Bovine In Vitro Production Research Articles

In vitro development of IVF-derived bovine embryos following cytoplasmic microinjection for the episomal expression of the IGF2 gene.

Important genomic imprinting changes usually occur following the in vitro production (IVP) of bovine embryos, especially in the imprinting pattern of components of the IGF system. This study aimed to evaluate the effects of a transient episomal overexpression of the IGF2 gene in bovine IVP embryos following embryo cytoplasmic microinjection (CMI) at the 1-cell stage on embryo survival, early and late developmental kinetics and morphological quality up to Day 7 of development. Selected cumulus-oocyte complexes (COCs) were matured and fertilized in vitro and subsequently segregated into six experimental groups: non-CMI control group and five CMI groups at increasing doses (0, 10, 20, 40 and 80ng/μl) of a GFP vector built for the episomal expression of bovine IGF2. Zygote CMI was effective in delivering the expression vector into the ooplasm, irrespective of the groups, with 58% of positive GFP fluorescence in Day7 blastocysts. Considering developmental rates and late embryo kinetics, the 10-ng/μl CMI vector dose promoted a lower blastocyst rate (10.4%), but for blastocysts at more advanced stages of development (93.0% blastocysts and expanded blastocysts), and higher number of cells (116.0±3.0) than non-CMI controls (23.3%, 75.0% and 75.0±6.8 were obtained, respectively). In conclusion, CMI at the 1-cell stage did not compromise subsequent in vitro development of surviving embryos, with the 10-ng/μl group demonstrating a possible growth-promoting effect of the IGF2 gene on embryo development, from the 1-cell to the blastocyst stage.

58 Effect of different light sources on the developmental capacity of bovine embryos produced invitro

Under natural conditions, mammalian oocytes and zygotes are never exposed to daylight or artificial light. During assisted reproductive procedures such as invitro production (IVP), oocytes and pre-implantation embryos are exposed to light from time to time before being transferred to recipients. The detrimental effect of visible light is not directly related to the intensity and exposure time, but is also a function of the spectral composition of the light. Recently, a green pass light filter was used during all steps of bovine IVP. Results indicated the protective effect of the filter against harmful blue and infrared regions of the light (Korhonen et al. 2009 Hum Reprod. 24, 308-314; https://doi.org/10.1093/humrep/den432). Data regarding either blue or infrared light are missing. Therefore, the effect of environmental light will be examined on the developmental capacity of bovine oocytes and pre-implantation embryos produced invitro. Three different experimental conditions were set during all IVP steps: (i) light from the microscope filtered with a red pass filter, no artificial light, and no daylight, (ii) artificial light without day light, light from the microscope without a filter, and (iii) artificial and daylight, light from the microscope without a filter, as normally used in the laboratory (control). Bovine cumulus-oocyte complexes were recovered from abattoir-derived ovaries by the slicing method. After 24h of maturation, fertilisation was realised and after 19h of co-culture of cumulus-oocyte complexes and sperm, presumptive zygotes were cultured in SOFaa for 8 days. Cleavage and developmental rates were recorded at Day 3 and Day 7/8 (Day 0=IVF, 10 IVP runs). Blastocysts from all groups were individually stored at −80°C until analyses. Then RT-qPCR (at least 12 replicates) was performed as described previously (Stinshoff et al. 2014 Reprod. Fertil. Dev. 26, 502-510; https://doi.org/10.1071/RD12372) for gene transcripts related to cellular stress (BAX, BCL2L1, HSPA1A, SOD1) and embryo developmental capacity (SLC2A3). The morphological quality of expanded blastocyst was assessed via a differential staining procedure (with propidium iodide and Hoechst, 4 replicates) to determine the number of inner cell masses (ICM), trophectoderms (TE), and total cells and to calculate the ICM/TE ratio. Data were analysed by analysis of variance followed by a Tukey test. Cleavage and developmental rates were similar in all groups of embryos (P>0.05). The relative abundance of all analysed gene transcripts was significantly increased (P ≤ 0.05) in blastocysts stemming from settings (i) and (ii) compared with those from the control group. Blastocysts generated using only artificial light or artificial and daylight did show a significantly increased number of ICM and total cells (86.3±3.2, 56.3±1.4; 183.6±4.0, 161.8±2.8) compared with embryos produced under filtered microscope light without any other light (35.3±0.8; 133.8±1.4; P ≤ 0.05). The number of TE cells was similar (P>0.05) in blastocysts of all groups (i: 98.5±1.3; ii: 97.3±2.6; iii (control): 105.5±2.4). These data suggest that day light exposure might influence bovine embryos at the molecular level, whereas the morphological quality seems to be unaffected when compared with embryos exposed to only artificial light.

Gene expression analysis and in vitro production procedures for bovine preimplantation embryos: Past highlights, present concepts and future prospects.

Over the past decades in vitro production (IVP) of bovine embryos has been significantly improved and in particular bovine IVP is now widely applied under field conditions. This in vitro technique provides new opportunities for cattle producers, particularly in the dairy industry, to overcome infertility and to increase dissemination of animals with high genetic merit. Improvements in OPU/IVP resulted in large-scale international commercialization. More than half a million IVP embryos are generated on the yearly basis demonstrating the enormous potential of this technology. These advances and the fact that bovine and human early development is remarkably similar have also prompted the use of bovine embryos as a model system to study early mammalian embryogenesis including humans. Despite all the improvements, embryos generated in vitro still differ from their in vivo derived counterparts. Embryos must adjust to multiple microenvironments at preimplantation stages. Consequently, maintaining or mimicking the in vivo situation in vitro will aid to improve the quality and developmental competence of the resulting embryo. The successful clinical application of the techniques in reproductive biotechnology requires both species-specific clinical skills and extensive laboratory experience. The recent advances in transcriptome profiling have also provided deeper insight into RNA expression and regulation at an unprecedented resolution. The development of these high-throughput DNA sequencing methods has resulted in new approaches for both mapping and quantifying transcriptomes. The aim of this review is therefore to summarize the available data related to gene expression analyses as well as in vitro embryo production procedures and to provide ideas about future concepts.

Improvements to In Vitro Culture Media for Use in Bovine IVF

In assisted reproduction of cattle the design and preparation of in vitro culture media has been instrumental in supporting the development of bovine oocytes and embryos. In vitro production (IVP) involves three main sequential steps: oocyte aspiration and in vitro maturation (IVM); in vitro fertilization (IVF); and in vitro culture (IVC) of early pre-implantation embryos. Research on IVC media has aimed to emulate the crucial elements that are present in the oviduct and uterus of female cattle by supplementing with protein and growth factors. As a result, IVP has proven a reliable methodology with an acceptable, if far from remarkable, blastocyst transfer rate of typically 40%. Moreover, the addition to media of defined extracts has gradually replaced that of fetal calf serum, leading to improved embryo viability and cryotolerance. Nevertheless, major advances in the composition of novel media that may facilitate a further increase of blastocyst rate have yet to be realized. Currently, there is considerable variation in the constituents of IVC media used in bovine assisted reproduction. Hence, significant innovations are required in order to not only achieve a greater success rate of IVP and to enhance the cryotolerance of bovine IVF-derived embryos but also to provide a defined IVC medium that may be recognized as a point of reference across the cattle breeding industry. This review examines the progress made to date in IVP to enhance embryo quality and points to future areas for investigation of a ‘gold standard’ IVC medium. This is a fully defined medium that supports consistently the attainment of a bovine IVP blastocyst rate similar to that achieved using a conventional medium supplemented with serum.

272 A NEW APPROACH TO IN VITRO MATURATION (IVM) AND EMBRYO IN VITRO PRODUCTION: INDUCED IVM SUBSTANTIALLY IMPROVES EMBRYO YIELD AND PREGNANCY OUTCOMES

Oocyte in vitro maturation (IVM) is the rate-limiting step in the in vitro production (IVP) of embryos. Oocyte maturation in vivo is a highly orchestrated, induced process, whereby cAMP-mediated meiotic arrest is overridden by the gonadotrophin surge prior to ovulation. However, aspirated oocytes resume maturation spontaneously compromising developmental competence. Hence, we hypothesized that establishing an induced system in vitro would synchronize oocyte-somatic cell communication leading to improved oocyte quality. Abattoir-collected bovine or 129/Sv mouse oocytes were treated for the first 1 to 2 h in vitro (pre-IVM) with the adenylate cyclase activator forskolin (100 μM, 50 μM, respectively) and a nonspecific phosphodiesterase (PDE) inhibitor, IBMX (500 μM, 50 μM), which substantially increased cumulus-oocyte complex (COC) cAMP (bovine, 180 v. 2 fmol/COC, treated v. control; P < 0.001). To maintain oocyte cAMP levels and prevent precocious oocyte maturation, IVM media (VitroMat + BSA) contained an oocyte-specific (type 3) PDE inhibitor, cilostamide (20 μM, 0.1 μM), plus FSH to induce maturation. The net effect of this system (induced-IVM) was to increase oocyte-cumulus cell gap-junctional communication (bovine: 1000 ± 148 v. 340 ± 73 unit, treated v. control; P < 0.05) and to slow meiotic progression through prophase I to metaphase II, extending the normal IVM interval (bovine: 30 v. 24 h, mouse: 22 v. 18 h; treated v. control). FSH was required to complete maturation and FSH-induced maturation was prevented by an epidermal growth factor receptor inhibitor, AG1478 (2.5 μM), demonstrating induced oocyte maturation functions via secondary autocrine signaling within the cumulus cell compartment. These effects on COC functions had profound consequences for oocyte developmental potential. In completely serum-free bovine IVP, induced-IVM more than doubled blastocyst yield (69 v. 27%, treated v. control; P < 0.05) and improved blastocyst quality (186 v. 132 blastomeres). To achieve these rates, the pre-IVM phase, the modified IVM conditions, and delayed IVF were all required. Adapting the system to the mouse, induced-IVM increased blastocyst rate (86 v. 55%, treated v. control; P < 0.05), implantation rate (51 v. 25%; P < 0.01), fetal survival rate (29 v. 5%; P < 0.01) and fetal weight (0.9 v. 0.5 g; P < 0.01). All these embryonic and fetal outcomes in mice were equivalent (P > 0.05) using induced-IVM to levels obtained from in vivo-matured control oocytes (conventional IVF). Data were analyzed by ANOVA. In conclusion, induced-IVM mimics some of the characteristics of oocyte maturation in vivo and substantially improves oocyte developmental outcomes in 2 disparate mammalian species. Adaption of this new approach to clinical/field conditions should lead to new opportunities for a wide range of reproductive biotechnologies. Such a notable increase in IVM efficiency could see IVP as the preferred embryo production technology in future livestock artificial breeding programs. Funded by an Australian Research Council Linkage Grant and Cook Australia. Thanks to M. Sasseville, M. Lane, and D. T. Armstrong.

74 EFFECT OF BETA-MERCAPTOETHANOL ON THE VITRIFICATION CRYOTOLERANCE OF BOVINE IN VITRO-PRODUCED EMBRYOS

The control of oxidative processes in in vitro production (IVP) systems by the use of additives may be an alternative approach to improve embryo cryotolerance. The aim of this study was to verify the effect of β-mercaptoethanol (βME) on the cryotolerance of bovine IVP embryos. In 7 replications, and following IVM-IVF, presumptive zygotes (n = 3735) were in vitro-cultured in SOF medium supplemented or not with 100 μm βME (IVC treatment), at 38.5°C and high humidity. The initial 24 h of IVC was performed in 5% CO2 in air, with the remaining 6 days of IVC carried out in 5% CO2, 5% O2, and 90% N2. On Day 7, resulting blastocysts and expanded blastocysts were vitrified in glass micropipettes in a solution with 20% ethylene glycol + 20% propylene glycol. After warming, embryos were randomly allocated to 1 of 2 sub-groups for an additional 72 h of IVC to the hatching blastocyst (HBL) stage, in fresh SOF medium supplemented or not with 100 μm βME (PVC treatment), at 38.5°C, high humidity and 5% CO2. Experimental groups were as follows: G1 (βME-free medium during IVC and PVC); G2 (βME only during PVC); G3 (βME only during IVC); and G4 (βME during IVC and PVC). Cleavage (Day 2) and blastocyst (Day 7) rates in the IVC treatment and hatching rates (Days 7 to 9) for the PVC treatment were analyzed by the chi-square test, for P < 0.05. Total cell number (TCN) estimated by fluorescence staining in HBL derived from vitrified and nonvitrified embryos was analyzed by ANOVA. The use of βME during IVC did not affect cleavage rates (βME-free, 1491/1858, 80.2% v. βME, 1522/1877, 81.1%), but negatively affected development to the blastocyst stage (βME-free, 813/1858, 43.8% v. βME, 525/1877, 28.0%). Following vitrification, however, βME supplementation during PVC improved hatching rates (G2, 58.1% and G4, 63.8%) compared with groups without the additive (G1, 36.6% and G3, 42.0%). In addition, the presence of βME either during IVC or PVC, or during both culture periods, increased TCN in HBL from vitrified embryos (Table 1). The use of βME during IVC, irrespective of the presence of βME during the PCV period, caused an increase in TCN in HBL in G3 + G4, with no effects on hatching rates (Table 1b), whereas the addition of βME during PVC, irrespective of the presence of βME during the IVC period, resulted in greater hatching rates and TCN in HBL in G2 + G4 than in G1 + G3 (Table 1). In conclusion, the addition of βME during the IVC period did not affect cleavage, but reduced blastocyst yield. Despite that, βME supplementation during the IVC period appeared to have increased the cryotolerance of the resulting blastocysts, expressed by greater TCN in HBL, whereas βME supplementation during the PVC period also improved embryo survival to the vitrification process, manifested by greater hatching rates and TCN in HBL. Table 1.Effect of βME on the cryotolerance of bovine IVP embryos This study was supported by a grant from CNPq/Brazil.

139 OXIDATIVE STRESS INDUCED CHANGES OF GLUTATHIONE CONCENTRATION IN IN VITRO-PRODUCED BOVINE BLASTOCYSTS

Glutathione (GSH) plays a critical role in the elimination of reactive oxygen species (ROS) by acting as substrate for GSH peroxidase (GPX) which results in the oxidation of GSH. Glutathione reductase (GSR) regenerates GSH from oxidized glutathione (GSSG). In bovine oocytes, GSH concentration increases and is thought to have beneficial effects on embryo development. The goal of the present study was to analyze the GSH content in bovine blastocysts during blastocoel expansion and to determine the effects of tertiary-butyl hydroperoxide (tBH) in causing oxidative stress. It is metabolized by GPX with conversion of GSH to GSSG. Blastocysts were generated with standard protocols of in vitro production (IVP) using SOF medium supplemented with FAF-BSA and were collected at Days 7/8 of culture (Day 0 = IVF). Diameters of blastocysts were measured and embryos were allocated to 4 groups: early blastocysts (ebla, less than 135 �m), blastocysts (bla, 135–180 �m), expanded blastocysts (exbla, greater than 180 �m), and hatched blastocysts (hbla). Pools of the different categories (3 replicates, 36 embryos in total) were frozen at -80�C until assayed for GSH by HPLC. Total cell numbers were determined using the Hoechst 33342 stain. Blastocysts from the 4 groups were cultured together for 30 min in tBH (20 �M) containing SOF medium to induce oxidative stress. This was followed by another culture period for 1 or 8 h. Samples of blastocysts were frozen at -80�C at 0 h, 30 min, 1 h, and 8 h to assess GSH levels (Cereser et al. 2001 J. Chromatogr. B. Biomed. Sci. Appl. 752, 123–132) and transcript abundance. Semi-quantitative real-time RT-PCR was used to determine the relative abundance (RA) of GPX and GSR transcripts. Differences between groups were tested using ANOVA followed by a Tukey or Student's test (P ≤ 0.05). The grade of expansion significantly increased GSH concentration in bovine blastocysts. Expanded blastocysts (0.38 ± 0.04 ng) and hatched blastocysts (0.46 ± 0.05 ng) had a higher GSH content than early blastocysts (0.21 ± 0.01 ng) and blastocysts (0.27 ± 0.02 ng). However, GSH concentration remained constant in blastomeres during expansion and hatching as the number of cells increased from 54.9 ± 3.5 (ebla) to 152.3 ± 4.7 (hbla). After induction of oxidative stress, blastocysts showed a significant decrease in GSH content after a 1-h recovery period, followed by an increase in their GSH content after a period of 8 h (0 h: 0.26 ± 0.01 ng; 30 min: 0.25 ± 0.01 ng; 1 h: 0.20 ± 0.01 ng; 8 h: 0.32 ± 0.01 ng, respectively). This was accompanied by a significant increase of the RA of GSR transcripts in blastocysts collected after the 8-h recovery period compared to embryos at the other time points of the treatment. GPX mRNA abundance remained stable during the entire culture period. In the present study, GSH concentration was determined in bovine embryos during blastocoel expansion for the first time. The increased GSH content might to some extent be due to de novo GSH synthesis. GSSG is reduced to GSH most likely by GSR. Results show that bovine IVP embryos may serve as a new model for the study of oxidative stress.

5 ASSESSMENT OF HSP70.1 TRANSCRIPTION LEVELS IN BOVINE EMBRYOS AFTER CUMULUS REMOVAL BY DIFFERENT TECHNIQUES

Previous results indicated that there was a difference in transcriptional activity depending on the method used to remove the cumulus cells after IVF (Zeringue et al. 2005 LabChip 5, 86-90). However, specific gene expression was not examined and therefore was the goal of the present study. The objective of this study was to compare the transcription levels of the HSP70.1 gene in bovine in vitro production (IVP) embryos that underwent cumulus removal by vortexing, hand stripping, or microfluidic treatment. Quantitative real-time PCR was used to estimate transcription levels of the HSP70.1 gene in bovine IVP zygotes and two-cell embryos. Transcription levels were compared between embryos that underwent three methods of cumulus removal. Presumptive zygotes were harvested at 2 or 24 h after cumulus removal by vortexing, by hand stripping, or by microfluidic means in order to compare the relative embryonic stress of these three treatments. Bovine in vitro embryos were produced by standard means with the only variable being the cumulus removal technique 24 h after fertilization. At 2 and 24 h post-cumulus removal, randomly selected presumptive zygotes were taken out of culture, preserved in RNAlater (Ambion, Inc., Austin, TX, USA) and stored at -20�C. RNA was extracted from single embryos via Qiagen's RNeasy Micro kit (Valencia, CA, USA). RNA was amplified and PCR products were detected with SYBR Green 1 (Applied Biosystems, Foster City, CA, USA) using an Opticon Monitor 3 real-time PCR machine (Bio-Rad Laboratories, Inc., Waltham, MA, USA). The threshold cycle (CT) numbers were determined for the amplified cDNA of the bovine HSP70.1 mRNA and for the housekeeping gene, acidic ribosomal phosphoprotein (PO), used as a reference. Then the amount of HSP70.1 was divided by the amount of PO to obtain a normalized HSP70.1 value expressed as the ratio of HSP70.1/PO. Ratios were analyzed by the GENMOD procedure in SAS (SAS Institute, Inc., Cary, NC, USA) accounting for replicates and treatments. Transcription levels of the HSP70.1 gene did not differ significantly between the microfluidically treated and vortexed groups of zygotes at 2 h post-cumulus removal (P = 0.1032) or between the hand-stripped and vortexed groups (P = 0.7567). In contrast, at 24 h post-cumulus removal, the embryos in the microfluidically treated group showed significantly higher levels of HSP70.1 transcription than the vortexed group (P = 0.0115). The transcription levels did not differ significantly between the hand-stripped and vortexed groups (P = 0.7875). This work strongly suggests that there is de novo RNA transcription in the early embryonic stages of the bovine. In addition to previously described improved developmental kinetics, the use of microfluidics in IVP leads to statistically significant differences in RNA transcription levels of HSP70.1.

155OXYGEN MEASUREMENTS AS INDICATORS OF CULTURE CONDITIONS AND VIABILITY OF CATTLE EMBRYOS

Experiments were performed to study oxygen and temperature conditions experienced by embryos during routine in vitro production (IVP) (Exp. 1) and to evaluate oxygen consumption of single embryos as a viability indicator (Exp. 2). In both experiments, bovine IVP embryos were used (Holm et al. 1999, Theriogenology 52: 683–700), and a combined oxygen-temperature microsensor (tip diameter approx. 50μm) was used to attain oxygen partial pressure gradients and temperature profiles. In Exp. 1, 4-well dishes with in vitro culture (IVC) medium (400-μL or 50-μL droplets) covered with 400μL oil were taken from the incubator (5% CO2 , 5% O2 , 90% N) and measured under atmospheric air conditions at room temperature (24–25°C). The profiles were determined over 2min, starting 2min after the dishes were taken out of the incubator. The 400μL medium was measured from the top layer and gradually 3/4 down into the well (seven replicates). The oxygen partial pressure decreased from 21.0±0.66% O2 at the top layer to 15.0±1.6% O2 at the bottom layer, while the average temperature increased from 27.2±3.0°C to 31.7±0.7°C. In the 50-μL droplets, the profiles were obtained in the middle of the droplet (five replicates). The oxygen partial pressure was 17.1±2.44% O2 and the temperature 31.0±1.11°C. Consequently, routine handling of culture dishes outside the incubator seems to have a strong influence on both temperature and oxygen conditions. In Exp. 2, Day 3 and Day 7 embryos were evaluated morphologically before being loaded consecutively into one of the holes in a measuring block developed for this purpose. The block was previously covered with 40mL IVC medium before being incubated for 3 days under 5% CO2 , 95% N (38.5°C, 100% humidity). The oxygen consumption of each embryo was measured from 75±5min after removal from the incubator. Each measurement lasted approx. 5min, was repeated three times for each embryo, and was performed under atmospheric conditions on a warming stage held at 37±1°C. The average oxygen consumption by Day 3 embryos was 0.25±0.14nL/embryo/h (n=20), and no clear relation between respiration rate and embryo morphology could be demonstrated. For Day 7 embryos, the average oxygen consumption was 0.90±0.56nL/embryo/h (n=22), and this varied according to their morphological quality (1.17±0.70 (n=7), 0.98±0.49 (n=6) and 0.46±0.38 (n=9) nL/embryo/h for good, fair and poor quality embryos, respectively). In conclusion, this study illustrated the changes in O2 partial pressure that embryos can temporarily be exposed to under routine handling, even for rapid procedures such as cleavage inspection. The results also show that respiration rate is lower in Day 3 than in Day 7 embryos. Furthermore, oxygen consumption values of Day 7 embryos seem to be in agreement with the morphological embryo quality, supporting the idea that oxygen consumption can be a valuable parameter for the evaluation of embryo viability.

Apoptosis and cell proliferation potential of bovine embryos stimulated with insulin-like growth factor I during in vitro maturation and culture.

Reliable estimation and improvement of the developmental potential of in vitro production (IVP) embryos requires functional criteria of embryo quality. Antiapoptotic and mitogenic effects of insulin-like growth factor I (IGF-I), applied during bovine IVP, were studied. Day 6.5 blastocysts were fixed and processed for TUNEL to detect apoptotic cells, for immunocytochemical detection of proliferating cell nuclear antigen (PCNA), and for propidium iodide (PI) staining to detect all nuclei. Laser scanning confocal microscopy was used to determine apoptotic (TUNEL/PI) and proliferative (PCNA/PI) indices. Addition of IGF-I to the culture but not to the maturation medium increased the morula/blastocyst yield (P = 0.03), but the cleavage rate was not affected. During culture, IGF-I significantly lowered the apoptotic index by decreasing the number of apoptotic cells per embryo and elevated the total cell number of the blastocysts. The frequency of blastocysts with apoptotic cells was not affected. IGF-I increased the proportion of blastocysts with apoptotic cells in the inner cell mass area only by reducing apoptosis in the trophectoderm area. The PCNA index was not affected by IGF-I. A positive correlation observed between apoptotic and PCNA-positive cells was significant in groups stimulated with IGF-I during in vitro culture. Of TUNEL-positive cells, 30%-40% per embryo were also positive for PCNA. This colocalization may indirectly suggest an activation of DNA repair process in TUNEL-positive cells in response to DNA fragmentation. IGF-I reduces apoptosis in bovine IVP embryos. The requirement of IGF-I is more critical during embryo culture than during oocyte maturation. Our data suggest that an assay for TUNEL in conjunction with cell proliferation analysis can provide useful information about the quality of IVP embryos.

Effects of different reproduction techniques: AI, moet or IVP, on health and welfare of bovine offspring

Since the introduction of in vitro production (IVP) of bovine and sheep pre-implantation embryos, increased birth weights and other deviations of IVP calves and lambs compared with AI or MOET offspring have been reported. Study 1 of the present paper, a comparison between AI, MOET and IVP (co-culture/serum) calves with respect to calf and calving characteristics in large-scale field conditions, confirms these reports. In addition, it is shown that MOET calves tend towards higher birth weights and have significantly longer gestations and more difficult calvings than AI calves. It is presently unknown if the effect of IVP is also observed later in life. In this paper, data on reproduction characteristics of bovine IVP co-culture/serum offspring are presented. Semen production — and non return data of one year old IVP bulls and superovulation-, AI- and OPU/IVP results of one year old IVP heifers are compared with those of one year old AI and MOET animals producing semen or embryos in the same time period. So far, there are no indications that the use of IVP is reflected in deviate reproduction characteristics of bovine IVP offspring. It has been suggested that use of co-culture cells and serum during in vitro culture of bovine (and sheep) embryos may partially explain the increased birth weights and other deviations of bovine and sheep IVP offspring. Deletion of these factors in semi-defined culture media, e.g. Synthetic Oviductal Fluid (SOF), could result in more normal offspring. Study 2 investigates this hypothesis in both field conditions (Study 2a, comparing AI, IVP co-culture/serum and IVP SOF calves) and in semi-standardized conditions (Study 2b, comparing MOET, IVP co-culture/serum and IVP SOF calves at one herd). In Study 2a, although IVP SOF calves showed (non-significant) shorter gestations, easier calvings and lower percentages of perinatal mortality and congenital malformations than IVP co-culture calves, birth weights were not decreased. In Study 2b however, the difference between IVP co-culture and IVP SOF calves in birth weight and ease of calving was significant (P<0.05), IVP SOF calves resembling MOET calves more. IVP calves differed significantly from MOET calves with respect to several physiological parameters, such as blood oxygen saturation level, heart beat frequency and some measures of the heart. In addition, in Study 2b, recipients receiving an IVP SOF embryo showed a more regular return to estrus than those receiving an IVP co-culture embryo. From Study 2 it can be concluded that using a semi-defined medium for in vitro culture (SOF) may improve characteristics of IVP calves born.