IVM), Fertilization Research Articles

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)

Generation of live piglets from cryopreserved oocytes for the first time using a defined system for in vitro embryo production.

We report the successful piglet production from cryopreserved oocytes for the first time by using a simple, high capacity vitrification protocol for preservation and a defined system for in vitro embryo production. Immature cumulus-oocyte complexes (COCs) from prepubertal gilts were vitrified in microdrops and stored in liquid nitrogen. After warming, COCs were subjected to in vitro maturation (IVM), fertilization (IVF), and subsequent culture (IVC). Adjusting warmplate temperature to 42°C during warming prevented temperature drops in a medium below 34.0°C and significantly increased the percentage of oocyte survival and thus blastocyst yields obtained from total vitrified oocytes compared with that of warming at 38°C (87.1% vs 66.9% and 4.4% vs 2.7%, respectively). Nuclear maturation and fertilization of oocytes were not affected by vitrification and warming temperature. Blastocyst development on day 7 (day 0 = IVF) of the surviving oocytes after warming at 38°C and 42°C was not different but lower (P<0.05) than those of non-vitrified control oocytes (4.6%, 5.2% and 17.9%, respectively). However, blastocyst cell numbers in the control and vitrified groups were similar irrespective of warming temperature. Omitting porcine follicular fluid (pFF) from IVM medium (POM) did not affect maturation, fertilization and embryo development of vitrified-warmed oocytes. Transfer of blastocysts obtained on day 5 from vitrified oocytes matured either with or without pFF into 4 recipients (2 for each group) resulted in 4 pregnancies and the delivery of a total of 18 piglets. In conclusion, optimization of warming temperature was a key factor for achieving high survival rates, and surviving oocytes could be utilized in vitro using defined media. Using these modifications, live piglets could be obtained from cryopreserved oocytes for the first time.

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.

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.

Developmental potential of bovine oocytes cultured in different maturation and culture conditions

Diverse groups of chemicals in culture media are needed for successful bovine oocyte maturation and embryo development during which dramatic cytoplasmic and nuclear reprogramming events take place. In vitro embryo production (IVP) procedures frequently include supplements such as serum and/or co-culture with various types of somatic cells. However, the presence of undefined serum in culture media introduces a variation from batch to batch, increases viral or prion contamination risk, and leads to problems during fetal development. The aim of the present study was to investigate the possibility of using chemically defined-synthetic serum substitute (SSS) in place of fetal calf serum (FCS) during maturation and long-term culture to stimulate in vitro maturation (IVM), fertilization (IVF) and subsequent embryo development. In Experiment I, the effect of the protein source on in vitro maturation was tested by maturing oocytes in culture media supplemented with 10% FCS (Control Group), 10% SSS (Group I) and 10% SSS + 10 ng/ml epidermal growth factor (EGF) (Group II). In Experiment II, effects of SSS on both oocyte maturation and embryo development during in vitro culture (IVC) were tested by maturing oocytes in media supplemented with 10% FCS (FCS Group) or 10% SSS + 10 ng/ml EGF (SSS Group), followed by IVF and IVC in SOF media supplemented with 10% FCS and 10% SSS on day 4 for FCS and SSS Groups, respectively. Even though rates for cleavage and development to blastocyst stage were not different, blastocyst cell numbers were higher in Group II containing SSS and EGF. The SSS supplementation group had higher apoptotic nuclei as compared to the FCS Group in Experiment II. Transcripts for heat shock protein 70 (Hsp70), interferon tau (IF-τ), DNA methyltransferase 3a (Dnmt3a), desmosomal glycoprotein desmocollin III (DcIII) and insulin-like growth factor II receptor (Igf-2r) were altered in different culture conditions in Experiment I. However, only glucose transporter-1 (Glut-1) mRNA was different in the SSS and FCS Groups in the second experiment. In summary, SSS and EGF in maturation medium and replacement of FCS with SSS alone in culture medium on day 4 of IVC support oocyte maturation and embryo development in vitro. However, significance of culture condition induced changes on the genome-wide abundance of messenger ribonucleic acid and the significance of the apoptotic nuclei during fetal development still remain to be determined.

355 DEVELOPMENTAL COMPETENCE OF IMMATURE BUBALINE CUMULUS - OOCYTE COMPLEXES VITRIFIED BY THE OPEN PULLED STRAW AND CONVENTIONAL STRAW METHODS

The in vitro maturation (IVM), fertilization (IVF), and morphological changes in buffalo cumulus–oocyte complexes (COCs) cryopreserved by ultrarapid freezing using conventional (CON) and open pulled staw (OPS) methods were tested. COCs were cryopreserved using a vitrification solution comprised of Dulbecco&amp;apos;s phosphate-buffered saline+0.5 M sucrose+0.4% BSA and two concentrations (4.5 or 5.5 M) of each cryoprotectant ethylene glycol (EG) and dimethylsulfoxide (DMSO) by either the CON or the OPS method. Vitrified COCs were stored in LN for 7 days and then thawed; morphologically normal COCs were used for IVM (n = 1070) and IVF (n = 933) in 2 separate experiments to record morphological damage of COCs due to vitrification, nuclear maturation 24 h after culture (9 replicates), and fertilization 24 h after insemination (10 replicates). The COCs were matured in vitro in TCM-199 media with hormone supplements and fertilized using TALP-BSA as described previously (Purohit et al. 2005 Anim. Reprod. Sci. 87, 229–239). Freshly collected COCs were separately used for IVM (n = 110) and IVF (n = 130) and kept as controls. The arcsin transformed data of the proportions of oocytes matured or fertilized was compared by Duncan&amp;apos;s new multiple range test. The highest proportion of morphologically normal oocytes was seen in 5.5 M EG with the CON method (94.5%) and the lowest was seen in 4.5 M DMSO with the OPS method (82.4%). At the end of experiment 1, it was apparent that IVM in all vitrification groups was significantly lower (P &amp;lt; 0.05) compared to the control group (66.4%). Among the various vitrification treatments, the highest IVM occurred in 5.5 M EG with the OPS method (39.2%) and the lowest in 4.5 DMSO with the CON method (19.3%). Comparison of both concentrations of EG and DMSO showed that the proportion of COCs attaining Metaphase-II (M-II) increased with increasing concentration of both of the cryoprotectants. However, at equal concentration of EG and DMSO, the proportion of COCs attaining M-II was significantly higher in the OPS method compared to the CON method. In experiment 2, a significantly higher (P &amp;lt; 0.05) IVF was seen for fresh COCs (45.4%) compared to vitrified COCs. Among the vitrification treatments, the highest fertilization was seen in 5.5 M EG with the OPS method (33.6%) and the lowest in 4.5 M DMSO with the CON method (15.17%). A dose-dependent increase in the proportion of oocytes fertilized was seen with increasing concentration of both EG and DMSO [CON: 4.5 M (15.2%), 5.5 M (25.6%); OPS: 4.5 M (21.3%), 5.5 M (27.5%)] in both CON and OPS methods. Comparison of the 2 cryoprotectants revealed that EG was better compared to DMSO.At equal concentrations of EG or DMSO, a significantly higher (P &amp;lt; 0.05) proportion of fertilized oocytes was seen in the OPS method compared to the CON method. It was concluded that vitrification results in some damage to oocytes, with decrease in their subsequent IVM and IVF. Developmental capacity of vitrified buffalo oocytes can be improved by using OPS instead of conventional straws.

Successful use of oviduct epithelial cell coculture for in vitro production of viable red deer ( Cervus elaphus) embryos

Techniques for in vitro production (IVP) of viable embryos have been thoroughly developed in several domestic species in view to improve breeding efficiency. When applied to wild life, these techniques may also help the maintenance of biodiversity through amplification of sparse animals offspring and facilitation of genetic material exchange. During the successive steps of IVP, i.e. oocyte in vitro maturation (IVM), fertilization (IVF) and early embryo development (IVD) to the blastocyst stage, gametes and embryos are faced with unusual environment, including oxidative stress, known to be detrimental to their survival. In the present study, starting from methods developed in domestic species, we have adapted IVP to produce viable red deer embryos. In a first experiment, cumulus cells were removed from in vitro matured oocytes either before or after IVF. The presence of cumulus cells during IVF did not affect final cleavage or development rates. In a second experiment, in vitro matured oocytes were fertilized in the presence of cumulus cells and cultured in SOFaaBSA medium alone or in the presence of ovine oviduct epithelial cell (oOEC) monolayer. Whereas, oviduct cells did not improve the cleavage rate, they significantly increased the rate of embryos reaching the blastocyst stage (from 3 to 25% of total oocytes). Ten blastocysts from oOEC coculture were transferred after freezing and thawing to five recipient hinds and gave rise to three pregnancies. The three pregnant hinds gave birth to three live and normal calves.

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.

A novel approach for in vitro production of bovine embryos: use of the oxoid atmosphere generating system

The importance of the incubator type is often overlooked when protocols for in vitro production of embryos are evaluated. In this study the ability of a standard CO 2 Heraeus incubator and the Oxoid CO 2Gen TM atmosphere-generating system to support bovine in vitro oocyte maturation, fertilization and embryo development is described for the first time. The Oxoid CO 2Gen TM gas generating system, originally designed for the growth of bacteria, is based on the chemical reaction of ascorbic acid and air. When the sachet with ascorbic acid is placed in the confined volume of the airtight AnaeroJar TM, an atmosphere of 6% CO 2 in 15% O 2 is created, which is comparable to the 5% CO 2 and 20% O 2 used for standard in vitro production of bovine embryos. In the first set of experiments oocyte in vitro maturation (IVM), fertilization (IVF) and embryo culture (IVC) were allocated to one or the other of the culture systems. In the second set of experiments IVM and IVF took place in the Heraeus incubator, while IVC was allocated either to the Heraeus or to the AnaeroJar TM. During experiments the AnaeroJar TM was placed in the Heraeus incubator to ensure identical incubation temperatures of 38.8°C. A standard protocol was used for production of embryos: 23 h of IVM in TCM-199, 20 h of IVF with frozen-thawed washed spermatozoa in TALP medium and 7 days of IVC (8 days after insemination) in B2 medium with bovine oviduct epithelial cells. In the first set of experiments, based on a total of 766 inseminated oocytes, the Day 8 blastocyst rates were the same in the Heraeus incubator and the AnaeroJar TM: 30% vs. 30% with oviduct cell coculture, and 21% vs. 18% without coculture. In the second set of experiments, based on 1963 inseminated oocytes, the average blastocyst rates were 27% vs. 32% from the Heraeus incubator and the AnaeroJar TM. In 2 of 6 replicates blastocyst rates were lower in the Heraeus incubator than in the jar; in the remaining replicates they were alike. No differences were noted in blastocyst kinetics or morphology. In conclusion, the Oxoid gas generating system seems to be a cheap, convenient and stable alternative to expensive CO 2 incubators, not only for the growth of bacteria, but also for in vitro production of bovine embryos.

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.

Production, freezing and transfer of bovine IVF embryos and subsequent calving results

Ultrasound-guided oocyte aspirations were performed repeatedly, on a weekly basis, on 155 different cows. An average of 4.9 oocytes with 4.1 classified as usable were collected. Following in vitro maturation (IVM), fertilization (IVF) and culture (IVC), a few Day 7 morulae, and all Day 7 and most Day 8 blastocysts were either transferred or frozen. The transfer of 2268 fresh IVF embryos resulted in 1220 pregnancies (53 8%). Day 7 embryos resulted in a higher (56%) pregnancy rate than did Day 8 (43%) or Day 9 (41%). In addition, the transfer of embryos classified as grade 1 on either Day 7 or 8 resulted in a higher pregnancy rate than did grade 2 embryos. Although there was no difference in pregnancy rate, embryos resulting from co-culture with Buffalo Rat Liver Cells n Menezo's B2 medium developed faster than embryos co-cultured with TCM 199 culture medium. Pregnancy rate following transfer of IVF blastocysts frozen on Day 7 was 42% and dropped to 20% for blastocysts frozen on Day 8. Pregnancy rate was not influenced when synchrony between the IVF embryo and recipient was within ± 36 h. The sex ratio of the resulting calves did not deviate from normal but there was a higher than normal incidence of early abortions and dystocias. There appeared to be some increase in calf birth weights.

Effect of follicle size on bovine oocyte quality and developmental competence following maturation, fertilization, and culture in vitro.

The aim of the present series of experiments was to investigate the effect of the size of follicle from which the oocytes originate on their subsequent in vitro developmental ability. Ovarian follicles were isolated and grouped according to size (2-6 mm, > 6 mm). Primary oocytes were carefully liberated and grouped according to morphology into one of five categories: denuded; expanded; with two or three layers of cumulus; with four or five layers; and with many (six or more) layers. Following in vitro maturation (IVM), fertilization (IVF), and culture (IVC), more oocytes with many layers of cumulus (P < 0.01, 70.2%, 73/104 vs. 46.8%, 87/186, respectively) and a higher proportion of blastocysts were obtained from follicles > 6 mm compared to 2-6 mm follicles (P < 0.01, 65.9%, 60/91 from > 6 mm follicles vs. 34.3%, 34/99 from 2-6 mm follicles, respectively). Use of follicular fluid (BFF) from follicles of different sizes in the IVM medium did not significantly increase the cleavage rate or blastocyst yield compared to controls. Administration of porcine follicle-stimulating hormone (pFSH) to donors prior to slaughter was investigated as a possible means of increasing the number of larger sized follicles in the ovaries and, thereby, the quality of the recovered oocytes. It was found that administration of six injections of pFSH beginning 3 days prior to slaughter resulted in a significant increase (P < 0.001) in the proportion of follicles > 6 mm in diameter (31.6%) compared to that in nontreated controls (6.6%) and to animals that received only four injection groups (9.4%).(ABSTRACT TRUNCATED AT 250 WORDS)

Production of chimeric calves by aggregation of in vitro-fertilized bovine embryos without zonae pellucidae

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.

Development of in vitro fertilized oocytes from pregnant and nonpregnant cows in oviductal epithelial and cumulus cell co-culture systems

The objectives of this study were 1) to measure cleavage, blastocyst formation, and blastocyst hatching after in vitro maturation (IVM), fertilization (IVF) and culture (IVC) of oocytes aspirated from pregnant versus nonpregnant cows, and 2) to compare embryo development in co-culture with bovine oviductal epithelial cells versus cumulus cells. No differences in cleavage (38 versus 40%), blastocyst formation (13 versus 13%), or blastocyst hatching (53 versus 51%) were observed for in vitro-matured, fertilized, and cultured oocytes from pregnant versus nonpregnant cows, respectively (P > 0.05), indicating that nonpregnant and early-pregnant cows are equally acceptable donors of oocytes for IVM/IVF/IVC procedures. Cleavage (36 versus 40%), blastocyst formation (11 versus 12%), and blastocyst hatching (50 versus 55%) were not different for embryos co-cultured with oviductal epithelial cells versus cumulus cells (P > 0.05). Thus, equivalent embryo development can be obtained with co-culture systems commonly used for in vitro-derived bovine embryos. These results help to define variables that affect comparison of results across laboratories and that are relevant to the practical application of IVM/IVF/IVC procedures to cattle.