% and 47-54% respectively. There was also no significant different (P > 0.05) in the percentage of cytoplasmic membrane integrity between the extenders of both ejaculated and epididymal sperms. This result suggested that epididymal sperms were reliable for artificial insemination as good as ejaculated sperms.

In vitro maturation is the crucial step for in vitro embryo production. It needs a large number of oocytes as source gamet cells recovered. The present study is aimed to assess the influence of corpus luteum on the average number oocytes harvested, COCs quality and subsequent maturation of immature oocytes recovered from sheep ovaries. Sheep ovaries were collected from local slaughterhouse and COCs were collected by using slicing method. Collected COCs were graded into three categories dependent upon cumulus cells surrounding them and the homogenous of cytoplasm. COCs were maturated in maturation media at 5% CO2 for 24 hours. Maturation of oocytes evaluated base on the expansion of cumulus cells and extrusion of the first polar body. There was significantly higher on average of COCs harvested from ovaries with corpus luteum compared without corpus luteum. The presence of Corpus luteum did not affect the COCs quality and ability to reach the maturation stage. However, there was a dramatic effect of cultured COCs quality on maturation rate both groups. Collectively, these results indicate that COCs quality is the main factor affecting the subsequent of oocytes matured in vitro. Keywords: Corpus luteum; cumulus oocyte complex; in vitro maturation; maturation rate; ovaries

Sex determination in turtle species is not only based on genotype, but also rely on the incubation temperature. In addition, sexual differentiation takes place during the thermo-sensitive period (TSP). This study was conducted to determine the effects of incubation temperature on sex expression profile of determination gene, bioreproduction, phenotype, and locomotory performances of olive ridley turtle hatchlings. Fertile eggs incubated at two temperatures, namely feminine temperature (30-33°C), and masculine temperature (26-27°C). Value of cycle threshold (CT) measured during TSP, i.e 23-25 embryonic development stage, and after TSP, i.e 26-27 embryonic development stage using real time PCR techniques. Comparison of gene expression at both incubation temperatures were analyzed by ANOVA, and Student’s t test. Hatchling bioreproduction and phenotype measurement consist of the incubation period, embryo growth, morphometrics, and locomotori performances hatchlings were analyzed with regression analysis and Student’s t test. The results showed expression of both aromatase and Rspond 1 genes (which plays a role in ovarian differentiation) after the TSP that incubated at feminine temperature higher and different with masculine temperature. In conjunction with the bioreproduction and phenotype, the incubation period of feminine temperature shorter than that of masculine. Likewise, growth of the embryo of feminine temperature was faster than that of masculine. Incubation at feminine temperature significantly affect to carapace width, length and width of the plastron, long flippers and rear arms, long neck, and the frequency of the swing flippers. Keywords: thermo-sensitive period (TSP), gene expression, phenotype, Lepidochelys olivacea

The aim of the present study was to determine the number of follicles, oocyte quality and maturation rate of oocytes from pairs of ovary with different reproductive status in two maturation medium, TCM-199 as control and CR1aa as treatment. The pairs of ovary were classified into four groups: (i) ovaries with corpus luteum (CL) and dominant follicle (DF), (ii) ovaries with CL, without DF, (iii) ovaries with DF, without CL, (iv) ovaries without both CL and DF. Results of the experiment revealed that the greatest number of follicles was observed from ovary with CL without DF (15.88 ± 10.68), although not significantly different (P > 0.05) with other status of ovaries. The lowest number (P < 0.05) of A grade oocytes was found from ovary with DF without CL (1.20 ± 1.10). The percentage of Metaphase II was highest in TCM-199 (75.51%) with oocytes from ovaries with CL and DF, and the lowest with oocytes from ovaries with DF without CL in TCM-199 and CR1aa (42.86 and 30.95%). The study suggested that the number of oocytes with A grade were influenced by the reproductive status of ovaries. The maturation rate of A grade oocytes was influenced by the quality of oocytes and the composition of maturation medium.

The significant decline of spotted buffalo population nowadays brought this species into an endangered situation. To perform an integrated conservation project, we need some basic information and data related to the reproductive and genetics potency of this buffalo. The purpose of this study was to observe the effect of coat color variation to the sperm quality, in order to get focus on specific candidate gene that allegedly bring the causative mutation(s) and responsible for the different pigmentation expression. In this study, we compare the quality of fresh and frozen-thawed epididymal sperm from 12 spotted bulls (that classified in 3 different spotted types, i.e Saleko, Bonga , and Lotong Boko ) with five solid bulls. The results showed that there were no significant differences (P > 0.05) in all parameters of fresh and frozen-thawed epididymal sperm among those groups. The percentage of frozen-thawed progressive motility from Saleko, Bonga, Lotong Boko , and Solid was 44%, 42%, 40% and 42%, respectively. Moreover, the percentage of livability and membrane integrity of frozen-thawed sperm from each groups were 64.9%; 65.2%; 62.6%; 62.7% and 64.6%; 67.1%; 64.5%; 64.1%. In conclusion, it suggested that the coat color/phenotype difference has no effects on the quality of fresh and frozen-thawed epididymal sperm of spotted buffalo. Key Words : Epididymal Sperm, Spotted Buffalo

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live offspring were produced at each station from transplanting these embryos. The objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8-cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF-derived blastomeres (method 1) and by aggregation of a whole parthenogenetic embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (method 2). Similarly in experiment II, chimeric embryos were constructed by aggregating IVF-derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogenetic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed from 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or eight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were constructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV). In the blastocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopreserved in ethylene glycol (EG) plus trehalose (T) with different concentrations of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%). In experiment I, the aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derived embryos cultured without agar than when cultured with agar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively). The development rate to blastocysts, however, was not different among the treatments. In experiment II, the developmental rates to the morula and blastocyst stages were 81%, 89%, and 28% for the chimeric embryos with parthenogenetic:IVF blastomere ratios of 2:6, 4:4, and 6:2, respectively. In experiment III, the developmental rate to the morula and blastocyst stages was 60% and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell parthenogenetic embryos. To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric embryos at 24 hr following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP (89% vs. 56%). Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male (stillbirths) and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transferred 40 chimeric embryos at the Louisiana station. Two pregnancies were lost prior to 4 months and one phenotypically- chimeric viable male calf was born. We conclude that the IVF-derived blastomeres were able to stimulate the development of bovine parthenogenetic blastomeres and that the chimeric parthenogenetic bovine embryos were developmentally competent. Mol. Reprod. Dev. 53:159–170, 1999. © 1999 Wiley-Liss, Inc.

This study examined morphological appearance, viability and hatching rates in relation to the total cell number following vitrification of in vitro produced bovine blastocysts and expanded blastocysts. In Experiment 1, embryos obtained after 7, 8 or 9 d of culture were pooled and equilibrated in either 10% ethylene glycol (EG) or 10% EG plus 0.3M trehalose in Dulbecco's phosphate buffered saline (DPBS) supplemented with 10% calf serum and 0.6% BSA for 5 min each, at room temperature, and then vitrified together in precooled vitrification solutions consisting of 40% EG (Treatment 1), 40% EG plus 0.3M trehalose (Treatment 2), 40% EG plus 0.3M trehalose and 20% polyvinylpyrrolidone (PVP, Treatment 3) in DPBS. The embryo viability and hatching rates of Treatment 1 (19 and 3%) differed significantly (P < 0.05) from those of Treatment 2 (56 and 31%) and Treatment 3 (70 and 43%). There was a significant difference (P < 0.05) in embryo viability between Treatment 2 (31%) and Treatment 3 (43%). In Experiment 2, Day 7, 8 and 9 embryos were vitrified separately, with higher viability and hatching rates in Experiment 1 than in Experiment 2. The viabilities of Day 7 (87%), 8 (71%) and 9 (46%) embryos differed significantly (P < 0.05). Again, there were significant differences (P < 0.01) among the hatching rates of Day 7 (75%), 8 (38%) and 9 (9%) embryos. The total cell number of hatched blastocysts was then determined by differential fluorochrome staining. The total cell number of Day 7, 8 and 9 embryos differed significantly (P < 0.05).

Bovine embryos produced by in vitro maturation (IVM), fertilization (IVF) and culture (IVC) were used to produce aggregation chimeras. An aggregated chimera was produced by combining bovine IVF embryos (Holstein × Japanese Black and Japanese Brown × Limousin breeds) which were cultured in vitro without the zonae pellucidae. Forty-eight hours after IVF, embryos at the 8 cell-stage were used to produce aggregation chimeras. In Experiment I, the zonae pellucidae was removed by a microsurgical method using a microblade or by treatment with 0.25% pronase. Holstein × Japanese Black embryos were aggregated with Japanese Brown × Limousin embryos after zonae removal by hand manipulation in culture medium. In Experiment II, the viability of the aggregated embryos developing into blastocysts was examined by measuring the extent of development. The number of aggregated embryos and embryos developed into blastocysts was 34 (91.9%) and 24 (70.6%), respectively, when the zonae pellucidae was removed by the microsurgical method; and 12 (92.3%) and 6 (50.0%), respectively, when the zonae pellucidae was removed using the 0.25% pronase treatment. The size of the aggregated embryos was significantly different from that of the normal embryos when cultured in vitro until Day 10, but not different thereafter. Five aggregated embryos were transferred nonsurgically to the recipients, resulting in 1 pregnancy and the birth of 2 chimeric calves. Skin color was used as evidence of chimerism.

Thorax ganglion was reported as a source of stimulating hormone for gonad development. The aim of this research was to study the development of mud crab ( Scylla olivaceous ) oocytes cultured in in vitro medium supplemented with thoracic ganglion extracts of Neopisesarma lafondi . Immature crabs with 110 mm carapace width were collected from the mouth of Bawana Marana Rivers, Maros Regency of South Sulawesi, Indonesia. Pieces of ovarian tissues from those crabs were incubated within 24 hours in culture medium (Medium-199 with 100 IU/ml Penicilin-G) supplemented with 2 mg/ml thoracicganglion extract. Result showed that the diameter of mud crab oocytes was increased in congruent with the incubation time. The development was prominent after in vitro incubation for 8 hours (P

In an attempt to produce the interspecies embryo transfer, this study was conducted to evaluate the efficacy of the production of reconstructed blastocyst by transferring the donor ICM into the recipient trophoblast. ICM cells were isolated from the donor blastocyst by immunosurgery method. Zona-free blastocysts were incubated in the medium (TCM-199) containing 20% of the heat-inactivated rabbit anti-bovine-serum. The embryo reconstruction was produced by three different methods. Recipient blastocyst was maintained on the holding pipette by gentle suction, with the ICM in a 9 o'clock position to have the possibility of developing incor porate ICMs (Method I), the ICM was in a 3 o'clock position to break the original ICM during injection (Method 2); cutting the original recipient ICM followed by insertion of the donor ICM (Method 3). Reconstructed blastocysts were then cultured overnight and examined morphologically according to the re-expansion of the reconstructed blastocyst with or without developed donor ICM. According to morphological observation in this study, 37.9% of the reconstructed blastocyst developed with the incorporation of two ICM originally from recipient and donor (Method I), 66.7% of the reconstructed blastocysts developed with a single ICM (Method 2), and 80.0% of the reconstructed blastocyst developed from the ICM originally from donor ICM (Method 3). These results showed that the reconstructed blastocyst is better produced by cutting the original recipient ICM followed by the insertion of the donor ICM (Method 3).

Presence of placental tissues from more normal noncloned embryos could reduce the pregnancy failure of somatic cloning in cattle. In this study, inner cell mass (ICM) cells of in vitro-produced (IVP) embryos was replaced with those of nuclear transfer (NT) embryos to reconstruct bovine blastocysts with ICM and trophoblast cells from NT and IVP embryos, respectively. A total of 65 of these reconstructed embryos were nonsurgically transferred to 20 recipient beef females. Of those, two females were diagnosed pregnant by ultrasonography on day 30 of gestation. One pregnancy was lost at 60-90 days of gestation, and the other recipient cow remained pregnant at day 240 of gestation; however, this female died on day 252 of gestation. Gross pathology of the internal organs of the recipient female, a large fetus, and a large placental tissue mass suggested the massive size of the fetus and placental tissue were likely involved in terminating the life of the recipient female. Biopsy samples were harvested from the skin of the dead recipient cow, the fetus and from cotyledonary tissue. Microsatellite DNA analysis of these samples revealed that the genotype of the fetus was the same as that of the NT donor cells and different from that of the recipient cow. Correspondingly, neither the fetus nor recipient cow had the same genotype with that of the fetal cotyledonary tissue. These results present the first known documented case of a bovine somatic NT pregnancy with nonclone placental tissues after transfer of a blastocyst reconstructed by a microsurgical method to exchange of ICM cells and trophoblast tissue between NT and IVP blastocysts.

This study was conducted to examine the quality of ejaculated sperm by Garut rams to be used for artificial insemination (AI) and viability of sperm that were collected from preserved cauda epididymis (4°C up to 12 days) for assisted reproductive technology. The semen was collected by artificial vagina, with the sperm motility, live sperm, acrosomal intact, and intact plasma membrane observed. Sperm motility was 75%, while for the live sperm, intact plasma membrane and sperm abnormality were 91.5%, 90.0%, and 1.8%, respectively. In the other study, sperm was collected from cauda epididymis by aspiration method and diluted in different media: 1) Brackett Oliphant (BO) media and 2) modified Phosphate Buffer Saline (mPBS). Evaluation of sperm motility and intact plasma membrane were conducted after washing, counting and dilution of the sperm. The results of this study showed that the sperm motility and intact plasma membrane could be maintained better in BO rather than PBS medium although they were not statistically different (P>0.05). At day 12 of preservation, the motility and intact plasma membrane of sperm collected from cauda epididymis were 0.7% and 1.33% for motility and plasma membrane intact, respectively. These findings showed that the Garut rams semen was qualified for AI and frozen processing; in vitro embryo production by introducing the assisted reproductive technology such as intracytoplasmic sperm injection (ICSI) could be applied by using the sperm collected from preserved cauda epididymis until 12 days of preservation at 4°C. Keywords : Reproduction/spcrmatogenesis/inscmination/Garut rams/small ruminant

Frozen-thawed spermatozoa collected from a beef bull (Japanese Black) were used for in vitro fertilization (IVF) of matured oocytes obtained from dairy (Holstein) and beef (Japanese Black) females. Embryos were examined for fertilization, cleavage rate, interval between insemination and blastocyst production (experiment I), total cell number per embryo and sex ratio during blastocyst formation (experiment II), and blastocyst production rate of zygotes that developed to 2-, 4-, and 8-cell stages at 48h post-fertilization (experiment III). Fertilized oocytes were cultured in vitro on a cumulus cell co-culture system. The fertilization and cleavage rate of oocytes groups were similar, however, the blastocyst production rate was greater (P0.05) in hybrid than from purebred embryos (27% versus 20%). Development of blastocysts produced from hybrid embryos developed at a faster rate than blastocysts produced from the straightbred embryos. In hybrid embryos, blastocyst production was significantly greater on day 7 (56%) and gradually decreased from 20% on day 8 to 17% on day 9. In contrast, blastocyst production rate from the purebred embryos was lower on day 7 (17%), increasing on day 8 to 59% and then decreased on day 9 to 24%. The total number of cells per embryo and sex ratio of in vitro-produced blastocysts were not different between hybrid and purebred embryos. The number of blastocysts obtained from embryos at the 8-cell stage of development by 48h post-fertilization (94%) was greater (P0.01) than the number of zygotes producing blastocysts that had developed to the 4-cell stage (4%) and the 2-cell stage (2%) during the same interval. These results show that the blastocyst production rate and developmental rate to the blastocyst stage were different between hybrid and purebred embryos, and that almost all of the in vitro-produced blastocysts were obtained from zygotes that had developed to the 8-cell stage 48h post-fertilization.

The present study was designed as 5 X 4 factorial to investigate the effects of using sperm from 5 bulls, and varied sperm-oocyte incubation times (5, 10, 15 and 20 h) on the fertilization, cleavage rates and blastocyst formation on an in vitro bovine embryo production system. The bulls included a tetraparental Chimera, its sires (Japanese Black and Limousin), its maternal grand-sires (Japanese Brown and Holstein). The proportion of polyspermy, 2-pronuclei formation, fertilization, cleavage and development to blastocyst were affected (p < 0.01) by the duration of sperm-oocyte incubation, as well as by the interaction between bulls and their corresponding sperm-oocyte incubation time. Blastocyst rate observed after 5 h in oocytes inseminated with Chimera, Japanese Black and Limousin were higher (p < 0.05) than those observed at 20 h incubation. The proportion of blastocysts from oocytes inseminated with Japanese Black observed at 10 h of incubation did not differ from that of Chimera, but both were higher (p < 0.05) than those observed for the Limousin, Japanese Brown and Holstein sires. The present study showed that there was an effect by the duration of sperm-oocyte incubation on in vitro embryo development. The optimal time of sperm-oocyte incubation for the Chimera was similar to that of its sires (Japanese Black and Limousin) but differed from its maternal grand-sires (Japanese Brown and Holstein). The fertilization rates for the sperm from the Holstein bull increased up to 15 h suggesting that this might be the only bull that would benefit from a long incubation period for insemination. 0 1997 Elsevier Science B.V.

The purpose of the present study was to examine the ability of a tetraparental Chimera in producing IVF embryos. Cumulus oocytes complexes (COCs) were matured in vitro for 22 h. Frozen-thawed sperm of a Chimera (CH), as well as Japanese Black (JB), Limousin (L), Japanese Brown (JBr), Holstein (H) bulls were used for IVF. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h of incubation was higher (P < 0.01) with the Chimera (CH) than with the Holstein and in Japanese Brown bulls, but did not differ from Japanese Black and Limousin bull sperm (79.0%, 71.2%, 72.5%, 57.8% and 57.0% for CH, JB, L, JBr and H sperm, respectively). Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera (O-CH) sperm were higher (P < 0.05) than with Japanese Brown (O-JBr) and (P < 0.01) than with Holstein (O-H) sperm, but did not differ from Japanese Black (O-JB) and Limousin (O-L) sperm (3644, 81.8%; 2835, 80.0%; 2536, 69.4%; 1943, 44.2% and 633, 18.2% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The cleavage rates of IVM oocytes inseminated with Chimera sperm were also higher (P < 0.001) than in Holstein, (P < 0.01) Japanese Brown and (P < 0.05) Limousin, but did not differ from Japanese Black sperm (181239, 75.7%; 123171, 71.9%; 108186, 58.1%; 80196, 40.8% and 30186, 16.1% for O-CH, O-JB, O-L, O-JBr and O-H, respectively). The blastocyst rates of IVM oocytes inseminated with sperm were higher (P < 0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black (69181, 38.1%; 48123, 39.0%; 27108, 25.0%; 730, 23.3% and 1680, 17.8% for O-CH, O-JB, O-L, O.-JBr and O-H, respectively). These findings suggested the sperm from this tetraparental Chimeric bull were able to be used for producing bovine IVF embryos.

In the present study, IVF bovine embryos were vitrified using as the cryoprotectants, ethylene glycol plus trehalose plus the polymer, polyvinylpyrrolidone (PVP). In Experiment I, toxicity of the vitrification solution (VS) containing 20% PVP was tested in relation to temperature and exposure time. One hundred percent embryo development was observed with treatment at 5 degrees C for 5 min, whereas only 55.5% embryos were developed when the treatment was carried out at 20 degrees C for 5 min. In Experiment II, embryos were vitrified using one of the three treatments (Treatment A, 40% ethylene glycol (EG); treatment B, 40% EG + 11.3% trehalose; and treatment C, 40% EG + 11.3% trehalose + 20% PVP and rehydrations) was performed directly in mPBS. Highest development (84.1%) and hatching rate (68.2%) were obtained when embryos were vitrified with the vitrification solution used in treatment C. In Experiment III, embryos were vitrified as in Experiment II (treatment C). The development and hatching rates were compared after rehydration in different rehydration solutions. No significant difference was observed among the development and hatching rates when rehydration was carried out in different concentrations of trehalose. Five vitrified-warmed bovine embryos were transferred directly to five recipients and three recipients gave birth to three normal calves.

A study was conducted to develop a better freezing protocol for in vitro developed biopsied bovine blastocysts. Biopsied blastocysts were exposed to 1.8 M ethylene glycol (EG) + 0.05 M trehalose (T) and different concentration (5, 10, and 20%) of polyvinylpyrrolidone (PVP). Exposure to the solutions alone did not affect their in vitro development (Experiment 1). Experiments 2, 3, and 4 tested the viability of biopsied blastocysts cryopreserved in 1.8 M EG + different concentrations of T (0, 0.05, 0.1, and 0.3 M), 1.8 M EG + different concentrations of PVP (0, 5, 10, and 20%), and 1.8 M EG + 0.05 M T + different concentrations of PVP (0, 5, 10, and 20%), respectively. The proportion of biopsied blastocysts that reexpanded following cryopreservation in 1.8 M EG + 0.05 M T (38.5%) and 1.8 M EG + 0.1 M T (36.1%) was significantly (P0.05) higher than the proportion that reexpanded in 1.8 M EG + 0.3 M T (13.9%) (Experiment 2). The viability and the percentage of embryos that developed to250 microns in diameter in the 5, 10, and 20% PVP groups (77.8 and 50.0%, 78.1 and 43.8%, 76.9 and 65.4%, respectively) were significantly higher than those that developed cryopreserved without PVP (55.1 and 20.7%) (Experiment 3). Optimum development of in vitro culture of frozen-thawed biopsied blastocysts was obtained using 1.8 M EG + 0.05 M T and 20% PVP. Analysis of blastocysts250 microns in diameter showed that the number of ICM cells of biopsied blastocysts cryopreserved in 1.8 M EG + 0.05 M T with or without PVP was not different from the number of unfrozen biopsied blastocysts. These results indicate that PVP has some beneficial effect on freezing of biopsied bovine blastocysts.

The relationship between cryoprotectant and exposure time on the development of IVF-derived embryos in culture after freezing and thawing was examined. Good to excellent quality Day 7 to 8 blastocysts and expanded blastocysts were suspended in 1.6 M propylene glycol (PG), 1.8 M ethylene glycol (EG), 1.1 M diethylene glycol, and 1.3 M ethylene glycol monomethyl ether (EME). In Experiment I, after 30 and 120 min of exposure, embryos were placed directly into culture medium and washed three times. Embryos were cocultured with cumulus cells in culture medium. After a 48-h culture, there were no significant differences in fully expanded, hatching, and hatched rates between the 30- and 120-min exposure to all four cryoprotectants except for the fully expanded rate with PG (55 vs 83%). In Experiment II, to compare the cryoprotective effects of the four cryoprotectants, embryos were exposed to each cryoprotectant for variable times from 10 to 120 min, seeded, and cooled to -30 degrees C at 0.3 degree C/min, plunged into liquid nitrogen for storage, and then thawed. Survival was measured by coculturing the embryos with cumulus cells in culture medium. There were no significant differences in development to hatched blastocysts of embryos exposed to either PG (27-42%) or EG (37-56%). The number of embryos developing after exposure to EME was similar to that in PG or EG when exposure time did not exceed 40 min (43-49%). Our findings suggest that the toxicity of different cryoprotectants is related to the exposure time and that PG and EG were relatively nontoxic as is exposure to EME for short periods.

Bovine oocytes surrounded with compact cumulus cells were cultured for 20 to 22 hours (38.5 degrees C, 5% CO(2)) in modified TCM-199 medium supplemented with 5% superovulated cow serum (SCS) and inseminated by in vitro capacitated spermatozoa. Day 7 to 8 embryos were equilibrated for 10 minutes in 1.3 M methyl cellosolve (MC), 1.1 M diethylene glycol (DEG), 1.8 M ethylene glycol (EG), 1.6 M propylene glycol (PG) and 1.1 M 1, 3-butylene glycol (BG) solutions. They were then loaded into 0.25-ml straws, placed into an alcohol bath freezer at 0 degrees C, cooled from 0 degrees C to -6 degrees C at -1 degrees C/minute, seeded, held for 10 minutes, and cooled again at -0.3 degrees C or -0.5 degrees C/minute to -30 degrees C. Straws were then plunged and stored in liquid nitrogen. After thawing in 30 degrees C water, the embryos were rehydrated in TCM-199 medium and then cultured for 48 hours in TCM-199 plus 5% SCS. Embryos were considered viable if they progressed to later developmental stages with good morphology. Some of the embryos frozen in each cryoprotectant were thawed and transferred nonsurgically without removing the cryoprotectant. Hatched embryos survived freezing and one-step dilution as follows: EG (50.0%), MC (53.6%), DEG (56.9%), PG (58.0%) and BG (11.5%). The survival rate of embryos cooled at -0.3 degrees C vs -0.5 degrees C/minute was not significantly different (P0.05), however, blastocysts hatched most often (P0.01) in vitro when cooled at a rate of -0.3 degrees C/minute (64.6%, 31/48) than at -0.5 degrees C/minute (22.6%, 12/53). Pregnancy rates resulting from embryos frozen in the different cryoprotectants were as follows: MC (48%, 10/21); DEG (30%, 3/10); EG (74%, 20/27); and PG (40%, 4/10). These results indicate that MC, DEG, EG and PG have utility as cryoprotectants for the freezing and thawing of IVF bovine embryos.

more common in older women (P 38 years were significantly lower ( P < 0.05). Endometrial thickness was similar in both groups on the day of embryo transfer. Pregnancy rates were significantly higher in the younger group. Wide perivitelline space with debris, abnormal zona pellucida and vacuoles in the oocytes were the abnormalities that were associated with lower pregnancy rates in both groups. These abnormalities were more common in older women. Conclusion: Poor oocyte morphology is associated with poor pregnancy outcome following ICSI. Older women have a higher incidence of poor quality oocytes, especially those with abnormal zona pellucida, wide perivitelline space with debris and vacuoles, which are found to adversely affect the pregnancy rate.

Mature bovine oocytes were activated with 7% ethanol followed by cytochalasin B or D treatment. Most oocytes extruded a second polar body and formed one pronucleus when treated with 7% ethanol alone [35/43 (81%)]. With ethanol followed by cytochalasin B or D, overall activation frequency was 70% (309/441), with activated oocytes containing two pronuclei. The cleavage rate was not significantly different between treatment with ethanol alone and ethanol followed by 5 micrograms mL-1 cytochalasin B, but it was significantly lower than in fertilized oocytes (P < 0.01). However, the blastocyst production rate was significantly different (P < 0.01) among the treatments. The incidence of parthenogenetic embryos with normal (diploid) complements and with chromosome anomalies (2N/4N) was 68% (17/25) and 32% (8/25) respectively, and this was not affected by cryopreservation treatment. The longitudinal diameter of aggregated-four embryos cultured in vitro was greater (P < 0.01) than aggregated-two or single embryos. One of the aggregated-four parthenogenetic embryos was further cultured in vitro and developed up to Day 27 after activation, with a diameter of 2980 microns. The aggregated-four parthenogenetic embryos were transferred to five recipients. The oestrus was prolonged in three recipients and they returned to oestrus on Day 57, 62 and 67 after the previous oestrus. These results indicate that aggregating parthenogenetic embryos can prolong their survival in vitro and in vivo.

Experiments were conducted on the morphology, fertilization and embryo development rate of vitrified ovine oocytes matured in vitro. Three vitrification solutions were used for vitrification. PBS supplemented with 1% BSA, 30% ethylene glycol was added by one of three different sucrose concentrations, 1.00 M (VS 1 ), 0.50 M (VS 2 ), and 0.25 M (VS 3 ). The results showed that the percentages of normal vitrified oocytes after warming were 78 and 63% in VS 1 and VS 2 , respectively, which was significantly higher as compared for VS 3 . The fertilization rates were 59 and 66% in VS 1 and VS 2 , respectively, which were also significantly higher as compared with VS 3 (35%). Zygote viability after 18 h was 57; 43; and 40%, for VS 1 ,VS 2 , and VS 3 , respectively, which was not significantly different. The incidence of polyspermic penetration increased with increasing sucrose concentration, i.e 23, 11, and 9% in VS 1 , VS 2 , and VS 3 , respectively, as compared with unvitrified oocytes (4%). The cleavage rate of vitrified oocytes in VS 1 was 13.2% which was significantly lower (p

The ability to mature and fertilize oocytes of endangered species may allow us to sustain genetic and global biodiversity. Epididymis sperms may be the last chance to ensure preservation of genetic materials after injury or death of a valuable animal. Studies have been conducted to determine wether both epididymis sperms and oocytes can be used to produce viable embryos and offspring. The purpose of this study was to determine how long cats sperms contained in epididymis were remain motile and had intact membranes when preserved at 4 ° C, and to determine whether such those preserved sperms are able to fertilize oocytes. Epididymis was preserved immediately in phosphate buffer saline at 4 ° C for 1, 3, and 6 days. The observation of sperm quality and viability after preservation was performed by vital staining acrosom and Hoechst-Propidium Iodine. Biological functions of sperms were evaluated by in vitro culture technique for fertilization, micro fertilization and embryonic development rate in CR1aa medium. The results showed that average motility of sperms collected from ductus deferens, cauda and corpus epididymis decreased not significantly (P > 0.05) from 0, 1, 3, and 6 days of preservation times (from 83.0%, 80.2%, 79.0%; 80.9%, 75.0%, 75.5%; 52.0%, 63.2%, 55.0% to 34.6%, 34.6%, 33.3%, respectively). The general results showed that sperms from epididymis preserved for 1, 3, and 6 days can be used for IVF. The rate of embryonal cleavage produced by IVF technique using sperms collected from epididymis preserved for 1-, 3- and 6-days were 33.3, 26.7, and 20.0%, respectively and significantly different (p < 0.05) from that of controll (50.0%). In conclusion, sperms contained in epididyimis preserved at 4 ° C in PBS (Phospate Buffer Saline) for 1-6 days can be used to IVF and in vitro production of cat embryos.