Modified dietary fiber from cassava pulp affects the cecal microbial population, short-chain fatty acid, and ammonia production in broiler chickens.

The objective of this study was to investigate the effects of modified dietary fiber from cassava pulp (M-DFCP) supplementation in broiler diets on cecal microbial populations, short-chain fatty acids (SCFAs), ammonia production, and immune responses. A total of 336, one-day-old male broiler chicks (Ross 308) were distributed over 4 dietary treatments in 7 replicate pens (n = 12 chicks) using a completely randomized design. Chicks were fed the control diet and 3 levels of M-DFCP (0.5, 1.0, and 1.5%) for an experimental duration of 42 d. The M-DFCP contained total dietary fiber (TDF), soluble dietary fiber (SDF), insoluble dietary fiber (IDF), cello-oligosaccharides (COS), and xylo-oligosaccharides (XOS) of approximately 280.70, 22.20, 258.50, 23.93, and 157.55 g/kg, respectively. The 1.0 and 1.5% M-DFCP supplementation diets showed positive effects on stimulating the growth of Lactobacillus spp. and Bifidobacterium spp., enhancing SCFAs (acetic, propionic, butyric acid, and branched SCFAs) and lactic acid concentrations during growing periods. Broilers fed 1.0 and 1.5% M-DFCP also exhibited a significant increase in caecal Lactobacillus spp. and lactic acid concentrations during the finisher period as well. In addition, M-DFCP also reduced cecal digesta and excreta ammonia production in broilers over both periods (0-21 and 22-42 d of age). However, M-DFCP did not exhibit any effect on total serum immunoglobulin (Ig) or lysozyme activity. In conclusion, this study shows that M-DFCP can be used as a dietary fiber source in broiler diets, with a recommended level of approximately 1.0%.

Unveiling how vitrification affects the porcine blastocyst: clues from a transcriptomic study.

BACKGROUND: Currently, there is a high demand for efficient pig embryo cryopreservation procedures in the porcine industry as well as for genetic diversity preservation and research purposes. To date, vitrification (VIT) is the most efficient method for pig embryo cryopreservation. Despite a high number of embryos survives in vitro after vitrification/warming procedures, the in vivo embryo survival rates after embryo transfer are variable among laboratories. So far, most studies have focused on cryoprotective agents and devices, while the VIT effects on porcine embryonic gene expression remained unclear. The few studies performed were based on vitrified/warmed embryos that were cultured in vitro (IVC) to allow them to re-expand. Thus, the specific alterations of VIT, IVC, and the cumulative effect of both remained unknown. To unveil the VIT-specific embryonic alterations, gene expression in VIT versus (vs.) IVC embryos was analyzed. Additionally, changes derived from both VIT and IVC vs. control embryos (CO) were analyzed to confirm the VIT embryonic alterations. Three groups of in vivo embryos at the blastocyst stage were analyzed by RNA-sequencing: (1) VIT embryos (vitrified/warmed and cultured in vitro), (2) IVC embryos and (3) CO embryos. RESULTS: RNA-sequencing revealed three clearly different mRNA profiles for VIT, IVC and CO embryos. Comparative analysis of mRNA profiles between VIT and IVC identified 321, differentially expressed genes (DEG) (FDR < 0.006). In VIT vs. CO and IVC vs. CO, 1901 and 1519 DEG were found, respectively, with an overlap of 1045 genes. VIT-specific functional alterations were associated to response to osmotic stress, response to hormones, and developmental growth. While alterations in response to hypoxia and mitophagy were related to the sum of VIT and IVC effects. CONCLUSIONS: Our findings revealed new insights into the VIT procedure-specific alterations of embryonic gene expression by first comparing differences in VIT vs. IVC embryos and second by an integrative transcriptome analysis including in vivo control embryos. The identified VIT alterations might reflect the transcriptional signature of the embryo cryodamage but also the embryo healing process overcoming the VIT impacts. Selected validated genes were pointed as potential biomarkers that may help to improve vitrification.

Oviduct fluid during IVF moderately modulates polyspermy in in vitro-produced goat embryos during the non-breeding season.

The present study determined i) the presence of proteins (oviduct-specific glycoprotein, OVGP1; heat shock protein-70A, HSPA1A; heat shock protein-A8, HSPA8; annexin A1, ANXA1; annexin A5, ANXA5; and myosin-9, MYH9) known to be involved in early reproduction in the oviduct fluid (OF) of anestrous goats; and ii) the functional effect of during IVF on polyspermy modulation and embryonic development. In vitro-matured oocytes were co-cultured with spermatozoa (1.0, 2.0, or 4.0 x 106 cells/mL) for 18 h in SOF medium supplemented with 5 µg/mL of heparin, 4 µg/mL gentamicin, and 10% estrus sheep serum (CTRL1, CTRL2, and CTRL4 groups) or the same medium plus 10% OF (OF1, OF2, and OF4 groups) obtained from anestrus goats. The analysis of OF by western blotting confirmed the presence of the six proteins tested for. The increase in sperm concentration had no effect (P > 0.05) on the penetration rate in any group; however, monospermy rate decreased as sperm concentration was increased in both OF and CTRL. Regardless of the concentration used, when data were pooled, OF supplementation improved (P < 0.05) monospermy and tended (P = 0.057) to enhance IVF efficiency. Additionally, IVF efficiency was higher (P < 0.05) in OF1 than in OF4 [60 ± 13 vs 37 ± 5%). The development capacity was not affected (P > 0.05) by the sperm concentration and OF treatment, and the average values were cleavage (72 ± 2.6%), blastocyst (37 ± 3.0%), blastocyst in relation to the cleaved (51 ± 4.8%), hatched (62 ± 1.2%), and number of cells per blastocyst (174 ± 1.8%). In conclusion, the six proteins analyzed are present in the OF of anestrous goats, and the supplementation of this OF during IVF may modulate the polyspermy incidence and enhance IVF efficiency, especially when 1x106 sperm per mL is used.

Review: Recent advances in bovine in vitro embryo production: reproductive biotechnology history and methods.

In vitro production (IVP) of embryos and associated technologies in cattle have shown significant progress in recent years, in part driven by a better understanding of the full potential of these tools by end users. The combination of IVP with sexed semen (SS) and genomic selection (GS) is being successfully and widely used in North America, South America and Europe. The main advantages offered by these technologies include a higher number of embryos and pregnancies per unit of time, and a wider range of potential female donors from which to retrieve oocytes (including open cyclic females and ones up to 3 months pregnant), including high index genomic calves, a reduced number of sperm required to produce embryos and increased chances of obtaining the desired sex of offspring. However, there are still unresolved aspects of IVP of embryos that limit a wider implementation of the technology, including potentially reduced fertility from the use of SS, reduced oocyte quality after in vitro oocyte maturation and lower embryo cryotolerance, resulting in reduced pregnancy rates compared to in vivo-produced embryos. Nevertheless, promising research results have been reported, and work is in progress to address current deficiencies. The combination of GS, IVP and SS has proven successful in the commercial field in several countries assisting practitioners and cattle producers to improve reproductive performance, efficiency and genetic gain.

Oviduct fluid extracellular vesicles regulate polyspermy during porcine in vitro fertilisation.

High polyspermy is one of the major limitations of porcine invitro fertilisation (IVF). The addition of oviductal fluid (OF) during IVF reduces polyspermy without decreasing the fertilisation rate. Because extracellular vesicles (EVs) have been described as important OF components, the aim of this study was to evaluate the effect of porcine oviductal EVs (poEVs) on IVF efficiency compared with porcine OF (fresh and lyophilised). OF was collected from abattoir oviducts by phosphate-buffered saline flush, and poEVs were isolated by serial ultracentrifugation. Four IVF treatments were conducted: poEVs (0.2mgmL-1), OF (10%), lyophilized and reconstituted pure OF (LOF; 1%) and IVF without supplementation (control). Penetration, monospermy and IVF efficiency were evaluated. Transmission electron microscopy showed an EVs population primarily composed of exosomes (83%; 30-150nm). Supplementation with poEVs during IVF increased monospermy compared with control (44% vs 17%) while maintaining an acceptable penetration rate (61% vs 78% respectively) in a similar way to OF and LOF. Western blotting revealed poEVs proteins involved in early reproductive events, including zona pellucida hardening. In conclusion, our finding show that poEVs are key components of porcine OF and may play roles in porcine fertilisation and polyspermy regulation, suggesting that supplementation with poEVs is a reliable strategy to decrease porcine polyspermy and improve invitro embryo production outcomes.

In vitro survival of follicles in prepubertal ewe ovarian cortex cryopreserved by slow freezing or non-equilibrium vitrification.

PURPOSE: Vitrification is a well-accepted fertility preservation procedure for cryopreservation of oocytes and embryos but little is known regarding ovarian tissue, for which slow freezing is the current convention. The aim of the present study was to assess the efficiency of non-equilibrium vitrification compared to conventional slow freezing for ovarian cortex cryopreservation. METHODS: Using prepubertal sheep ovaries, the capacity of the tissue to sustain folliculogenesis following cryopreservation and in vitro culture was evaluated. Ovarian cortex fragments were cultured in wells for 9 days, immediately or after cryopreservation by conventional slow freezing or non-equilibrium vitrification in straws. During culture, follicular populations within cortex were evaluated by histology and immunohistochemistry for PCNA and TUNEL. Steroidogenic activity of the tissue was monitored by assay for progesterone and estradiol in spent media. RESULTS: No significant differences in follicle morphology, PCNA, or TUNEL labeling were observed between cryopreservation methods at the initiation of culture. Similar decreases in the proportion of primordial follicle population, and increases in the proportion of growing follicles, were observed following culture of fresh or cryopreserved ovarian tissue regardless of cryopreservation method. At the end of culture, PCNA and TUNEL-positive follicles were not statistically altered by slow freezing or vitrification in comparison to fresh cultured fragments. CONCLUSIONS: Overall, for both cryopreservation methods, the cryopreserved tissue showed equal capacity to fresh tissue for supporting basal folliculogenesis in vitro. Taken together, these data confirm that both non-equilibrium vitrification and slow-freezing methods are both efficient for the cryopreservation of sheep ovarian cortex fragments.

Deciphering the oviductal extracellular vesicles content across the estrous cycle: implications for the gametes-oviduct interactions and the environment of the potential embryo.

BACKGROUND: The success of early reproductive events depends on an appropriate communication between gametes/embryos and the oviduct. Extracellular vesicles (EVs) contained in oviductal secretions have been suggested as new players in mediating this crucial cross-talk by transferring their cargo (proteins, mRNA and small ncRNA) from cell to cell. However, little is known about the oviductal EVs (oEVS) composition and their implications in the reproductive success. The aim of the study was to determine the oEVs content at protein, mRNA and small RNA level and to examine whether the oEVs content is under the hormonal influence of the estrous cycle. RESULTS: We identified the presence of oEVs, exosomes and microvesicles, in the bovine oviductal fluid at different stages of the estrous cycle (postovulatory-stage, early luteal phase, late luteal phase and pre-ovulatory stage) and demonstrated that their composition is under hormonal regulation. RNA-sequencing identified 903 differentially expressed transcripts (FDR < 0.001) in oEVs across the estrous cycle. Moreover, small RNA-Seq identified the presence of different types of ncRNAs (miRNAs, rRNA fragments, tRNA fragments, snRNA, snoRNA, and other ncRNAs), which were partially also under hormonal influence. Major differences were found between post-ovulatory and the rest of the stages analyzed for mRNAs. Interesting miRNAs identified in oEVs and showing differential abundance among stages, miR-34c and miR-449a, have been associated with defective cilia in the oviduct and infertility. Furthermore, functional annotation of the differentially abundant mRNAs identified functions related to exosome/vesicles, cilia expression, embryo development and many transcripts encoding ribosomal proteins. Moreover, the analysis of oEVs protein content also revealed changes across the estrous cycle. Mass spectrometry identified 336 clusters of proteins in oEVs, of which 170 were differentially abundant across the estrous cycle (p-value< 0.05, ratio < 0.5 or ratio > 2). Our data revealed proteins related to early embryo development and gamete-oviduct interactions as well as numerous ribosomal proteins. CONCLUSIONS: Our study provides with the first molecular signature of oEVs across the bovine estrous cycle, revealing marked differences between post- and pre-ovulatory stages. Our findings contribute to a better understanding of the potential role of oEVs as modulators of gamete/embryo-maternal interactions and their implications for the reproductive success.

Oral propylene glycol modifies follicular fluid and gene expression profiles in cumulus-oocyte complexes and embryos in feed-restricted heifers.

Dietary supplementation with propylene glycol (PG) increases in vitro production of high-quality embryos in feed-restricted heifers. The aim of the present study was to evaluate the effects of PG in feed-restricted heifers on follicular fluid insulin and insulin-like growth factor (IGF) 1 concentrations, expression of IGF system genes in oocytes and cumulus cells and the expression of selected genes in blastocysts. Feed-restricted (R) heifers were drenched with water or PG during induced oestrous cycles (400mL of PG or water/drench, daily drenching at 1600 hours for the first 9 days of the oestrous cycle). Ovum pick-up (OPU) was performed after superovulation to produce in vitro embryos and without superovulation to recover oocytes, cumulus cells and follicular fluid. OPU was also performed in a control group (not feed restricted and no drenching). Follicular fluid IGF1 concentrations were reduced by R, and PG restored IGF1 concentrations to those seen in the control group. In cumulus cells, expression of IGF1, IGF1 receptor (IGF1R) and IGF binding protein 4 (IGFBP4) was decreased in the R group, and fully (IGF1 and IGF1R) or partially (IGFBP4) restored to control levels by PG. Blastocyst perilipin 2 (PLIN2; also known as adipophilin), Bcl-2-associated X protein (BAX), SCL2A1 (facilitated glucose/fructose transporter GLUT1), aquaporin 3 (AQP3), DNA (cytosine-5)-methyltransferase 3A (DNMT3A) and heat shock 70-kDa protein 9 (HSPA9B) expression were decreased in R heifers; PG restored the expression of the last four genes to control levels. In conclusion, these results suggest that, during follicular growth, PG exerts epigenetic regulatory effects on gene expression in blastocyst stage embryos.

Attachment of Coxiella burnetii to the zona pellucida of in vitro produced goat embryos.

Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 109Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.

Different co-culture systems have the same impact on bovine embryo transcriptome.

During the last few years, several co-culture systems using either BOEC or VERO feeder cells have been developed to improve bovine embryo development and these systems give better results at high oxygen concentration (20%). In parallel, the SOF medium, used at 5% O2, has been developed to mimic the oviduct fluid. Since 2010s, the SOF medium has become popular in improving bovine embryo development and authors have started to associate this medium to co-culture systems. Nevertheless, little is known about the putative benefit of this association on early development. To address this question, we have compared embryo transcriptomes in four different culture conditions: SOF with BOEC or VERO at 20% O2, and SOF without feeders at 5% or 20% O2 Embryos have been analyzed at 16-cell and blastocyst stages. Co-culture systems did not improve the developmental rate when compared to 5% O2 Direct comparison of the two co-culture systems failed to highlight major differences in embryo transcriptome at both developmental stages. Both feeder cell types appear to regulate the same cytokines and growth factors pathways, and thus to influence embryo physiology in the same way. In blastocysts, when compared to culture in SOF at 5% O2, BOEC or VERO seems to reduce cell survival and differentiation by, at least, negatively regulating STAT3 and STAT5 pathways. Collectively, in SOF medium both blastocysts rate and embryo transcriptome suggest no influence of feeder origin on bovine early development and no beneficial impact of co-culture systems when compared to 5% O2.

The activity of three glycosidases (ß-Ν-acetyloglucosaminidase, α-mannosidase, and ß-galactosidase) in the follicular fluid and in the maturation medium affects bovine oocyte maturation.

We studied the role of follicular fluid's (FF) glycosidase (α-mannosidase [α-ΜΑΝ], ß-Ν-acetyloglucosaminidase [NAGASE], ß-galactosidase [ß-GAL]) activity during IVM of bovine oocytes. Oocytes were allocated into two groups according to the follicular size (small follicle [SF]: 2-5 mm, large follicle [LF]: >5-8 mm). In experiment 1, cumulus-oocyte complexes (COCs) quality was evaluated according to morphologic criteria (grades A, B-C, D); oocyte (n = 801) nuclear maturation was assessed after 24 hours of incubation. Bovine embryos were produced in vitro in groups (experiment 2, n = 1503 oocytes) or individually (experiment 3, n = 50 oocytes). More grade-A and -BC COCs were collected from SF and LF groups, respectively (P < 0.05). Maturation rate (experiment 1) and cleavage rate (experiments 2 and 3) were similar in SF and LF groups. Activity of all glycosidases in FF was higher (P < 0.05) in SF group than in LF group, whereas in maturation medium of SF group it was, overall, significantly lower than in that of LF (experiments 2 and 3). In FF of SF group, NAGASE positively associated with grade-A oocytes and negatively with BC oocytes; increased ß-GAL was associated with degenerated oocytes. Cleavage rate in LF group, related negatively to NAGASE and positively to α-MAN in maturation medium. These results indicate that during maturation, COCs release NAGASE and consume ß-GAL, but differences probably exist between individual and group maturation.

Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation.

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 µm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

Bovine Oviduct Epithelial Cells Dedifferentiate Partly in Culture, While Maintaining their Ability to Improve Early Embryo Development Rate and Quality.

There are convincing arguments to suggest that the success of early reproductive events is reliant on a satisfactory dialogue between gametes-embryo and the oviduct epithelium. The aim of this study was to develop and characterize an in vitro model to study these interactions. Cattle zygotes produced in vitro were cultured in either SOF or TCM-199 in the presence or absence of bovine oviduct cell monolayers (BOEC), under 20% or 5% O2 . The embryonic development rate and its quality (cell numbers, cryosurvival) were evaluated, as were the BOEC contents in 11 candidate transcripts (real-time PCR) at different time points. A BOEC co-culture did indeed increase the rate of development in both media under 5% O2 (41 vs 27% and 28 vs 10% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). The effect of BOEC on the developmental rate was more pronounced under 20% O2 (35 vs 6% and 27 vs 4% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). BOEC significantly increased the embryonic cell count in TCM-199 (122.5 ± 11.1 vs 70.3 ± 9.6; p < 0.05) and embryonic cryosurvival in both media. The expression levels of SOD, FGF2 and TGF-ß1 in BOEC remained steady during culture, although mRNA levels of OGP, C3, PGR and ESR2 were clearly reduced, suggesting a dedifferentiation of BOEC during culture. However, SSP1 and GPX4 transcripts were slightly increased during culture, this rise becoming significant by the end of the culture period. In conclusion, our co-culture system with bovine oviduct epithelial cells used for the development of bovine zygotes produced in vitro enhanced blastocyst formation and above all the quality of the resulting embryos, which was associated with specific transcriptomic changes.

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-ß) and their effect on in vitro development of caprine preantral follicles.

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-ß) and its receptors (TGF-ßRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-ß, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-ß and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-ß receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-ß (10 ng/ml), or TGF-ß + FSH for 18 d. TGF-ß increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-ß in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-ß and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.

Development rate and gene expression of IVP bovine embryos cocultured with bovine oviduct epithelial cells at early or late stage of preimplantation development.

The use of somatic cells for coculture with embryos has been amply investigated to study embryo maternal interactions. The use of bovine oviduct epithelial cells (BOEC) has been shown to improve the blastocyst rate and quality, affecting their gene expression profile. In this study, we evaluated different timings of BOEC coculture for the development of in-vitro-produced embryos and their effects on blastocysts rate and mRNA abundance of some genes that are important for embryo development. Our results confirmed the positive effects of BOEC on early development of bovine embryos. The presence of the cells during the first four days or during the last four days of development was enough to produce the full BOEC effect. When the presence of BOEC was restricted to the four first days, the kinetics of blastocyst development was accelerated, with significantly more blastocysts at Days 6 and 7 than when the cells were present all along the culture or only during the last four days. Older cells used at early stage were not active anymore. Using young cells at late stage did not improve the cell effect, compared with the older ones. Therefore, the lower effect of BOEC at late stage, compared with early period, may not be explained by cell aging. In addition, the presence of BOEC, at early or late stages, induced changes in the embryos expression profile of genes known to be related to embryo quality, suggesting reduced apoptosis and increased capacity to struggle against oxidative stress after coculture. In conclusion, we confirmed the effect of BOEC on the rate and quality of bovine IVP embryos development. We found for the first time that the presence of BOEC during the four first days of the 8-days development is enough to produce these effects. These first four days represent the period of the presence of the embryos in the oviduct in vivo, highlighting the physiological relevance of this in vitro model of coculture. In addition, we found that the presence of BOEC at early stages of development induced modification of transcription profile in the blastocyst, four days later, suggesting an epigenetic regulation induced by BOEC in growing embryos.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos.

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10µM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

[Use of an in vitro model in bovine to evidence a functional and molecular dialogue between preimplantation embryo and oviduct epithelial cells]. / Mise en évidence d'un dialogue moléculaire et fonctionnel entre l'embryon préimplantatoire bovin et l'oviducte grâce à un modèle in vitro.

Beyond being a pipe between ovary and uterus, the oviduct is an active player in different aspects of early reproductive processes, in particular in the transport of embryos to the site of implantation and the regulation of its early development. Different studies evidenced a communication between oviduct and early embryo at the molecular and functional levels. Since the study of these interactions is difficult in vivo, different in vitro systems have been developed to mimic the maternal milieu during early development. These systems allowed to confirm the action of the cells on the quality of early development (blastocyst rate and viability). In turn, the embryos are producing signals that are able to modify and adapt the activity of maternal cells.

[Regulating pre-antral follicle development: a brake on depletion of the ovarian reserve]. / Régulation de la croissance des follicules pré-antraux, un frein à l'épuisement de la réserve ovarienne.

The process of folliculogenesis is a complex and dynamic process that is regulated by positive and negative factors. During folliculogenesis in the mammalian ovary, the primordial follicle pool is gradually reduced through successive waves of follicle growth. It is important that this pool is carefully regulated, otherwise premature ovarian failure will occur. This process results in the ovulation of an ovocyte or more commonly, the loss of ovocytes through atresia. The majority of research has concentrated on the positive regulators of follicle growth with very little attention on the negative regulators. Nonetheless, there are several factors that have been observed to inhibit follicle activation and development. In this review, we summarize those here.