Characterization of preovulatory follicular fluid secretome and its effects on equine oocytes during in vitro maturation.

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.

Description of a new quantitative method to assess mitochondrial distribution pattern in mature equine oocytes.

The Mitochondrial distribution pattern or MDP in mammalian oocytes serves as an indicator of their cytoplasmic maturity, with a heterogeneous pattern associated with mature cytoplasm. Currently, MDP assessment involves fluorescent labelling of mitochondria followed by visual evaluation, as no quantitative method exists. Our objective was to develop a quantitative approach to assess MDP in mature equine oocytes. Equine oocytes, obtained by ovum pick up (OPU) were matured in vitro, and only metaphase II oocytes were used in the study (n = 56). Following denudation, oocytes were fixed, stained with MitoTracker™ Red CMXRos (50 nM in TCM-199 with Hank´s salts and 10% FBS) for 15 min at 38 °C, and then incubated with 2.5 µg/ml Hoechst 33342 for 10 min at 38 °C. Confocal microscope images were acquired, and the oocyte's MDP was visually classified as either homogeneous (HoD; n = 17) or heterogeneous (HeD; n = 39). For quantitative analysis, Fiji-ImageJ software was employed. Background subtraction was performed, and a 1-pixel line along the diameter was drawn to calculate the intensity profile. Fluorescence intensities were normalized, and ratios of peripheral to central fluorescence intensity were determined. Student´s t-test was used for comparations; MDP ratio was (mean ± standard error of the mean): 0.8 ± 0.02 for HoD and 0.3 ± 0.02 for HeD (p < 0.001). These results demonstrate concordance between quantitative and qualitative MDP assessment in mature equine oocytes. Our study describes a new approach to quantify mitochondrial distribution pattern and cytoplasmic maturation in mature equine oocytes.

Resolution of two cases of ovarian abscesses in mares subjected to ovum pick up.

BACKGROUND: Currently, for in vitro embryo production in live mares, immature oocytes are retrieved by transvaginal follicular aspiration or ovum pick up (OPU). Occasionally, ovarian abscesses have been described after OPU, but no current consensus exists on how to treat this condition. OBJECTIVES: To describe diagnosis and successful treatment of ovarian abscesses in two mares subjected to OPU. STUDY DESIGN: Case report. METHODS: Case records were reviewed and summarised. RESULTS: In the first case, a pony mare showed tachypnoea, tachycardia, high temperature, leukocytosis, left hindlimb lameness and slight increase in concentration of serum amyloid A. Ultrasonography revealed an increase in the size of the left ovary and two well defined structures suggestive of ovarian abscess. A left ovariectomy by standing laparoscopy was the treatment of choice: the diagnosis was confirmed, and bacterial culture produced heavy growth of Streptococcus equi Zooepidemicus. In the second mare, an abnormal structure was observed in the left ovary in a routine transrectal ultrasonographic exam in the absence of any clinical signs or abnormal blood parameters. A medical approach was chosen and a sample of the purulent material was aspirated with a transvaginal ultrasound-guided approach. The sample yielded a heavy growth of Streptococcus equi Zooepidemicus after culture. Treatment was initiated with rifampicin and trimethoprim-sulfadiazine based on the antibiogram results and the abscess completely resolved after 40 days. MAIN LIMITATIONS: Limited to two cases. CONCLUSIONS: Ovarian abscesses in mares can be successfully treated both surgically and medically.

Selected Metabolites Found in Equine Oviductal Fluid do not Modify the Parameters Associated to Capacitation of the Frozen-thawed Equine Spermatozoa In Vitro.

In the horse, a repeatable protocol for in vitro fertilization has not been developed, possibly due to incomplete sperm capacitation. We have previously identified the metabolites present in equine oviductal fluid (OF). We aimed to test the effects of different metabolites found in equine oviductal fluid on quality parameters of frozen-thawed spermatozoa. Different concentrations of myoinositol (5-25 mM), lactate (6-60 mM), glycine (0.1-5 mM), ß-alanine (1-6 mM), and histamine (0.05-0.4 mM) were added independently to modified Whitten's medium (pH = 7.25). Thawed equine spermatozoa (three stallions, one ejaculate per stallion, n = 3) were incubated for 2 hours at 37˚C in presence of the selected metabolites. After sperm incubation, total motility (TM), and progressive motility (PM) were evaluated by computer-assisted sperm analysis. Viability (SYBR-14+/PI-), mitochondrial membrane potential (ΔΨm) (JC-1), acrosome reaction (PNA+/PI-) and reactive oxygen species (ROS) production (CellRox+/PI-), were evaluated by flow cytometry. Protein tyrosine phosphorylation (PY) was evaluated by indirect immunofluorescence. Our results show that the addition of the metabolites at the dosages tested does not exert any effect on the sperm parameters analyzed. More research is needed to ascertain if metabolite addition at the dosages found in the equine OF exerts any remarkable effect on in vitro equine sperm capacitation.

The Proteome of Equine Oviductal Fluid Varies Before and After Ovulation: A Comparative Study.

Equine fertilization cannot be performed in the laboratory as equine spermatozoa do not cross the oocyte's zona pellucida in vitro. Hence, a more profound study of equine oviductal fluid (OF) composition at the pre-ovulatory and post-ovulatory stages could help in understanding what components are required to achieve fertilization in horses. Our work aimed to elucidate the proteomic composition of equine OF at both stages. To do this, OF was obtained postmortem from oviducts of slaughtered mares ipsilateral to a pre-ovulatory follicle (n = 4) or a recent ovulation (n = 4); the samples were kept at -80°C until analysis. After protein extraction and isobaric tags for relative and absolute quantification (iTRAQ) labeling, the samples were analyzed by nano-liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The analysis of the spectra resulted in the identification of a total of 1,173 proteins present in pre-ovulatory and post-ovulatory samples; among these, 691 were unique for Equus caballus. Proteins from post-ovulatory oviductal fluid were compared with the proteins from pre-ovulatory oviductal fluid and were categorized as upregulated (positive log fold change) or downregulated (negative log fold change). Fifteen proteins were found to be downregulated in the post-ovulatory fluid and 156 were upregulated in the post-ovulatory OF compared to the pre-ovulatory fluid; among the upregulated proteins, 87 were included in the metabolism of proteins pathway. The identified proteins were related to sperm-oviduct interaction, fertilization, and metabolism, among others. Our data reveal consistent differences in the proteome of equine OF prior to and after ovulation, helping to increase our understanding in the factors that promote fertilization and early embryo development in horses.

Study of the Metabolomics of Equine Preovulatory Follicular Fluid: A Way to Improve Current In Vitro Maturation Media.

Production of equine embryos in vitro is currently a commercial technique and a reliable way of obtaining offspring. In order to produce those embryos, immature oocytes are retrieved from postmortem ovaries or live mares by ovum pick-up (OPU), matured in vitro (IVM), fertilized by intracytoplasmic sperm injection (ICSI), and cultured until day 8-10 of development. However, at best, roughly 10% of the oocytes matured in vitro and followed by ICSI end up in successful pregnancy and foaling, and this could be due to suboptimal IVM conditions. Hence, in the present work, we aimed to elucidate the major metabolites present in equine preovulatory follicular fluid (FF) obtained from postmortem mares using proton nuclear magnetic resonance spectroscopy (1H-NMR). The results were contrasted against the composition of the most commonly used media for equine oocyte IVM: tissue culture medium 199 (TCM-199) and Dulbecco's modified eagle medium/nutrient mixture F-12 Ham (DMEM/F-12). Twenty-two metabolites were identified in equine FF; among these, nine of them are not included in the composition of DMEM/F-12 or TCM-199 media, including (mean ± SEM): acetylcarnitine (0.37 ± 0.2 mM), carnitine (0.09 ± 0.01 mM), citrate (0.4 ± 0.04 mM), creatine (0.36 ± 0.14 mM), creatine phosphate (0.36 ± 0.05 mM), fumarate (0.05 ± 0.007 mM), glucose-1-phosphate (6.9 ± 0.4 mM), histamine (0.25 ± 0.01 mM), or lactate (27.3 ± 2.2 mM). Besides, the mean concentration of core metabolites such as glucose varied (4.3 mM in FF vs. 5.55 mM in TCM-199 vs. 17.5 mM in DMEM/F-12). Hence, our data suggest that the currently used media for equine oocyte IVM can be further improved.

Stage-specific metabolomic changes in equine oviductal fluid: New insights into the equine fertilization environment.

A repeatable protocol for equine in vitro fertilization (IVF) has remained elusive. This is likely, in part, due to suboptimal composition of capacitation or IVF media that are currently in use. Hence, we aimed to analyse the metabolome of equine oviductal fluid (OF) at the pre- (PRE) and immediate post-ovulatory (PST) stages using proton magnetic resonance spectroscopy (1H NMR). Oviductal fluid from eight PRE and six PST mares were used to prepare a total of five samples per group. A total of 18 metabolites were identified. The five metabolites with the highest concentrations in the OF samples were lactate, myoinositol, creatine, alanine and carnitine. Only fumarate and glycine showed significant differences in their concentrations between PRE and PST OF samples, with higher concentrations in the PST samples. In a preliminary study, stallion spermatozoa (n = 3 ejaculates) were incubated with different concentrations of PST OF from one mare (0, 0.0625, 0.125, 0.25, 0.5 or 1%; v:v). After 4 h of sperm incubation, protein tyrosine phosphorylation (PY) by western blotting, sperm motility, and acrosomal status were evaluated. An increase of PY was observed in sperm from two stallions when treated with 0.0625% and 0.125% of OF; however no change in PY was noted in the other stallion. There were no effects of OF on spermatozoa motility or acrosome status. These results provide the first information on the metabolomics of equine OF at different stages of the estrus cycle, and present the possibility that OF may affect PY in stallion spermatozoa.

Hábitos preferenciales de los consumidores de frijol común (Phaseolus vulgaris L) en México / Preferential habits of consumers of common bean (Phaseolus vulgaris L) in Mexico

El conocimiento de los hábitos preferenciales de los consumidores de frijol es fundamental para definir los objetos en los programas de mejoramiento genético y para diseñar las estrategias de mercadeo en una región o país determinados. El presente trabajo se basó en la aplicación de 1514 encuestas a consumidores de frijol de 14 entidades federativas de la República Mexicana. Para la interpretación de los resultados el país se dividió en cuatro regiones: Noroeste, Centro y Sur. En la región Noroeste el 98 por ciento de los encuestados consume frijol "Azufrado" (amarillo azufre); en el Noreste el 70 por ciento concume "Pinto" (beige con motas cafés) y "Bayo" (Beige); en la zona Sur el 90 por ciento consume frijol "Negro", mientras que en la zona Centro se consume todas las clases comerciales. Se detectó que dentro de cada clase comercial existen preferencias específicas en relación al tamaño y brillantez del grano; sin embargo, en la clase comercial Negro los consumidores prefieren el grano de testa opaca y tamaño de 18-22/100 semillas mientras que en la clase "Flor de Mayo" (beige con motas rosas) los consumidores prefieren grano de testa brillante y tamaño de 30-35/100 semillas. La principal característica que utilizan los consumidores para definir sus preferencias es el tiempo de cocción y el sabor. Se detectó que entre los consumidores de frijol los hábitos preferenciales están muy arraigadas pues el 70 por ciento declaró no estar dispuesto a cambiar el frijol de su preferencia aún cuando la clase alternativa fuese más barata. Por otro lado, los consumidores normalmente no remojan el grano en agua ni agregan sal al inicio del proceso de cocción para no afectar el sabor y apariencia del frijol. Estos resultados fueron confirmados con pruebas sensoriales. En el presente trabajo también se discuten aspectos relacionados con formas de procesamiento y consumo y algunos aspectos de mercadeo del grano de frijol