IVF) And Culture Research Articles

Lipid availability during in vitro maturation alters oocyte lipid content and blastocyst development and metabolism.

Lipids play a crucial role in various biological functions, including membrane composition, energy storage, cell signalling, and metabolic and epigenetic processes. Abnormal lipid accumulation and metabolism during in vitro maturation (IVM) of oocytes have been linked to the use of fetal bovine serum (FBS), even though it provides several beneficial molecules, contributing to the oocyte competence. Delipidating agents have been used to mitigate these deleterious effects, but they can have adverse effects on embryonic development. In this study, we explored how lipids present in fetal bovine serum (FBS) can impact the composition of oocytes and their resulting blastocysts in vitro. For that, we used organic solvents to separate the polar and nonpolar (lipid enriched) phase of FBS. Oocytes were in vitro matured in the presence of 10% whole FBS (control), 10% FBS plus 10% nonpolar lipids (lipid enriched - OL) or 10% polar lipids only (partially delipidated - ODL). After 24 h, part of the matured oocytes was collected and those remaining in each group underwent in vitro fertilization (IVF) and culture (IVC) under the same conditions and expanded blastocysts were collected at day 7 (control, BL and BDL). Oocytes and embryos were analysed by Multiple Reaction Monitoring mass spectrometry (MRM-MS) to determine their lipid composition. Interestingly, principal component analysis (PCA) revealed a clear distinction in the lipid profile of oocytes and blastocysts from both treatments compared to the control group. Control oocytes and blastocysts had higher triacylglycerol and cholesterol ester enrichment while the OL, ODL, BL and BDL groups had higher amounts of free fatty acids (FFAs). The structural and signalling phospholipids also differed among groups. Our findings suggest that the lipid-enriched fraction of FBS can be manipulated for IVM to ensure proper maturation, resulting in oocytes and blastocysts with less accumulated intracellular lipids and an improved metabolic status.

Effect of Culture Medium on in vitro Fertilization in Local Iraqi Ewes.

in vitro fertilization (IVF) is considered to be the most important reproductive biotechnological method having great potential to accelerate genetic improvement in ruminants as well as for research on embryonic development. The present study aimed to investigate the effect of culture medium and the addition of natural and synthetic antioxidants on in vitro maturation (IVM), fertilization (IVF), and culture (IVC) in local Iraqi ewes. A total of 304 reproductive systems of local ewes were collected from a slaughterhouse in Fallujah, Anbar Province, Iraq from 3, January to 1, July 2021. The study was conducted in the Reproductive Biotechnology Laboratory, Department of Surgery and Theriogenology, College of Veterinary Medicine, University of Fallujah, Iraq. A total of 1368 oocytes were recovered from 608 ovaries surrounded by cumulus cells. The method of collection was aspiration and oocytes were divided into eight treatments. The first (T1), the second (T2), the third (T3), the fourth (T4), the fifth (T5), the sixth (T6), the seventh (T7), and the eighth (T8) treatments were MEM + Capparis spinosa extract 50µmol, MEM + Silymarin extract 100µmol, MEM + Coenzyme Q10 5 µmol, MEM only serves as a control, DMEM + Capparis spinosa extract 50 µmol, DMEM + Silymarin extract 100µmol, DMEM + Coenzyme Q10 5µmol and DMEM only serves as a control, respectively. The results indicated a significant difference (P≤0.05) between T5 (DMEM + Capparis spinosa extract) and other controls or treatments. Cultural medium DMEM with Capparis spinosa extract (as an antioxidant) presents the best results in the morula and blastocyst stage.

79 Effect of Polydatin on Development and Cryotolerance of In Vitro-Produced Bovine Embryos

In vitro-produced (IVP) cattle embryos have high reactive oxygen species levels resulting in poor development and cryotolerance. Polydatin, a naturally occurring antioxidant, improves embryonic metabolism when added to maturation media; however, it has not been evaluated at other stages of embryo production. We hypothesised that embryos cultured with polydatin during maturation and fertilization would have increased development and cryotolerance. Therefore, IVP embryos were produced in 8 treatment groups supplemented with 1 µM polydatin during in vitro maturation, fertilization, and culture, or a combination of the different production stages, and each assigned a letter (Table 1). Embryos were produced in 7 replicates by oocytes (n = 3320) aspirated from abattoir ovaries, matured for 23 h in TCM-199 plus 10% fetal bovine serum and gonadotropins, fertilized with semen from 1 of 3 bulls, and cultured in SCF1 (SOF for Conventional Freezing 1; Owen et al. 2017 Reprod. Fertil. Dev. 29, 129-130) in 38.5°C in 5% O2, 5% CO2, and 90% N2. Stage 7 blastocysts were stained with 1 µg mL−1 Nile Red for lipid content or 300 nM Mitotracker Red CMX-Rosamine (Thermo Fisher Scientific, Waltham, MA, USA) for mitochondrial activity. Ten images per embryo were acquired using confocal microscopy at 5 µM step size at 40× magnification, and fluorescence was measured by Image Pro software (Media Cybernetics, Rockville, MD, USA). Remaining blastocysts were slow frozen following a 20-min equilibration in conventional freezing medium (1.5 M ethylene glycol and 0.5 M sucrose in holding medium) with 1 mm l-ascorbic acid. Embryos were thawed and assessed for re-expansion at 48 h. Blastocyst rate, Nile Red, Mitotracker, and re-expansion data were analysed by one-way ANOVA and means separated by least significant difference. Results indicate that treatment B had a higher blastocyst rate than treatment H (P < 0.01), lower lipid content than all other treatment groups (P < 0.01 or 0.05), and higher level of mitochondrial polarity than treatments A, D, E, and G (P < 0.01 or 0.05), suggesting enhanced metabolic activity. Additionally, this treatment enhanced cryotolerance compared with treatment H (P < 0.01). These results suggest that adding polydatin to maturation media has the most effect on embryo developmental competence and cryotolerance. Table 1.Effect of polydatin addition during in vitro maturation (IVM), fertilization (IVF), and culture (IVC) on blastocyst rate, lipid content, Mitotracker, and cryotolerance (± SEM)

Effect of Physiologically Relevant Heat Shock on Development, Apoptosis and Expression of Some Genes in Buffalo (Bubalus bubalis) Embryos Produced In Vitro

For investigating the effects of physiologically relevant heat shock, buffalo oocytes/embryos were cultured at 38.5°C (control) or were exposed to 39.5°C (Group II) or 40.5°C (Group III) for 2h once every day throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC). Percentage of oocytes that developed to 8-cell, 16-cell or blastocyst stage was lower (p<0.05) and the number of apoptotic nuclei was higher (p<0.05) for Group III>Group II>controls. At both 8-16-cell and blastocyst stages, relative mRNA abundance of stress-related genes HSP 70.1 and HSP 70.2 and pro-apoptotic genes CASPASE-3, BID and BAX was higher (p<0.05) in Groups III and II than that in controls with the exception of stress-related gene HSF1. Expression level of anti-apoptotic genes BCL-XL and MCL-1 was also higher (p<0.05) in Groups III and II than that in controls at both 8-16-cell and blastocyst stages. Among the genes related to embryonic development, at 8-16-cell stage, the expression level of GDF9 was higher (p<0.05) in Group III than that in controls, whereas that of GLUT1, ZAR1 and BMP15 was not significantly different among the three groups. At the blastocyst stage, relative mRNA abundance of GLUT1 and GDF9 was higher (p<0.05) in Group II than that in controls, whereas that of ZAR-1 and BMP15 was not affected. The results of this study demonstrate that exposure of buffalo oocytes and embryos to elevated temperatures for duration of time that is physiologically relevant severely compromises their developmental competence, increases apoptosis and affects stress-, apoptosis- and development-related genes.

Comparative Study on Goat Oocyte Recovery Methods and Factors Affecting the Quantity and Quality of Oocytes

A strategize step in oocyte recovery is important and beneficial in producing successful production of high-quality Cumulus Oocyte-Complexes (COC) for in vitro maturation, fertilization and culture (IVMFC), nuclear transfer and other emerging technologies. Experiments were conducted to compare Oocyte Recovery (OR) between Laparoscopic Ovum Pick-Up (LOPU) and ovarian slicing and to evaluate the effects of OR cycle, hormone stimulation, oestrus synchronization, breeds, liveweight and age of goats on the quantity and quality of oocytes obtained. In Experiment 1, oocytes were recovered from abattoir-derived ovaries by the slicing method. The ovaries were transported to the laboratory for immediate harvesting. The slicing method yielded significantly (p<0.05) larger number of oocytes per ovary than LOPU (22.53±2.78 vs. 6.10±0.46). However, the proportion of Grade A oocytes was highest from the LOPU technique (30.51±4.14) compared to the slicing method (19.07±3.57). LOPU resulted in good quality Cumulus Oocytes Complexes (COC) with more than 5 layers of cumulus cells. Experiment 2 consisted of three OR cycles (OR-1, OR-2 or OR-3); four durations of oestrus synchronization using CIDR (Day-10, -14, -17 or -21); three goat breeds (Boer Crossbred, Mixed breed or Katjang); 4 age groups (Young, Mature, Old or Very Old) and 4 levels of body weight (20, 21-29, 30-37 or 38 kg). Goats were synchronized using Control Internal Drug Release (CIDR) combined with 125 jig Estrumate and hyperstimulated with 70 mg FSH and 400 IU hCG after the removal of CIDR 24 h later. There was no significant effect of OR cycle, CIDR removal goat breed, age or body weight on the total number of oocytes recovered per ovary. Katjang goats yielded a higher proportion of Grade A oocytes compared to Boer Crossbreds and mixed breeds which had a higher proportion of Grade C oocytes (p<0.05). Age and weight of animals had no significant effect on the quality of oocytes recovered. Grade A and B oocytes were obtained in OR-1, whilst the repetition of LOPU resulted in an increment of Grade C oocytes in OR-2 and OR-3, respectively. Day-10 and -21 gave the highest cumulative percentage of 58 and 64% for Grade A and B oocytes, respectively. Under the conditions of the experiment, LOPU yielded better quality oocytes from the Katj ang goats using less or no OR repetition, with oestrus synchronization at 10 or 21-Day. © 2013 Academic Journals Inc.

Purification of cattle oviduct specific proteins and their effect on in vitro embryo development

The purpose of this study was to determine the effect of oviduct specific proteins as a media supplement for in vitro embryo development in cattle. The proteins were extracted from oviducts of cows and precipitated by ammonium sulfate (30%, 40%, 50% and 60%) followed by dialysis in 50mM Tris–HCl (pH 7.0) buffer. The dialyzed proteins were fractionated into acidic, basic and neutral fractions using SP sephadex cation exchange and DEAE sephadex anion exchange column chromatography respectively. Cow oviduct specific proteins (cOSPs) constituting all the extracted proteins were used as media supplement in three different concentrations (10, 50 and 100μg/ml) for in vitro maturation, fertilization and culture (IVMFC) of cow oocytes. Acidic, basic and neutral (unbound) fractions were also used as media supplement in three different concentrations (10, 30 and 50μg/ml) for IVMFC. Cumulus oocytes complexes were collected from slaughterhouse ovaries, washed thoroughly and cultured in maturation media for 24h in 5% CO2 at 38.5°C with maximum humidity. In vitro matured oocytes were co-incubated with in vitro capacitated sperm in Fert-BO media at 38.5°C for 18h in 5% CO2. The fertilized oocytes were washed and cultured in embryo development media for cleavage. After 40–42h cleavage was observed and embryos were put in the replacement media for further development. The cleavage rates (%) for cOSPs were observed as 68.24±2.46, 69.28±2.05, 61.77±0.93 and 42.62±1.31 at concentrations of 0, 10, 50 and 100μg/ml respectively. Rates of blastocyst stage development were 14.49±3.61, 21.17±2.77, 14.66±1.06 and 11.98±1.84. These results indicate that addition of cOSP at10μg/ml increased blastocyst formation as compared to other concentrations (0, 50 and 100μg/ml). Although acidic, basic and neutral fractions seemed to have no major effect on cleavage rate, but both acidic and neutral fraction of oviduct specific proteins improved the cleavage rate at 30μg/ml concentration and basic fraction improved the blastocyst formation at 10μg/ml concentration.

Oxygen Concentration and Cysteamine Supplementation During In vitro Production of Buffalo (Bubalus bubalis) Embryos Affect mRNA Expression of BCL‐2, BCL‐XL, MCL‐1, BAX and BID

This study examined the effects of O(2) concentration (5% vs 20%) during in vitro maturation (IVM), fertilization (IVF) and culture (IVC) or supplementation of IVM and IVC media with cysteamine (50 and 100 μm, respectively; IVM, IVF and IVC carried out in 20% O(2)), on blastocyst rate and relative mRNA abundance of some apoptosis-related genes measured by real-time qPCR in immature and in vitro-matured buffalo oocytes and in embryos at 2-, 4-, 8- to 16-cell, morula and blastocyst stages. The blastocyst rate was significantly higher (p < 0.05) while the percentage of TUNEL-positive cells was significantly lower (p < 0.05) under 5% O(2) than that under 20% O(2). The mRNA expression of anti-apoptotic genes BCL-2 and MCL-1 was significantly higher (p < 0.05) and that of pro-apoptotic genes BAX and BID was lower (p < 0.05) under 5% O(2) than that under 20% O(2) concentration at many embryonic stages. Following cysteamine supplementation, the blastocyst rate and the relative mRNA abundance of BCL-XL and MCL-1 was significantly higher (p < 0.05) and that of BAX but not BID was lower (p < 0.05) at many stages of embryonic development, although it did not affect the percentage of TUNEL positive cells in the blastocysts significantly. The mRNA expression pattern of these genes during embryonic development was different in 5% vs 20% O(2) groups and in cysteamine supplemented vs controls. At the 8- to 16-cell stage, where developmental block occurs in buffalo, the relative mRNA abundance of BCL-2 and MCL-1 was highest under 5% O(2) concentration and that of BAX and BID was highest (p < 0.05) under 20% O(2) concentration. These results suggest that one of the mechanisms through which beneficial effects of low O(2) concentration and cysteamine supplementation are mediated during in vitro embryo production is through an increase in the expression of anti-apoptotic and a decrease in the expression of pro-apoptotic genes.

In Vitro Embryo Production in Buffalo: Basic Concepts

In vitro embryo production involves a combination of the techniques of in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes. The in vitro matured oocytes are incubated with suitably processed spermatozoa for an appropriate period of time for carrying out IVF. The presumptive zygotes are then cultured in vitro up to the blastocyst stage at which these could either be transferred to synchronized recipients for producing live offspring or cryopreserved for future use. The IVM, IVF and IVC are also integral parts of a number of other reproductive technologies like embryo or somatic cell cloning, production of transgenic animals or production of embryonic stem cells etc., which are expected to bring about a quantum jump in the population of superior genetic merit buffaloes or be useful to mankind in other ways.

The effects of ovalbumin as a protein source during the in vitro production of bovine embryos

Embryo quality is influenced by the culture conditions that affect in vitro maturation (IVM), fertilization (IVF) and culture (IVC) rates. The present study investigated the feasibility of producing bovine embryos after the replacement of fetal calf serum (FCS) and bovine serum albumin (BSA) by ovalbumin (OVA). The IVM and IVC medium were supplemented with 10% FCS, 4 mg/mL BSA, or 4 mg/mL OVA. The IVF medium was supplemented with 6 mg/mL BSA or OVA. For IVM, supplementation with FCS, BSA, and OVA did not affect nuclear maturation or cortical granule migration. Higher rates of formation of two pronuclei were obtained when FCS was employed for IVM (79.97%), regardless of the supplement used for IVF, and when BSA was used for IVF (59.4%), regardless of the supplement used for IVM. Supplementation with OVA for IVM+IVC (20.40%) and for IVF (22.15%) was inferior to supplementation with FCS for IVM+IVC (30.47%) and with BSA for IVF (28.91%) for blastocyst development. Hatching rates were lower using OVA for IVM+IVC (23.02%) and for IVF (28.93%) compared with FCS and BSA under the same conditions (40.78 and 34.82%, respectively) and BSA for IVF (36.82%). Supplementation with OVA for IVM+IVC and IVF resulted in reduced inner cell mass, trophectoderm cells and total blastocyst cell numbers (17.29, 37.88, and 55.17, respectively). In conclusion, OVA is a protein source for bovine in vitro embryo production, although the quantity and quality of bovine blastocysts using only ovalbumin in the entire in vitro production process are lower than those obtained in the presence of FCS and BSA, when used as supplements in any step of bovine in vitro embryo production.

Embryonic Development and Gene Expression in Oocytes Cultured In Vitro in Supplemented Pre-Maturation and Maturation Media

This study aimed at assessing the effect of the addition of brain-derived neurotrophic factor (10 ng/ml BDNF) and/or cysteamine (100 μm CYS) during pre-maturation and BDNF, CYS or leptin (10 ng/ml LEP) during maturation culture in vitro on embryo development and oocyte gene expression in cattle. Oocytes were obtained by the aspiration of 2- to 8-mm follicles from slaughtered cows. In Experiment 1, oocytes were pre-matured for 24 h with 10 μm butyrolactone I in the presence or not of BDNF and/or CYS followed by in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 2, oocytes were submitted to IVM with BDNF, CYS or LEP or no supplements followed by IVF and IVC. In Experiment 3, oocytes were pre-matured with BDNF and CYS followed by IVM or only in vitro matured with BDNF. Samples for quantitative PCR (qPCR) were collected after pre-maturation (BGV) and after IVM of pre-matured oocytes (BMII) or immediately after follicle aspiration (immature control = GV) and IVM (matured control = MII). Embryo production was not affected by the inclusion of the different factors either during pre-maturation or maturation culture (∼ 43% blastocysts, p>0.05). Transcripts analysis showed that most genes (NLRP5, ZAR1, GPX1, KEAP1, SPHK2, HSP70 and PSMP1) were downregulated (p<0.05) after IVM irrespective of being previously pre-matured. The relative abundance of BAX, BCL2, IGFBP3 and ARFRP1 transcripts was unaffected by pre-maturation or maturation (p>0.05). In conclusion, supplementation of in vitro pre-maturation (BDNF and/or CYS) or maturation media (BDNF, CYS or LEP) did not improve embryo development. Gene expression was not affected by pre-maturation treatment, but some genes were downregulated after maturation, probably related to selective and differential degradation.

Defined System for In Vitro Production of Porcine Embryos Using a Single Basic Medium

We have previously indicated that porcine blastocysts can be produced by in vitro fertilization (IVF) and culture (IVC) in chemically defined porcine gamete medium (PGM) and porcine zygote medium (PZM)-5, respectively, In the present study, the effects of basic media and macromolecular components on in vitro maturation (IVM) were investigated to develop a defined system for in vitro embryo production using a single basic medium through IVM, IVF and IVC. Porcine immature oocytes were matured in porcine oocyte medium (POM) or modified North Carolina State University (mNCSU) 37, which were supplemented with either 10% (v/v) porcine follicular fluid (pFF) or 3 mg/ml polyvinyl alcohol (PVA) as a macromolecular component (designated POM+pFF, POM+PVA, mNCSU37+pFF and mNCSU37+PVA). In the maturation with mNCSU37+PVA, the percentages of oocytes that reached the metaphase II stages were significantly lower than those in the other treatments. Following IVM with the above media, oocytes were treated with an electrical stimulus and cycloheximide for parthenogenetic activation and were cultured in PZM-5 for 5 days. The rates of cleavage and blastocyst formation of parthenogenetic oocytes were significantly lowered for maturation with mNCSU37+PVA compared with the other treatments, while there were no significant differences in the total numbers of cells in blastocysts among the treatments. Following IVF and IVC, the rates of penetration, male pronucleus formation, cleavage and blastocyst formation were significantly lower when oocytes were matured in mNCSU37+PVA than in other maturation media. The normal fertilization rate was significantly higher in POM+PVA compared with the other treatments, although the total number of cells in blastocysts was reduced with the addition of PVA to both POM and mNCSU37 compared with pFF supplementation. These results demonstrate that porcine blastocysts can be produced by the defined system using a single basic medium.

In vitro oocyte maturation, fertilization and culture after ovum pick-up in an endangered gazelle (Gazella dama mhorr)

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 of endangered species. Studies exist on sperm cryopreservation of endangered Mohor gazelle (Gazella dama mhorr), but no work has been carried out yet on oocyte collection, fertilization and culture in this or related species. The purpose of this study was to develop a protocol for ovarian stimulation for the recovery of oocytes and subsequent IVMFC in the Mohor gazelle using frozen-thawed spermatozoa. Ovum pick-up was performed after ovarian stimulation with a total dose of 5.28 mg of ovine FSH. A total of 35 oocytes were recovered from 56 punctured follicles (62%) (N=6 females). Out of 29 cumulus-oocyte complexes matured in vitro, 3% were found at germinal vesicle stage, 7% at metaphase I, 21% were degenerated, and 69% advanced to metaphase II. Fertilization and cleavage rates of matured oocytes were 40 and 30%, respectively. Embryos cleaved in vitro up to the 6-8 cell stage but none progressed to the blastocyst stage, suggesting the existence of a developmental block and the need to improve culture conditions. Although more studies are needed to improve hormonal stimulation and oocyte harvesting, as well as IVMFC conditions, this study demonstrates for the first time the feasibility of in vitro fertilization with frozen-thawed semen of in vitro matured oocytes collected by ovum pick-up from FSH-stimulated endangered gazelles.

A comparison of BoviPure ® and Percoll ® on bull sperm separation protocols for IVF

In the present study, we have examined the effect of density gradient preparations BoviPure ® and Percoll ® on bull sperm separation and the in vitro fertilization (IVF) and culture (IVC) results. Frozen/thawed semen from five simmental bulls were pooled. Sperm quality parameters such as sperm motility, concentration, membrane activity (HOS assay), membrane integrity (SYBR-14/PI assay) and acrosomal status (EthD-1/FITC-PSA assay) were evaluated before and after sperm processing for IVF using BoviPure ® and Percoll ® density gradient separations. The results of the evaluated parameters before sperm processing were: motility 50%, concentration 82.33 × 10 6 spz/mL, membrane activity 39.05%, membrane integrity 42.97% and the acrosomal status 46.90% of the live spermatozoa with intact acrosomes. After sperm processing with BoviPure ® and Percoll ® the motility was 66.67 and 64.17%, the concentration was 25.50 × 10 6 and 27.67 × 10 6 spz/mL, the membrane activity was 53.78 and 56.58%, the membrane integrity was 70.85 and 68.76% of and the acrosomal status was 74.16 and 67.46% of the live spermatozoa with intact acrosomes, respectively. Percentages were referred to the total number of spermatozoa. There were significant differences ( P < 0.05) between the evaluated parameters before and after sperm processing for both separation protocols. We found no significant differences ( P > 0.05) regarding sperm evaluation parameters between the protocols. A total of 492 oocytes were matured and fertilized in vitro and cultured in SOFaaBSA in six replicates. The cleavage (D2) and blastocysts (D7) rate were significantly higher ( P < 0.05) for the BoviPure ® group compared to the Percoll ® group: 75.80 and 28.21%; 61.58 and 20.83%, respectively. However, the number of hatched blastocysts (D10) did not differ significantly between sperm separation protocols ( P > 0.05). Our results indicate that both protocols gave suitable sperm for IVF. This finding and the similarity between those two density gradient preparations suggests that BoviPure ® is a good alternative for sperm separation in bovine IVF.

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

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).

292. Influence of the in vitro environment on rat gametes and pre-implantation embryos

Rat in vitro fertilization (IVF) and culture (IVC) is attempted by few because of its reputation for difficulty. Currently very few functional rat in vitro systems (IVS) exist for sperm–oocyte interaction research. Successful fertilization of rat metaphase II (MII) oocytes was achieved with two different media, Enriched Krebs Ringer Bicarbonate (EKRB) (70.2%) and M16 (57.4%). Using this IVS we have shown that the rat germinal vesicle-intact (GV-i) oocyte lacks the necessary maturity to interact with capacitated caudal epididymal spermatozoa, whether zona pellucida intact (ZP-i) or free (ZP-f). Proteomic analysis of the protein profile of the oolemma from the GV-i stage through to the MII stage in oocytes is being conducted to characterize any maturational changes that may occur. In addition we provide initial evidence to suggest that an acrosome-intact spermatozoa can fuse with the oolemma of a ZP-f MII oocyte during IVF. Although high percentages of polyspermic embryos in ZP-f IVF (64.8–100%) were observed, the possibility that the rat oolemma may undergo a post-fusion block to polyspermy was implied by a small proportion of normally fertilized embryos (3.8–17.0%) in M16 supplemented with different ratios of hyperactived spermatozoa. Despite successful culture to the blastocyst stage for in vivo fertilized zygotes (33.73%) and 2-cell stage embryos (79.3%), IVF embryos have repeatedly failed to develop in culture. Two dimensional analyses of the protein profile of oocytes/embryos immediately prior to fertilization (MII oocyte–101 spots) and the maternal to zygotic transition (MZT) (zygotes–59 spots and 2-cell embryos–84 spots) has shown a difference in patterns of protein expressed. Comparison of IVF zygotes (41 spots) obtained from EKRB displayed reduced protein expression suggesting that nuclear maturation and/or MZT is not being adequately supported. These data illustrate that rat IVF and IVC require suitable media if its problematic reputation is finally to be shed.

Improvement of the culture conditions for in vitro production of cattle embryos in a portable CO2 incubator.

The effects of different concentrations of growth hormone (GH) on in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocyte/embryos in CR1aa or CR2aa media using a simple CO2 incubator were investigated. The IVM/IVF/IVC of oocytes were carried out in the presence of 0, 50, 100 and 200 ng/ml GH in the medium. The proportion of metaphase II oocytes was significantly higher (p < 0.05) in 200 ng/ml compared with 0 ng/ml GH in CR1aa medium (59 versus 85%, respectively), but this effect was not observed under CR2aa. Higher concentrations of GH yielded lower rates of unfertilized ova and thus superior cleavage rates (36.5 +/- 0.2 and 63.5 +/- 2.0% versus 17.5 +/- 0.2 and 82.5 +/- 1.5% or 40.4 +/- 0.6 and 59.6 +/- 1.4% versus 16.6 +/- 1.2 and 83.4 +/- 6.2% for 0 and 200 ng/ml GH in portable or ordinary incubator, respectively) in CR1aa. This dose-dependent effect was also observed in the percentages of transferable embryos, although not statistically different (17.2 +/- 1.7 versus 27.3 +/- 1.8% and 16.6 +/- 3.1 versus 26.0 +/- 1.4%, for 0 versus 200 ng/ml GH in portable and ordinary incubator, respectively). In contrast to the CR1aa, different concentrations of GH in CR2aa medium did not increase either fertilization or cleavage rates. In fact, higher concentrations of GH in this medium negatively affected the rate of transferable embryos. Hence, percentages of transferable embryos obtained in the portable incubator under 0 or 50 ng/ml GH were higher (p < 0.05) compared with those obtained in 100 or 200 ng/ml GH (35.4 +/- 5.7 or 40.5 +/- 5.4% versus 22.4 +/- 2.4 or 15.5 +/- 2.1%, respectively). There was however, no significant difference in the rate of transferable embryos in an ordinary incubator employing CR2aa medium, but the trend was more or less similar to that observed in the portable incubator. Despite the fact that relatively fewer oocytes were employed for the culture in the ordinary incubator, overall results observed employing the simple portable CO2 incubator were within the range of those obtained in an ordinary incubator: implying that the simple portable incubator can effectively be employed for the in vitro production of bovine embryos under field conditions.

Fertilizability and developmental capacity of individually cultured bovine oocytes

Culture of single oocytes throughout in vitro maturation (IVM), fertilization (IVF) and culture (IVC) provides detailed information on maturity, fertilizability and developmental capacity of individual bovine oocytes and embryos. In the present study, effects of sperm concentration (Experiment 1), microdrop size (Experiment 2), and the addition of hypotaurine (HT) or glutathione (GSH; Experiment 3) during IVF were investigated. In Experiment 4, in vitro maturity and developmental capacity of bovine oocytes cultured for IVM in a medium supplemented with fetal calf serum (FCS), bovine serum albumin (BSA) or polyvinyl alcohol (PVA) during IVM were investigated. In Experiments 1 to 3, the percentages of normal (2 pronuclei with a spermtail) and polyspermic fertilization in singly cultured oocytes were similar to those of group IVF culture (5 oocytes/drop). The addition of GSH during single oocyte IVF significantly increased the proportion of normal fertilization and decreased the polyspermic fertilization compared with addition of HT or of the control. The rates of mature oocytes (62.4 and 67.7%) and blastocyst development (12.9 and 15.2%) for single oocyte IVM cultures (Experiment 4) were also similar compared with the group culture; PVA supplementation significantly increased the matured oocyte rate, but decreased blastocyst development significantly (7.1%) as compared with FCS (19.5%) or BSA (15.6%). These results indicate that a single oocyte culture system throughout in vitro production of bovine embryos provides similar maturity, fertilizability and developmental capacity to oocytes cultured in groups.

Development of a simple, portable carbon dioxide incubator for in vitro production of bovine embryos.

The objective of this study was to develop a simple and portable CO2 incubator using effervescent granules (EG) and to examine the effect of negative and positive air pressure for in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of bovine oocytes. In experiment 1, cumulus-oocyte complexes (COCs) were matured (22 h), fertilized (5 h) and cultured (7 days) using 0.25, 0.5 or 1.0 g of EG per 0.6 l added to maintain an optimum level of CO2 (approximately 3, 6 or 12%, respectively) for in vitro production of embryos. Control oocytes, zygotes and embryos were cultured in a standard CO2 incubator. The blastocyst production rates observed on Days 7 to 9 after insemination were 20.5+/-4.2%, 18.5+/-3.9% and 28.7+/-5.1% for the 0.25 g EG, 0.5 g EG treatments and control, respectively. These rates were significantly higher (P < 0.05) than that of the 1.0 g EG treatment (8.7+/-2.6%). The number of cells in the inner cell mass (ICM) and trophectoderm (TE) produced from blastocysts using the control procedure were 40.8+/-2.9 and 81.2+/-5.3, respectively, and were higher (P < 0.05) compared to the 0.50 g EG (34.6+/-2.9 and 66.8+/-5.7) and 1.0 g EG treatments (33.4+/-3.4 and 67.2+/-7.3). In experiment 2, COCs were placed in a small box with 0.25 g of EG so that the effects on IVM, IVF and IVC of positive or negative air pressure could be compared. The blastocyst production rate observed in the negative air pressure treatment (29.6+/-4.6%) was higher (P < 0.01) than that of the positive air pressure treatment (6.2+/-1.5%) or the normal treatment pressure (P < 0.05; 18.7+/-4.2%) but did not differ from that of the control (30.7+/-4.4%). These results indicate that this simple type of incubator with negative air pressure can be successfully used for in vitro production of bovine embryos and could be used at the field level.

Effects of resazurin on bovine oocyte fertilization and embryo development in vitro.

Resazurin is a redox dye (7-hydroxy-3H-phenoxazin-3-one-10-oxide) used for assessing potential fertility of spermatozoa and functional status of eukaryotic cells. In this study, the fertilizing capacity of spermatozoa treated with resazurin and effects of resazurin on bovine embryo development in vitro was examined. Abattoir-derived bovine oocytes were collected and subjected to in vitro maturation (IVM), fertilization (IVF) and culture (IVC). In Experiment 1, bovine oocytes (n = 2767) were fertilized with spermatozoa exposed to resazurin (17.6 micrograms/ml) for 0, 15, 30, 60 min, respectively. There was no significant (P > 0.05) difference in cleavage rates. However, the proportion of embryos that developed into blastocysts, expanded and hatched blastocysts in those groups in which oocytes/embryos were treated with resazurin during IVC or IVM/IVF/IVC was significantly (P < 0.05) less than those exposed to resazurin during IVM only, or during IVF only. We conclude that resazurin did not have significant adverse effects on fertilizing capability of bovine spermatozoa; however, extended treatment of embryos with resazurin may be detrimental to embryonic development.

Laboratory production of buffalo (Bubalus bubalis) embryos.

There is an increasing interest in large-scale in vitro production (IVP) of buffalo embryos through in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes for faster multiplication of superior germplasm. The recovery of total and usable quality oocytes from slaughterhouse ovaries is low in this species. The nuclear maturation rates of buffalo oocytes matured in the presence of follicular fluid or serum and hormones like luteinizing hormone, follicle-stimulating hormone and oestradiol vary from 70 to 80% and are comparable to those reported for cattle oocytes. However, with fertilization rates of 40-55%, and the yield of blastocysts at around 10-15%, the efficiency of IVP is much lower than that in cattle. The in vitro sperm preparation procedures and the systems employed for performing IVF and culture of zygotes up to blastocyst stage are suboptimal and need substantial improvements. The quality and viability of blastocysts produced need to be checked by cell count, and after transfer to synchronized recipients, for development of quality control standards.