Acquisition and maintenance of pluripotency are influenced by fibroblast growth factor, leukemia inhibitory factor, and 2i in bovine-induced pluripotent stem cells.

Several opportunities for embryo development, stem cell maintenance, cell fate, and differentiation have emerged using induced pluripotent stem cells (iPSCs). However, the difficulty in comparing bovine iPSCs (biPSCs) with embryonic stem cells (ESCs) was a challenge for many years. Here, we reprogrammed fetal fibroblasts by transient expression of the four transcription factors (Oct4, Sox2, Klf4, and c-Myc, collectively termed "OSKM" factors) and cultured in iPSC medium, supplemented with bFGF, bFGF2i, leukemia inhibitory factor (LIF), or LIF2i, and then compared these biPSC lines with bESC to evaluate the pluripotent state. biPSC lines were generated in all experimental groups. Particularly, reprogrammed cells treated with bFGF were more efficient in promoting the acquisition of pluripotency. However, LIF2i treatment did not promote continuous self-renewal. biPSCs (line 2) labeled with GFP were injected into early embryos (day 4.5) to assess the potential to contribute to chimeric blastocysts. The biPSC lines show a pluripotency state and are differentiated into three embryonic layers. Moreover, biPSCs and bESCs labeled with GFP were able to contribute to chimeric blastocysts. Additionally, biPSCs have shown promising potential for contributing to chimeric blastocysts and for future studies.

In vitro induced pluripotency from urine-derived cells in porcine.

BACKGROUND: The generation of induced pluripotent stem cells (iPSC) has been a game-changer in translational and regenerative medicine; however, their large-scale applicability is still hampered by the scarcity of accessible, safe, and reproducible protocols. The porcine model is a large biomedical model that enables translational applications, including gene editing, long term in vivo and offspring analysis; therefore, suitable for both medicine and animal production. AIM: To reprogramme in vitro into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine. METHODS: The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4 (OCT4), SRY-box2 (SOX2), Kruppel-like factor 4 (KLF4), and C-MYC, and cultured with basic fibroblast growth factor (bFGF) supplementation. To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase (AP), OCT4, SOX2, NANOG, TRA1 81 and SSEA 1 detection. Endogenous transcripts related to the pluripotency (OCT4, SOX2 and NANOG) were analyzed via reverse transcription quantitative real-time polymerase chain reaction in different time points during the culture, and all three lineages formed embryoid bodies (EBs) when cultured in suspension without bFGF supplementation. RESULTS: The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming. Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors (mOSKM), but not from human factors (hOSKM). Three clonal lineages were isolated and further cultured for at least 28 passages, all the lineages were positive for AP detection, the OCT4, SOX2, NANOG markers, albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1, similar results were observed in the abundance of the endogenous transcripts related to pluripotent state. All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts. CONCLUSION: For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotent-like state, enabling new numerous applications in both human or veterinary regenerative medicine.

Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions.

Culture conditions regulate the process of pluripotency acquisition and self-renewal. This study aimed to analyse the influence of the in vitro environment on the induction of porcine induced pluripotent stem cell (piPSCs) differentiation into primordial germ cell-like cells (pPGCLCs). piPSC culture with different supplementation strategies (LIF, bFGF, or LIF plus bFGF) promoted heterogeneous phenotypic profiles. Continuous bFGF supplementation during piPSCs culture was beneficial to support a pluripotent state and the differentiation of piPSCs into pPGCLCs. The pPGCLCs were positive for the gene and protein expression of pluripotent and germinative markers. This study can provide a suitable in vitro model for use in translational studies and to help answer numerous remaining questions about germ cells.

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro.

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.

Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients.

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

Actions and Roles of FSH in Germinative Cells.

Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic-pituitary-gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and ß. The FSH ß-subunit (FSHß) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH ß-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation.

The event of cellular reprogramming into pluripotency is influenced by several factors, such as in vitro culture conditions (e.g., culture medium and oxygen concentration). Herein, bovine iPSCs (biPSCs) were generated in different levels of oxygen tension (5% or 20% of oxygen) and supplementation (bFGF or bFGF + LIF + 2i-bFL2i) to evaluate the efficiency of pluripotency induction and maintenance in vitro. Initial reprogramming was observed in all groups and bFL2i supplementation initially resulted in a superior number of colonies. However, bFL2i supplementation in low oxygen led to a loss of self-renewal and pluripotency maintenance. All clonal lines were positive for alkaline phosphatase; they expressed endogenous pluripotency-related genes SOX2, OCT4 and STELLA. However, expression was decreased throughout the passages without the influence of oxygen tension. GLUT1 and GLUT3 were upregulated by low oxygen. The biPSCs were immunofluorescence-positive stained for OCT4 and SOX2 and they formed embryoid bodies which differentiated in ectoderm and mesoderm (all groups), as well as endoderm (one line from bFL2i in high oxygen). Our study is the first to compare high and low oxygen environments during and after induced reprogramming in cattle. In our conditions, a low oxygen environment did not favor the pluripotency maintenance of biPSCs.

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Abstract Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.

Effects of different stimulators of cGMP synthesis on lipid content in bovine oocytes matured in vitro

Bovine oocytes and blastocysts produced in vitro are frequently of lower quality and less cryotolerant than those produced in vivo, and greater accumulation of lipids in the cytoplasm has been pointed out as one of the reasons. In human adipocytes cGMP signaling through the activation of PKG appears to be involved in lipid metabolism, and components of this pathway have been detected in bovine cumulus-oocyte complexes (COCs). The aim of this study was to investigate the influence of this pathway on the lipid content in oocytes and expression of PLIN2 (a lipid metabolism-related gene) in cumulus cells. COCs were matured in vitro for 24 h with different stimulators of cGMP synthesis. The activation of soluble guanylyl cyclase (sGC) by Protoporphyrin IX reduced lipid content (22.7 FI) compared to control oocytes (36.45 FI; P <0.05). Stimulation of membrane guanylyl cyclase (mGC) with natriuretic peptides precursors A and C (NPPA and NPPC) had no effect (36.5 FI; P>0.05). When the PKG inhibitor KT5823 was associated with Protoporphyrin IX, its effect was reversed and lipid contents increased (52.71 FI; P<0.05). None of the stimulators of cGMP synthesis affected the expression of PLIN2 in cumulus cells. In conclusion, stimulation of sGC for cGMP synthesis promotes lipolytic activities in bovine oocytes matured in vitro and such effect is mediated by PKG. However, such effect may vary depending on the stimulus received and/or which synthesis enzyme was activated, as stimulation of mGC had no effects.(AU)

Ovarian follicular dynamics, progesterone concentrations, pregnancy rates and transcriptional patterns in Bos indicus females with a high or low antral follicle count.

We evaluated the effect of the antral follicle count (AFC) on ovarian follicular dynamics, pregnancy rates, progesterone concentrations, and transcriptional patterns of genes in Nelore cattle (Bos taurus indicus) after a timed artificial insemination (TAI) programme. Cows were separated based on the AFC, and those with a high AFC showed a larger (P < 0.0001) ovarian diameter and area than those with a very low AFC. Females with a very low AFC exhibited a larger (P < 0.01) diameter of the dominant follicle at TAI (13.6 ± 0.3 vs. 12.2 ± 0.4 mm) and a tendency (P = 0.06) to have different serum progesterone concentrations (2.9 ± 0.3 vs. 2.1 ± 0.3 ng/mL; on day 18, considering day 0 as the beginning of the synchronization protocol) than those with a high AFC. The pregnancy rate was higher (P ≤ 0.05) in animals with a very low (57.9%) and low (53.1%) AFC than in those with a high AFC (45.2%). The expression of genes related to intercellular communication, meiotic control, epigenetic modulation, cell division, follicular growth, cell maintenance, steroidogenesis and cellular stress response was assessed on day 5. In females with a low AFC, 8 and 21 genes in oocytes and cumulus cells, respectively, were upregulated (P < 0.05), while 3 and 6 genes in oocytes and cumulus cells, respectively, were downregulated. The results described here will help elucidate the differences in ovarian physiology and the reproductive success of Bos indicus females with a low or high AFC.

Generation of neural progenitor cells from porcine-induced pluripotent stem cells.

In this study, porcine embryonic fibroblasts (pEFs) were reprogrammed into porcine-induced pluripotent stem cells (piPSCs) using either human or mouse specific sequences for the OCT4, SOX2, c-Myc, and KLF4 transcription factors. In total, three pEFs lines were reprogrammed, cultured for at least 15 passages, and characterized regarding their pluripotency status (alkaline phosphatase expression, embryoid body formation, expression of exogenous and endogenous genes, and immunofluorescence). Two piPSC lines were further differentiated, using chemical inhibitors, into putative neural progenitor-like (NPC-like) cells with subsequent analyses of their morphology and expression of neural markers such as NESTIN and GFAP as well as immunofluorescent labeling of NESTIN, ß-TUBULIN III, and VIMENTIN. NPC-like cells were positive for all the neural markers tested. These results evidence of the generation of porcine NPC-like cells after in vitro induction with chemical inhibitors, representing an adequate model for future regenerative and translational medicine research.

Stem cells on regenerative and reproductive science in domestic animals.

Stem cells are undifferentiated and self-renewable cells that present new possibilities for both regenerative medicine and the understanding of early mammalian development. Adult multipotent stem cells are already widely used worldwide in human and veterinary medicine, and their therapeutic signalling, particularly with respect to immunomodulation, and their trophic properties have been intensively studied. The derivation of embryonic stem cells (ESCs) from domestic species, however, has been challenging, and the poor results do not reflect the successes obtained in mouse and human experiments. More recently, the generation of induced pluripotent stem cells (iPSCs) via the forced expression of specific transcription factors has been demonstrated in domestic species and has introduced new potentials in regenerative medicine and reproductive science based upon the ability of these cells to differentiate into a variety of cells types in vitro. For example, iPSCs have been differentiated into primordial germ-like cells (PGC-like cells, PGCLs) and functional gametes in mice. The possibility of using iPSCs from domestic species for this purpose would contribute significantly to reproductive technologies, offering unprecedented opportunities to restore fertility, to preserve endangered species and to generate transgenic animals for biomedical applications. Therefore, this review aims to provide an updated overview of adult multipotent stem cells and to discuss new possibilities introduced by the generation of iPSCs in domestic animals, highlighting the possibility of generating gametes in vitro via PGCL induction.

Células-tronco pluripotentes induzidas (células iPS) em animais domésticos e a possibilidade de geração in vitro de gametas / Induced pluripotent stem cells (iPS cells) in domestic animals and the possibility to generate gametes in vitro

Células-tronco são conhecidas pela característica de alto potencial de auto-renovação e podem ser classificadas segundo seu estágio de indiferenciação e perfil epigenético. Células-tronco embrionárias (CTEs) e células-tronco adultas são bastante estudadas e caracterizadas, principalmente nos modelos humano e em camundongos por apresentarem novas possibilidades tanto para a medicina regenerativa quanto para o entendimento do desenvolvimento inicial dos mamíferos. Contudo, a derivação e a manutenção de CTEs em espécies domésticas é desafiadora e apresenta resultados inconsistentes em relação à manutenção da pluripotência in vitro. Nesse contexto, a geração das células pluripotentes induzidas in vitro (células iPS ou iPSCs) é imprescindível para a geração de novas possibilidades na medicina veterinária regenerativa e reprodutiva devido à capacidade de diferenciação destas em uma variedade de outros tipos celulares, inclusive em animais. Assim, esta revisão apresenta a geração das células iPS em animais domésticos, e em especial, recentes estudos sobre as etapas necessárias para a possibilidade de geração de gametas funcionais in vitro, uma importante contribuição na correção de infertilidades, conservação de espécies ameaçadas de extinção e geração de indivíduos geneticamente superiores ou modificados para aplicações agropecuárias ou biomédicas.

Stem cells are widely known for their high potential for self-renewal, being classified according to their stage of undifferentiation and epigenetic profile. Embryonic stem cells (ES) and adult stem cells are already well studied and characterized, mainly in human and mouse models, once they present new possibilities both for regenerative medicine and for understanding the initial development of mammals. However, the derivation and maintenance of ES in domestic species is challenging and presents inconsistent results regarding maintenance of in vitro pluripotency. In this context, the derivation of in vitro induced pluripotent cells (iPS cells or iPSCs) enables new possibilities in regenerative and reproductive veterinary medicine due to their ability to differentiate into a variety of other cell types. Therefore, this review presents the generation of iPS cells in domestic animals, and focuses on recent studies on the steps necessary for the generation of functional gametes in vitro, an important contribution of stem cells aiming the correction of infertility, the conservation of species in risk of extinction and for the generation of genetically superior or modified organisms for agricultural and biomedical applications.

Células-tronco pluripotentes induzidas (células iPS) em animais domésticos e a possibilidade de geração in vitro de gametas / Induced pluripotent stem cells (iPS cells) in domestic animals and the possibility to generate gametes in vitro

Células-tronco são conhecidas pela característica de alto potencial de auto-renovação e podem ser classificadas segundo seu estágio de indiferenciação e perfil epigenético. Células-tronco embrionárias (CTEs) e células-tronco adultas são bastante estudadas e caracterizadas, principalmente nos modelos humano e em camundongos por apresentarem novas possibilidades tanto para a medicina regenerativa quanto para o entendimento do desenvolvimento inicial dos mamíferos. Contudo, a derivação e a manutenção de CTEs em espécies domésticas é desafiadora e apresenta resultados inconsistentes em relação à manutenção da pluripotência in vitro. Nesse contexto, a geração das células pluripotentes induzidas in vitro (células iPS ou iPSCs) é imprescindível para a geração de novas possibilidades na medicina veterinária regenerativa e reprodutiva devido à capacidade de diferenciação destas em uma variedade de outros tipos celulares, inclusive em animais. Assim, esta revisão apresenta a geração das células iPS em animais domésticos, e em especial, recentes estudos sobre as etapas necessárias para a possibilidade de geração de gametas funcionais in vitro, uma importante contribuição na correção de infertilidades, conservação de espécies ameaçadas de extinção e geração de indivíduos geneticamente superiores ou modificados para aplicações agropecuárias ou biomédicas.(AU)

Stem cells are widely known for their high potential for self-renewal, being classified according to their stage of undifferentiation and epigenetic profile. Embryonic stem cells (ES) and adult stem cells are already well studied and characterized, mainly in human and mouse models, once they present new possibilities both for regenerative medicine and for understanding the initial development of mammals. However, the derivation and maintenance of ES in domestic species is challenging and presents inconsistent results regarding maintenance of in vitro pluripotency. In this context, the derivation of in vitro induced pluripotent cells (iPS cells or iPSCs) enables new possibilities in regenerative and reproductive veterinary medicine due to their ability to differentiate into a variety of other cell types. Therefore, this review presents the generation of iPS cells in domestic animals, and focuses on recent studies on the steps necessary for the generation of functional gametes in vitro, an important contribution of stem cells aiming the correction of infertility, the conservation of species in risk of extinction and for the generation of genetically superior or modified organisms for agricultural and biomedical applications.(AU)

Influence of nitric oxide and phosphodiesterases during in vitro maturation of bovine oocytes on meiotic resumption and embryo production.

This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE) families on meiosis resumption, nucleotides levels and embryo production. Experiment I, COCs were matured in vitro with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) associated or not with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), meiotic resumption and nucleotides levels were assessed. SNAP delayed germinal vesicle breakdown (GVBD) (53.4 ± 1.2 versus 78.4 ± 2.4% for controls, P 0.05). Cyclic GMP levels were higher in SNAP (3.94 ± 0.18, P 0.05). Embryo development did not differ from the control for SNAP and cilostamide groups (38.7 ± 5.8, 37.9 ± 6.2 and 40.5 ± 5.8%, P > 0.05), but SNAP + cilostamide decreased embryo production (25.7 ± 6.9%, P < 0.05). In conclusion, SNAP was confirmed to delay meiosis resumption by the NO/sGC/cGMP pathway, by increasing cGMP, but not cAMP. Inhibiting different PDEs to further increase nucleotides in association with SNAP did not show any additive effects on meiosis resumption, indicating that other pathways are involved. Moreover, SNAP + cilostamide affected the meiosis progression and decreased embryo development.

New approaches regarding the in vitro maturation of oocytes: manipulating cyclic nucleotides and their partners in crime.

Several discoveries have been described recently (5-10 years) about the biology of ovarian follicles (oocyte, cumulus cells and granulosa cells), including new aspects of cellular communication, the control of oocyte maturation and the acquisition of oocyte competence for fertilization and further embryo development. These advances are nourishing assisted reproduction techniques (ART) with new possibilities, in which novel culture systems are being developed and tested to improve embryo yield and quality. This mini-review aims to describe how the recent knowledge on the physiological aspects of mammalian oocyte is reflecting as original or revisited approaches into the context of embryo production. These new insights include recent findings on the mechanisms that control oocyte maturation, especially modulating intraoocyte levels of cyclic nucleotides during in vitro maturation using endogenous or exogenous agents. In this mini-review we also discuss the positive and negative effects of these manipulations on the outcoming embryo.

Effects of fetal calf serum on cGMP pathway and oocyte lipid metabolism in vitro.

Intracellular levels of cyclic nucleotides, such as cGMP, are involved in the regulation of adipocyte lipolysis. Cumulus-oocyte complexes (COCs) express enzymes that both synthesise (guanylate cyclase) and degrade (phosphodiesterase (PDE) 5A) cGMP. Because serum interferes with lipid metabolism, its effects on the cGMP pathway and lipid content in bovine COCs were examined. COCs were matured in medium containing fetal calf serum (FCS; 2% or 10%) or 0.4% bovine serum albumin (BSA; control). At both 2% and 10%, FCS decreased cGMP levels in COCs compared with BSA (0.64 and 1.04 vs 9.46 fmol per COC respectively; P<0.05) and decreased transcript levels of guanylate cyclase 1, soluble, beta 3 (GUCY1B3), whereas PDE5A levels were increased. FCS also affected the expression of genes related to lipolysis, increasing relative expression of perilipin 2 (PLIN2) and carnitine palmitoyltransferase 1B (CPT1B) in cumulus cells. Effects of FCS and cGMP on the lipid content of oocytes and embryos were evaluated by Nile red staining. COCs were matured with 10% FCS, FCS+10-5 M sildenafil (SDF), a PDE5 inhibitor, or 0.4% BSA. The lipid content was increased in oocytes matured in FCS compared with BSA (fluorescence intensity 20.1 vs 17.61 respectively; P<0.05), whereas the lipid content in oocytes matured in FCS+SDF (fluorescence intensity 16.33) was similar to that in the BSA-treated group (P>0.05). In addition, lipid content was higher in embryos from oocytes matured with FCS than BSA (fluorescence intensity 31.12 vs 22.31 respectively; P<0.05), but was increased even further in the FCS+SDF-treated group (fluorescence intensity 40.35; P<0.05), possibly due to a compensatory mechanism during embryo culture without SDF for the reduction in lipid content during IVM. The present study provides, for the first time, evidence that the cGMP pathway may be involved in lipid metabolism in bovine COCs and that this pathway is affected by FCS.