284 COMPARATIVE FERTILITY OF FRESHLY-COLLECTED VERSUS FROZEN - THAWED SPERMATOZOA FOR IN VITRO FERTILIZATION IN THE FISHING CAT (PRIONAILURUS VIVERRINUS)

2006 ◽  
Vol 18 (2) ◽  
pp. 249
Author(s):  
G. Magarey ◽  
J. Herrick ◽  
K. Thiangtum ◽  
W. Tunwattana ◽  
W. Swanson
Keyword(s):  
Sperm Motility ◽  
Culture Medium ◽  
Culture Media ◽  
Egg Yolk ◽  
Ovarian Follicles ◽  
Blastocyst Stage ◽  
Motile Sperm ◽  
Vitro Fertilization ◽  
International Transport

Wild populations of fishing cats (Prionailurus viverrinus) in Southeast Asia are in decline, primarily due to habitat loss. Because the fishing cat population in North American zoos is small (n = 69) and inbred (F = 0.17) with relatively low genetic variation (86%), infusion of new founder genes from Asia is a conservation priority. Importation of cryopreserved semen for use with IVF and ET may offer one alternative to the international transport of living animals. In this study, our objectives were to (1) compare motility longevity of fresh vs. frozen-thawed fishing cat spermatozoa in two culture media, (2) evaluate ovarian responses to exogenous gonadotropins, and (3) assess development of IVF embryos produced with fresh vs. frozen-thawed spermatozoa. Raw semen was collected via electroejaculation from male fishing cats (n = 4), divided into groups, and washed. Two sperm pellets were resuspended in either Ham's F10 medium (HF10; with 5% FBS) or our feline optimized culture medium (FOCM; with 0.4% BSA); another pellet was diluted in TEST egg yolk, cooled to 5�C over 3 h, glycerated (4%), and cryopreserved in straws over LN2 vapor. Frozen sperm samples were thawed, washed, and diluted in either HF10 or FOCM. Fresh and frozen-thawed sperm motility (percent motile, rate of forward progress) in each medium (10 � 106 motile sperm/mL) was assessed (at 0, 1, 3, and 6 h) in microdrops under oil during culture (38�C; 6% CO2 in air). Female fishing cats (n = 10) were treated with exogenous gonadotropins (150 IU eCG, 100 IU hCG, 85-h interval) and ovarian follicles were aspirated laparoscopically. Recovered oocytes were inseminated with fresh (2 � 105 motile sperm/mL) or frozen-thawed (5 � 105 motile sperm/mL) spermatozoa in FOCM microdrops; resulting embryos were either cryopreserved or cultured in FOCM (with 5% FBS added at 72 h post-insemination) for 7 days. Sperm motility over time did not differ (P > 0.05) between media for either fresh or frozen-thawed samples; however, across media, frozen-thawed sperm motility was lower (P < 0.05) and declined faster (P < 0.05) compared to fresh spermatozoa. Females produced an average (�SEM) of 9.8 � 2.9 mature ovarian follicles, allowing recovery of 7.3 � 2.6 high-quality oocytes per female. Oocyte cleavage percentage at 42 h p.i. was lower (P < 0.05) with frozen-thawed spermatozoa (38%, 11/29) compared to freshly collected spermatozoa (68%, 17/25). Overall, 35% (6/17) of cultured embryos developed to blastocysts with no difference (P > 0.05) between embryos produced with frozen-thawed (4/11) vs. fresh (2/6) spermatozoa. Although fishing cat sperm motility and fertility appear compromised after cryopreservation, our results demonstrate the ability of frozen-thawed spermatozoa to produce IVF embryos that are capable of developing to blastocyst stage in vitro. This work was supported by (NIH RR015388).

In vitro culture and non-invasive metabolic profiling of single bovine embryos

2019 ◽  
Vol 31 (2) ◽  
pp. 306
Author(s):  
Monika Nõmm ◽  
Rando Porosk ◽  
Pille Pärn ◽  
Kalle Kilk ◽  
Ursel Soomets ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Metabolic Profiling ◽  
Culture Medium ◽  
Culture Media ◽  
Blastocyst Stage ◽  
Growth Media ◽  
Low Molecular Weight ◽  
Bovine Embryos ◽  
Non Invasive

Selecting high-quality embryos for transfer has been a difficult task when producing bovine embryos invitro. The most used non-invasive method is based on visual observation. Molecular characterisation of embryo growth media has been proposed as a complementary method. In this study we demonstrate a culture medium sampling method for identifying potential embryonic viability markers to predict normal or abnormal embryonic development. During single embryo culture, 20µL culture media was removed at Days 2, 5 and 8 after fertilisation from the same droplet (60µL). In all, 58 samples were analysed using liquid chromatography–mass spectrometry. We demonstrate that it is possible to remove samples from the same culture medium droplets and not significantly affect blastocyst rate (25.2%). Changes in any single low molecular weight compound were not predictive enough. Combining multiple low molecular weight signals made it possible to predict Day 2 and 5 embryo development to the blastocyst stage with an accuracy of 64%. Elevated concentrations of lysophosphatidylethanolamines (m/z=453, 566, 588) in the culture media of Day 8 well-developing embryos were observed. Choline (104m/z) and citrate (215m/z) concentrations were increased in embryos in which development was retarded. Metabolic profiling provides possibilities to identify well-developing embryos before transfer, thus improving pregnancy rates and the number of calves born.

135 EXPRESSION OF APOPTOSIS-RELATED GENES IN BUFFALO (BUBALUS BUBALIS) EMBRYOS PRODUCED THROUGH IN VITRO FERTILIZATION AND PARTHENOGENETIC ACTIVATION

2011 ◽  
Vol 23 (1) ◽  
pp. 172
Author(s):  
S. Saw ◽  
K. P. Singh ◽  
R. Kaushik ◽  
M. Muzaffar ◽  
M. S. Chauhan ◽  
...  
Keyword(s):  
Relative Abundance ◽  
Culture Medium ◽  
Mammalian Species ◽  
Control Cell ◽  
Bubalus Bubalis ◽  
Blastocyst Stage ◽  
Vitro Fertilization ◽  
Immature Oocytes ◽  
Tissue Size

Apoptosis, a highly conserved evolutionary mechanism that allows an organism to tightly control cell numbers, tissue size, and protect itself from dangerous cells and unfavourable environments that threaten homeostasis, is generally directed by specific genes involved in the regulation of a series of pro-apoptotic (BAX) and anti-apoptotic (BCL-XL) proteins that are expressed during early development. All mammalian species show the highest level of spontaneous apoptotic processes at the blastocyst stage. These proteins prevent apoptosis by maintaining the cell survival by interfering with the release of cytochrome-C from mitochondria. In this study, immature oocytes were obtained from buffalo slaughterhouse ovaries and were subjected to in vitro maturation (IVM) in TCM-199 + 10% FBS + 5 μg mL–1 porcine FSH for 24 h in a CO2 incubator (5% CO2, 90 to 95% relative humidity) at 38.5°C. The mature oocytes were used for IVF, and the cleaved embryos were cultured for 8 days in culture medium (CR2 medium containing 0.6% BSA and 10% FBS) for production of embryos at different stages. The parthenotes were produced with exposure of 7% ethanol, 6-dimethyl aminopurine and cultured for 8 days in culture medium. The total RNA was isolated from oocytes and embryos and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA), according to manufacturer protocol. The PCR cycle included heating to 94°C for 5 min, followed by 35 cycles of 94°C for 30 s, 60 (BAX) and 62°C (BCL) for 30 s, and 72°C for 45 s with a final extension at 72°C for 10 min. The amplified product of both genes were separated on agarose gel and densitometry data for band intensities were generated using AlphaDigiDocTM AD-1201 software under a WindowsTM environment and data analysed with the help of SYSTAT software. Relative abundance of BCL-XL transcripts in immature, mature oocytes and embryos produced through IVF (i.e. 2-cell, 4-cell, 8- to 16-cell, morula, and blastocyst stage) were 25.33 ± 0.90, 12.67 ± 1.20, 37.67 ± 0.90, 30.67 ± 0.30, 23.67 ± 0.90, 18.33 ± 0.90, and 27.00 ± 1.20, respectively, whereas in parthenogenesis these values were 23.67 ± 0.88, 13.67 ± 1.20, 23.67 ± 1.20, 22.34 ± 0.88, 24.34 ± 0.88, 33.67 ± 0.88, and 45.34 ± 1.20, respectively. Relative abundance of BAX transcripts by IVF were 23.0 ± 0.60, 0.33 ± 0.10, 4.00 ± 0.60, 5.00 ± 0.60, 0.37 ± 0.06, 13.0 ± 0.66, and 56.7 ± 0.90; and by parthenonenesis were 22.3 ± 0.90, 0.13 ± 0.03, 13.67 ± 0.90, 14.0 ± 0.60, 15.33 ± 0.90, 64.67 ± 2.20, and 55.0 ± 2.10, respectively. In conclusion, the expression pattern of the apoptosis-related genes revealed that the incidence of apoptosis was significantly higher in IVF and parthenogenetically produced buffalo embryos at stages such as immature oocytes, morula, and blastocyst than the early cleavage stage embryos.

154 COMPARISON OF DIFFERENT CULTURE MEDIA AND INCUBATION METHODS ON CULTURING MURINE EMBRYOS IN VITRO USING STRAW AS A RECEPTACLE

2017 ◽  
Vol 29 (1) ◽  
pp. 185
Author(s):  
L. R. Madzhie ◽  
M. A. Raseona ◽  
L. P. Nethenzheni ◽  
O. Ajao ◽  
M. L. Mphaphathi ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Culture Media ◽  
Uv Light ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Blastocyst Formation ◽  
Vitro Fertilization ◽  
Stage Of Development ◽  
Murine Embryos

In vitro fertilization in the straw system might increase the efficiency of fertilization and the quality of blastocyst formation as compared with micro-drops-IVF systems. The aim of the study was to in vitro fertilize mouse oocytes and culture the resulting zygotes in bi-gas incubator and in a goat vagina and compare the in vitro embryo developmental stages in TCM-199 and Ham’s F10 culture media until the blastocyst-stage of development. F1 generations (Balb C × C57) were used to harvest oocytes and spermatozoa. The fresh sperm were capacitated in different incubation methods (bi-gas incubator and in the vagina of a goat). A volume of 2–4 µL of Ham’s F10 containing capacitated sperm (~8 × 106 per mL) were placed into Ham’s F10 fertilization drops under the oil, containing 10 oocytes and penicillamine, hypotaurine, and epinephrine for enhancing sperm motility and penetration of oocytes. The same procedure was used with the TCM-199 medium and IVF drops without oil (both TCM-199 and Ham’s F10) for straw filling. The presumptive embryos in Ham’s F10 and TCM-199 were divided into different groups: first group were cultured in micro-drops, second group the embryos were aspirated in semen straws and placed in the incubator (incubator straws) for culture, and other straws were covered with a sponge and inserted in the vagina of a goat (vaginal straws) for culture. The resulted blastocysts were stained using Hoechst 33528 solution and blastomeres were counted on a fluorescent UV light inverted microscope at 400× magnification (Nikon Eclipse TI, Narishige Co., Ltd., Amityville, NY, USA). The results were analysed by 2 × 2 factorial designs and Student’s t-test was used to separate the mean. There was no statistical difference (P > 0.05) between the media and incubators on the stage of murine embryo development. The overall fertilization rate was 94 to 99%. The incubator straws with Ham’s F10 (80.5%) had the highest rate of embryos that reached the blastocyst stage, followed by incubator straws with TCM-199 (77.0%), and vaginal straws with Ham’s F10 (60.0%) had the lowest rate of embryos that reached the blastocyst stage. The overall mean number of blastomeres in the blastocyst stage of the embryos ranged from 85 ± 9 to 90 ± 9 cells in all receptacles and incubators. It was concluded that the fertilization and culturing of murine embryos are possible in straws incubated in a bi-gas incubator and in the goat vagina as an alternative method of fertilizing oocytes and culturing murine embryos. In addition, Ham’s F10 and TCM-199 can both be used to fertilize oocytes and culture murine embryos until blastocyst formation embryo in vitro, incubated in a bi-gas incubator or in the vagina.

Effect of protein supplementation on development to the hatching and hatched blastocyst stages of cat IVF embryos

2002 ◽  
Vol 14 (5) ◽  
pp. 291 ◽  
Author(s):  
N. W. Kurniani Karja ◽  
Takeshige Otoi ◽  
Masako Murakami ◽  
Minori Yuge ◽  
Mokhamad Fahrudin ◽  
...  
Keyword(s):  
Bovine Serum ◽  
Culture Medium ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Early Embryo ◽  
Protein Supplementation ◽  
Vitro Fertilization ◽  
Fetal Bovine ◽  
The Mean

The effects of protein supplementation in culture medium on development to the hatching and hatched blastocyst stages of cat in vitro-fertilized embryos were investigated. In the first experiment, presumptive zygotes derived from in vitro maturation and in vitro fertilization (IVF) were cultured in modified Earle's balanced salt solution (MK-1) supplemented with 0.4% bovine serum albumin (BSA) or 5% fetal bovine serum (FBS) for 9 days. There were no significant differences between the BSA and FBS groups with respect to the proportion of cleavage and development to the morula and blastocyst stages of zygotes. However, the presence of FBS in the medium enhanced development to the hatching blastocyst stage of zygotes compared with the BSA group (31.4% v. 7.8%). Moreover, 2.9% of zygotes cultured with FBS developed to the hatched blastocyst stage. The mean cell number of blastocysts derived from zygotes cultured with FBS was significantly higher (P&lt;0.01) than that from zygotes cultured with BSA (136.6 v.101.5). In the second experiment, embryos at the morula or blastocyst stage, which were produced by culturing in MK-1 supplemented with 0.4% BSA after IVF, were subsequently cultured in MK-1 with 0.4% BSA or 5% FBS. Significantly more morulae developed to the blastocyst (P&lt;0.05) and hatching blastocyst stages (P&lt;0.01) in the FBS group than in the BSA group (71.5% and 53.6% v. 44.9% and 6.0%, respectively). Although none of the morulae cultured with BSA developed to the hatched blastocyst stage, 11.5% of morulae cultured with FBS developed to the hatched blastocyst stage. Moreover, the proportion of development to the hatching blastocyst stage of blastocysts was significantly higher (P&lt;0.01) in the FBS group than in the BSA group (68.7% v. 9.8%). None of the blastocysts cultured with BSA developed to the hatched blastocyst stage, whereas 7.3% of blastocysts cultured with FBS developed to the hatched blastocyst stage. The results of the present study indicate that supplementation with FBS at different stages of early embryo development promotes development to the hatching and hatched blastocyst stages of cat IVF embryos.

scholarly journals Effect of conditioned medium of umbilical cord-derived mesenchymal stem cells as a culture medium for human granulosa cells: An experimental study

2022 ◽  
pp. 1037-1044
Author(s):  
Kanadi Sumapraja ◽  
Andon Hestiantoro ◽  
Isabella Kurnia Liem ◽  
Arief Boediono ◽  
Teuku Z Jacoeb
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Conditioned Medium ◽  
Umbilical Cord ◽  
Granulosa Cells ◽  
Culture Medium ◽  
Culture Media ◽  
Controlled Ovarian Hyperstimulation ◽  
Vitro Fertilization

Background: The umbilical cord-derived mesenchymal stem cells conditioned medium (UC-MSCs-CM) produces secretomes with anti-apoptotic properties, and has the potential to prevent apoptosis of granulosa cells (GC) during controlled ovarian hyperstimulation. Objective: To observe the effect of UC-MSCs-CM on the interaction between pro- and anti-apoptotic proteins and the influence of growth differentiation factor 9 (GDF9) production in GC. Materials and Methods: UC-MSCs-CM was collected from umbilical cord stem cell culture on passage 4. GC from 23 women who underwent in vitro fertilization were cultured and exposed to UC-MSCs-CM for 24 hr. Then RNA of the GC was extracted and the mRNA expression of BCL-2 associated X (BAX), survivin and GDF9 were analysed using quantitative real-time PCR. The spent culture media of the GC were collected for measurement of insulin growth factor 1 using ELISA. Results: The expression of BAX was significantly different after UC-MSCs-CM exposure (4.09E-7 vs. 3.74E-7, p = 0.02). No significant changes occurred in survivin, BAX/survivin ratio, and GDF9 expression after UC-MSCs-CM exposure (p > 0.05). The IGF-1 level of the CM was significantly higher after the CM was used as a culture medium for GC (2.28 vs. 3.07 ± 1.72, p ≤ 0.001). A significant positive correlation was found between survivin and GDF9 (r = 0.966, p ≤ 0.001). Conclusion: IGF-1 produced by UC-MSCs-CM can work in paracrine fashion through the IGF receptor, which can inhibit BAX and maintain GDF9 production. Moreover, under the influence of UC-MSCs-CM, GC are also capable of producing IGF-1, which can impact GC through autocrine processes. Key words: Conditioned medium, BAX, Survivin, GDF9, IGF-1.

216 IN VITRO OOCYTE MATURATION, FERTILIZATION, AND CULTURE AFTER LAPAROSCOPIC OVUM PICK-UP IN AN ENDANGERED GAZELLE (GAZELLA DAMA MHORR)

2006 ◽  
Vol 18 (2) ◽  
pp. 216 ◽  
Author(s):  
F. Berlinguer ◽  
S. Succu ◽  
A. del Olmo ◽  
R. Gonzalez ◽  
J. J. Garde ◽  
...  
Keyword(s):  
Sperm Concentration ◽  
Egg Yolk ◽  
Mature Female ◽  
Blastocyst Stage ◽  
Culture Conditions ◽  
Ministry Of Education ◽  
Hormonal Stimulation ◽  
Vitro Fertilization ◽  
Genetic Value

The recovery of immature oocytes followed by in vitro maturation, fertilization and culture (IVMFC) allows the rescue of biological material of great genetic value for the establishment of genetic resource banks. Studies have been carried out on endangered Mohor gazelle sperm cryopreservation (Garde et al. 2003 Biol. Reprod. 69, 602-611), but there are no studies on oocytes in this species. The purpose of this work was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC. The study was conducted using six reproductively mature female Mohor gazelles from the breeding herd at the Estacion Experimental de Zonas Aridas. Animals were synchronized by insertion of controlled progesterone internal drug release (CIDR) devices for 14 days and removal of the devices on the day of ovum pickup (OPU). Follicular growth was stimulated by a total of 5.28 mg of oFSH (Ovagen, ICP, Auckland, New Zealand) given in four equal doses every 12 h. OPUs were performed (Berlinguer et al. 2004 Theriogenology 61, 1477-1486) on Day 15 from the beginning of treatment, and follicles were aspirated with a syringe and a 25G needle using TCM199-HEPES with 50 �g/mL streptomycin, 50 IU/mL penicillin, 0.1% polyvinyl alcohol, and 15 IU/mL heparin. Degenerated oocytes and those with expanded cumulus were removed. Oocytes were cultured in TCM-199 plus 10% FCS, 10 �g/mL ovine FSH/LH, 1 �g/mL estradiol, and 0.1 mg/mL glutamine at 38.5�C under 5% CO2/air and maximum humidity. Spermatozoa were cryopreserved in Tes-Tris with 5% egg yolk and 6% glycerol, and selected by swim-up in SOF medium. After 24 h sperm-oocyte coincubation (sperm concentration: 1 � 106/mL) in SOF with 2% estrus sheep serum under 5% CO2 5% O2 90% N2, presumptive zygotes were transferred to SOF with 0.4% BSA and amino acids under 5% CO2, 5% O2 90% N2 and cultured for 4 days. Oocytes and embryos were stained with Hoechst 33342 and propidium iodide (1 �g/mL each) and visualized under a fluorescence microscope. A total of 35 oocytes were recovered from 56 punctured follicles (62.5%). This recovery rate was similar to those in wildlife in earlier reports, but more studies are needed to improve hormonal stimulation and oocyte harvesting. Out of 29 cumulus-oocyte complexes matured in vitro, 3.5% were found at GV and 6.9% at MI; 20.7% were degenerated and 68.9% had advanced to MII. Fertilization and cleavage rates were 40% and 30%, respectively, of matured oocytes. Out of eight zygotes, six showed cleavage (ranging from 2 to 8 cells). None of the developing embryos progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more trials will help to improve IVMFC, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by OPU from FSH-stimulated endangered gazelles. This work was supported by the Spanish Ministry of Education and Science (REN 2003-11587) and Acciones Integradas (HI20030336).

scholarly journals In vitro production of Sudanese camel (Camelus dromedarius) embryos from epididymal spermatozoa and follicular oocytes of slaughtered animals

2017 ◽  
Vol 20 (1) ◽  
pp. 95-101 ◽  
Author(s):  
A.E. Abdelkhalek ◽  
Sh.A. Gabr ◽  
W.A. Khalil ◽  
Sh.M. Shamiah ◽  
L. Pan ◽  
...  
Keyword(s):  
Culture Medium ◽  
Fertilization Rate ◽  
Blastocyst Stage ◽  
Dromedary Camel ◽  
In Vitro Production ◽  
Vitro Fertilization ◽  
Maturation Rate ◽  
Epididymal Spermatozoa ◽  
Vitro Production

Abstract Application of assisted reproductive technology in camelidea, such as artificial insemination (AI) and embryo transfer, has been slow in comparison to that for other livestock species. In Egypt, there are few attempts to establish in vitro maturation (IVM) and fertilization (IVF) techniques in dromedary camel. The present study was carried out to produce Sudanese camel embryos using in vitro matured oocytes and epididymal spermatozoa. Dromedary camel ovaries were collected from abattoirs and then, the oocytes were aspirated from all the visible follicles on the ovarian surface (~2-8 mm in a diameter). Meanwhile, Fetal Dromedary Camel Serum (FDCS) was obtained from camel fetuses after slaughtering. Thereafter, only Cumulus Oocyte Complexes (COCs) were matured in vitro in the Tissue Culture Medium (TCM-199) complemented with 10% FDCS. Spermatozoa required for in vitro fertilization were collected from testes (epididymal cauda) of the slaughtered camel bulls. The results clearly showed that the maturation rate of oocytes at metaphase II was about 59.5% while the fertilization rate was around 70.4%. Intriguingly, the embryo rates determined were 13.1%, in 2-cell; 0.0%, in 4-cell; 34.7%, in 8-16% cell; 39.1%, in morula and 13.1% in a blastocyst stage. This study represented a successful in vitro production of Sudanese dromedary camel embryos from epididymal sperm cells and in vitro matured oocytes recovered from slaughtered camels.

scholarly journals 248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

2005 ◽  
Vol 17 (2) ◽  
pp. 274
Author(s):  
A.S. Lima ◽  
C.E. Ferguson ◽  
M.B. Wheeler
Keyword(s):  
Embryo Development ◽  
Culture Medium ◽  
Culture Media ◽  
Cumulus Cells ◽  
Blastocyst Stage ◽  
The Other ◽  
Cell Stage ◽  
Development Rates ◽  
Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

297 BIRTH OF LIVE RATS THROUGH IN VITRO FERTILIZATION USING CRYOPRESERVED SPERMATOZOA: SPERM FERTILITY IMPROVED BY FREEZING AT - 150°C

2006 ◽  
Vol 18 (2) ◽  
pp. 256
Author(s):  
Y. Seita ◽  
Y. Okuda ◽  
A. Takizawa ◽  
S. Hisamatu ◽  
T. Inomata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
In Vitro Fertilization ◽  
Sperm Motility ◽  
Membrane Integrity ◽  
Egg Yolk ◽  
Chi Square ◽  
Vitro Fertilization ◽  
Plasma Membrane Integrity ◽  
Freezing Temperatures

We previously reported that damages to spermatozoa by cold shock can be avoided by cooling slowly at 0.5�C/min to 5.0�C (Seita et al. 2005 Reprod. Fertil. Dev. 17, 277-278). The objective of the present study was to develop an in vitro fertilization (IVF) system with frozen-thawed rat spermatozoa for more efficient reproduction of live offspring. We examined the effect of freezing temperatures (cooling 5.0�C to pre-plunging) on post-thaw sperm motility, plasma membrane integrity, and fertility in vitro. Epididymal spermatozoa of Wistar rats were collected in 2.0 mL of freezing medium containing 23% (v/v) egg yolk, 8.0% (w/v) lactose monohydrate, and 0.7% (v/v) Equex STM (Nova Animal Sales, Inc., Scituate, MA, USA) at room temperature. Samples were loaded into 0.25-mL straws and cooled to 5.0�C at 0.5�C/min in a programmable freezer. Next, the samples were exposed to liquid nitrogen (LN) vapor at various freezing temperatures (-120�C, -150�C or -180�C) above the LN level for 15 min and then plunged into LN. Straws were thawed in a 37�C water bath for 10 min. The thawed samples were diluted to 0.5-1.5 � 106 sperm/mL in a droplet of 200 �L of R1ECM and then pre-incubated for 5 h. Ovulated oocytes were introduced into the droplet and co-cultured for 10 h. The oocytes were denuded, fixed, and/or examined for two pronuclei (2PN) formation microscopically. The denuded oocytes, which were fertilized with spermatozoa frozen at -150�C and were microscopically confirmed to have 2PN formation, were transferred to pseudo-pregnant recipient females. IVF was also performed by the same method using fresh spermatozoa as the control. Differences in the sperm motility and plasma membrane integrity were analyzed by ANOVA, and the IVF data were analyzed by chi-square test. At 2 h after thawing the motility of spermatozoa frozen at -150�C was significantly higher than that of spermatozoa frozen at -180�C (19.8% and 11.1%; P < 0.05), although the sperm plasma membrane integrity was not significantly different among different freezing temperatures, -120�C, -150�C, and -180�C (18.2%, 23.5%, and 17.9%; P > 0.05). The percentage of oocytes with 2PN was not significantly different between the -150�C frozen and the control (fresh spermatozoa) groups [59% (131/221) and 62% (155/251); P > 0.05], although that of frozen spermatozoa at -120�C and -180�C [20% (38/188) and 23% (35/153)] were significantly lower than that of frozen spermatozoa at -150�C (P < 0.05). A total of 168 putative fertilized zygotes with 2PN were transferred to eleven recipients, and 87 live young were born. In conclusion, our results indicated that post-thaw motility of cryopreserved rat spermatozoa was improved by using a suitable cooling protocol, and the IVF system used in the present study would effectively produce offspring from the cryopreserved epididymal rat spermatozoa. To our knowledge, this procedure is the first successful production of live offspring from cryopreserved rat spermatozoa through in vitro fertilization.

122 EFFECTS OF EMBRYO SEX AND GLUCOSE OR FRUCTOSE IN CULTURE MEDIA ON BOVINE EMBRYO DEVELOPMENT

2008 ◽  
Vol 20 (1) ◽  
pp. 141
Author(s):  
M. Barceló-Fimbres ◽  
G. E. Seidel Jr
Keyword(s):  
Embryonic Development ◽  
Culture Medium ◽  
Culture Media ◽  
Defined Medium ◽  
Blastocyst Stage ◽  
Bovine Embryo ◽  
Energy Substrates ◽  
Sexed Semen ◽  
Embryo Sex

The objective of the present study was to determine if glucose, fructose, or both, in culture medium affects female embryos differently from male embryos. Embryos of predetermined sex were produced by use of sexed semen. Blastocysts were produced in vitro in a factorial design from 1851 oocytes fertilized with 3 sperm types [non-sexed control, X-bearing, and Y-bearing (~95% accuracy)]. Semen from 3 bulls (A, B, and C) was used, with 2 replicates for each bull. Zygotes were cultured with 4 energy substrates (no-hexose control, glucose, fructose, and glucose + fructose). Chemically defined medium (CDM) plus 0.5% fatty acid-free BSA (De La Torre-Sanchez et al. 2006 Mol. Reprod. Dev. 18, 585–596) was the culture medium to which the energy substrates were added. For the first 2.5 d of culture, the medium contained 0.5 mm hexose, and for the last 4.5 d, it contained 2.0 mm hexose. Data were analyzed by ANOVA. There was no effect of hexose treatments on cleavage rates (P > 0.1; Table 1). However, blastocyst rates per oocyte were higher for the fructose treatment than for the control, glucose, or glucose + fructose (P < 0.05) groups. Glucose treatment resulted in retarded development compared with no hexose or fructose (P < 0.05). Analysis of a subset of data with only the fructose and glucose treatments resulted in an interaction of hexose (glucose and fructose) and X-bearing andY-bearing sperm for blastocysts per oocyte: fructose X-sperm 20.4%a; fructoseY-sperm 17.7%ab; glucose X-sperm 10.6%bc, and glucoseY-sperm 14.7%c (means without common superscripts differ; P < 0.05). Glucose was detrimental to female embryos, and fructose improved development to the blastocyst stage for both sexes. Lower cleavage rates were found after fertilization with X-bearing sperm (Table 1); however, blastocyst rates per oocyte were not different after fertilization with X- or Y-bearing sperm or non-sexed sperm (P > 0.1). There was no bull effect on rates of cleavage or blastocysts per oocyte (P > 0.1). In conclusion, fructose improved embryonic development of embryos of both sexes; glucose retarded development of, and was toxic to, female embryos. Sexed sperm resulted in similar cleavage and blastocyst production as non-sexed sperm, and female embryos had retarded embryonic development relative to male embryos. Table 1. Main effect least-squares means for development of bovine embryos (±SE)

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