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During the luteal and follicular phases of the estrous cycle, cumulus-oocyte complexes (COC) and oviduct epithelial cells (OEC) undergo notable physiological and morphological changes. Maintaining proper zinc (Zn) homeostasis is crucial in both somatic and germinal mammalian cells. This study aimed to assess the impact of the estrous phase (luteal or follicular) on Zn transporter expression in bovine COC and OEC (BOEC). The expression of Zn transporters Slc39a6 (ZIP6), Slc39a8 (ZIP8), Slc39a14 (ZIP14), Slc30a3 (ZnT3), Slc30a7 (ZnT7), and Slc30a9 (ZnT9) was analyzed in COC and BOEC from cows during the luteal or follicular phases. Gene expression of ZIP6, ZIP14, and ZnT9 was quantified in COC and BOEC. The gene expression in the remaining transporters could not be quantified due to low mRNA levels (ZIP8 and ZnT3 in COC and BOEC; ZnT7 in BOEC) or absence of expression (ZnT7 in COC). In COC, the relative expression (RE) of all three transporters was higher in the luteal phase compared to the follicular phase (P ≤ 0.05). In BOEC, the luteal phase increased the RE of ZIP 6 (P ≤ 0.05), decreased the RE of ZnT9 (P ≤ 0.05), and did not modify the RE of ZIP14 (P > 0.05) compared to the follicular phase. In conclusion, the study reveals differences in the gene expression of ZIP6, ZIP14, and ZnT9 according to the estrous cycle phase in ex vivo samples of bovine COC and OEC.

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Fluazuron is a novel veterinary pour-on antitick formulation which can be applied simultaneously with bovine reproduction management strategies. Considering the economic importance of the livestock industry in many countries, it is important to know whether antiparasitics such as fluazuron may cause embryonic loss. The aim of this study was to evaluate the toxicological effect of fluazuron on bovine oocytes during in vitro maturation. The best fluazuron concentrations were determined in a preliminary experiment on Chinese hamster ovary (CHO)-K1 cells and further used to compare fluazuron toxicity in both study models. Results of the annexin V and alkaline single cell gel electrophoresis assays demonstrated that fluazuron caused cytotoxicity and genotoxicity in bovine cumulus cells at all the concentrations tested (50, 75 and 100 µg fluazuron/mL). The evaluation of cortical granules and mitochondria distribution showed that cytoplasmic maturation was not affected by fluazuron treatment. However, a decrease in metaphase II + polar body, degenerate oocytes as well as disorganized chromatin in polar body were observed at all concentrations tested. Whereas the fertilization process was not altered by 50 µg/mL fluazuron, the embryo development rate decreased significantly. No significant differences were observed in any of the oxidative stress parameters assessed. This study contributes to a better understanding of fluazuron in bovines, suggesting that the antiparasitic may affect bovine reproduction and might cause embryo loss.

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Zinc (Zn) plays essential roles in numerous cellular processes. However, there is limited understanding of Zn homeostasis within the bovine reproductive system. This study investigated the influence of estradiol (E2) and progesterone (P4) on Zn transporter expression and intracellular free Zn levels in bovine oviduct epithelial cells (BOEC). For this purpose, cells were harvested from slaughtered cows and cultured in vitro. Intracellular Zn concentrations were measured using FluoZin-3AM staining, while real-time polymerase chain reaction assessed Zn transporter gene expression and quantification. Overall, our results confirmed the gene expression of all the evaluated Zn transporters (ZIP6, ZIP8, ZIP14, ZnT3, ZnT7 and ZnT9), denoted and the active role of E2 and P4 in intracellular Zn regulation. Our findings suggest an interaction between Zn, E2 and P4.

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Veterinary drugs are potential environmental pollutants that interfere with male reproductive function. Infertility has increased, and it is known that environmental toxins contribute to declining sperm parameters. Amitraz {N,N-[(methylamino) dimeth-ylidyne] di-2,4-xylidine} (AMZ) is a formamidine pesticide widely used as an insecticide and an acaricide. The aim of this study was to evaluate the toxicity of AMZ in bovine sperm. Three experiments using frozen-thawed bovine semen incubated with AMZ for 2 h were carried out. Negative and solvent (dimethyl sulfoxide) controls were run simultaneously with treatments. In experiment 1, the AMZ concentrations used were 10, 15 and 25 µg AMZ/ml and the sperm parameters evaluated were viability, mitochondrial activity, acrosomal status, functional membrane integrity and apoptosis. In experiments 2 and 3, 25 µg AMZ/ml was used to evaluate fertilizing capacity, embryo development and blastocyst DNA damage. In experiment 1, 25 µg AMZ/ml decreased sperm viability (P = 0.01), reduced mitochondrial activity (P = 0.03) and induced apoptosis (P < 0.01). Also, 15 and 25 µg AMZ/ml affected functional membrane integrity (P < 0.01). In experiment 2, AMZ did not alter sperm-zona binding (P = 0.40) and pronucleus formation (P = 0.36). In experiment 3, 25 µg AMZ/ml decreased the rate of embryo development (P < 0.01) and increased apoptosis (P = 0.03). These results suggest that AMZ induced alterations in bovine sperm, probably affecting male fertility at concentrations that could be present in the environment.

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In vitro embryo production has grown in recent decades due to its great potential for cattle production. However, the quality of in vitro-produced embryos is lower compared with those produced in vivo. The postfertilization culture environment has a major influence on bovine embryo quality. We hypothesize that the inclusion of the inclusion of alpha-lipoic acid (ALA) in the in vitro culture (IVC) medium during the first 24 h would have positive effects on embryo development in vitro and cryotolerance. The aims of this study were to evaluate the antioxidant effect of ALA in IVC medium for 24 h on bovine zygotes (21 h post in vitro fertilization, IVF), day 2 cleaved embryos (46 h post-IVF), and to assess embryo quality, developmental competence, and cryotolerance after vitrification. In all experiments, IVC medium was the Control, and 2.5 µM ALA was the treatment implemented. Viability and reactive oxygen species (ROS) levels in zygotes and day 2 embryos did not differ from the Control (P > 0.05). Supplementation with ALA increased total blastocyst and hatching rates (P < 0.05). It also improved embryo quality, evidenced by the increased blastocyst total cell number and the percentage of excellent-quality embryos observed (P < 0.05). In embryos cultured with ALA and then vitrified, ALA reduced intracellular ROS levels in warmed blastocysts (P < 0.05). In conclusion, ALA supplementation to IVC medium during 24 h is a new advantage in improving embryo quality for assisted bovine reproduction.

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Trace minerals participate in reproductive processes and are crucial for oocyte maturation. The objective of the present study was to investigate the effect of combined supplementation with copper (Cu), manganese (Mn), selenium (Se) and zinc (Zn) during bovine in vitro maturation (IVM) on subsequent embryo development and quality. The IVM medium was supplemented as follows: a) Control (no mineral supplementation); b) MScz (6 ng/mL Mn + 100 ng/mL Se + 200 ng/mL Cu + 400 ng/mL Zn); c) MScZ (6 ng/mL Mn + 100 ng/mL Se + 200 ng/mL Cu + 1200 ng/mL Zn); d) MSCz (6 ng/mL Mn + 100 ng/mL Se + 600 ng/mL Cu + 400 ng/mL Zn). Supplementation with MScz and MSCz produced more blastocysts compared with the control. Total blastocyst cell number was higher when minerals were added at any combination. Day-8 blastocysts derived from oocytes treated with minerals had lower intracellular reactive oxygen species concentration and lipid content than the control. In conclusion, combined supplementation with Cu, Mn, Se and Zn during bovine oocyte IVM increased in vitro production performance, improving embryo developmental ability and quality.

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ABSTRACT Exogenous lipoid pneumonia (ELP) is an unusual disease, with an incidence of 1 to 2.5%, caused by aspiration and accumulation of exogenous lipids in the pulmonary alveoli. When the aspirated agent has an oily composition, a pulmonary inflammatory reaction called lipoid pneumonia is triggered, which over time can lead to irreversible parenchymal fibrosis. Sometimes it can appear as nodular lesions that require a broad differential diagnosis, in which case imaging studies have a fundamental role. Histopathological confirmation is required for definitive diagnosis1. We present a clinical case of lipoid pneumonia which appeared as a lung mass accompanied by bilateral pulmonary infiltrates.

RESUMEN La neumonía lipoidea exógena (NLE) es una patología inusual, que tiene una incidencia del 1% al 2,5%, y tiene su causa en la aspiración y la acumulación de lípidos de origen exógeno dentro de los alvéolos pulmonares. Cuando el agente aspirado es de composición oleosa, se desencadena una reacción inflamatoria pulmonar denominada neumonía lipoidea, que con el tiempo puede conducir a una fibrosis parenquimatosa irreversible. En ocasiones, se puede presentar en forma de lesiones nodulares, que obligan a un amplio diagnóstico diferencial, en el que desempeñan un papel fundamental los estudios por imágenes. Para el diagnóstico definitivo, se requiere confirmación histopatológica1. Presentamos un caso clínico de neumonía lipoidea que se presentó como masa pulmonar acompañada de infiltrados pulmonares bilaterales.

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La neumonía lipoidea exógena (NLE) es una patología inusual, que tiene una incidencia del 1% al 2,5%, y tiene su causa en la aspiración y la acumulación de lípidos de origen exógeno dentro de los alvéolos pulmonares. Cuando el agente aspirado es de composición oleosa, se desencadena una reacción inflamatoria pulmonar denominada neumonía lipoidea, que con el tiempo puede conducir a una fibrosis parenquimatosa irreversible. En ocasiones, se puede presentar en forma de lesiones nodulares, que obligan a un amplio diagnóstico diferencial, en el que desempeñan un papel fundamental los estudios por imágenes. Para el diagnóstico definitivo, se requiere confirmación histopatológica1. Presentamos un caso clínico de neumonía lipoidea que se presentó como masa pulmonar acompañada de infiltrados pulmonares bilaterales.

Exogenous lipoid pneumonia (ELP) is an unusual disease, with an incidence of 1 to 2.5%, caused by aspiration and accumulation of exogenous lipids in the pulmonary alveoli. When the aspirated agent has an oily composition, a pulmonary inflammatory reaction called lipoid pneumonia is triggered, which over time can lead to irreversible parenchymal fibrosis. Sometimes it can appear as nodular lesions that require a broad differential diagnosis, in which case imaging studies have a fundamental role. Histo pathological confirmation is required for definitive diagnosis1. We present a clinical case of lipoid pneumonia which appeared as a lung mass ac companied by bilateral pulmonary infiltrates.

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The aim of this study was to evaluate the association between plasma copper (Cu) concentration and ovarian function during a fixed-time artificial insemination (FTAI) protocol and the effect of parenteral Cu administration (100 mg) at the start of such protocol (day 0) on area of preovulatory follicle (APF); area of corpus luteum (ACL), plasma estradiol (E2), and progesterone (P4) concentrations; CL blood flow (CLBF); and pregnancy rate in beef heifers and cows. In cows, plasma Cu concentration on days 0 and 7 correlated positively with APF. Copper administration increased plasma Cu concentration and decreased APF and plasma E2 concentration (day 9), without modifying ACL, plasma P4 concentration, and CLBF (day 16) in cows. Pregnancy rate was higher in Cu-supplemented cattle on day 41 after FTAI as compared with controls (58.76 and 45.28%, respectively). In conclusion, Cu administration at the beginning of the FTAI protocol increased pregnancy rate in beef heifers and cows, modifying APF and plasma E2 concentration in the latter.

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Ghrelin is a gut hormone related to energy balance and reproductive functions. The aim of this study was to evaluate the effect of ghrelin antagonist D-Lys3-GHRP-6 (GA) as a potential agent that prevents ghrelin effects during bovine oocyte maturation on progesterone production, cumulus cell (CC) viability, CC DNA damage and embryo development and hatching rates. Ghrelin's potential to induce oxidative stress in cumulus-oocyte complexes (COC) was also evaluated. COCs were cultured for 24 hr in medium without supplementation (C) or supplemented with 60 pM ghrelin (Ghrelin60), Ghrelin60 + 20 pM GA (GA20), Ghrelin60 + 60 pM GA (GA60) or Ghrelin60 + 100 pM GA (GA100) for experiment I. For experiment II, C and Ghrelin60 treatments were used. Differences between C and Ghrelin60 and the linear or quadratic association between GAs on Ghrelin60 were evaluated. Results demonstrated that Ghrelin60 increased progesterone concentration, reduced CC viability, induced CC DNA damage and decreased blastocyst and hatching rate compared with C (p < .05). GA20, GA60 and GA100 had a linear effect on CC genetic damage index (p ≤ .05) and a quadratic effect on CC viability (p < .01). GA20 counteracted the low hatching rate produced by Ghrelin60. However, GAs did not counteract progesterone concentration and blastocyst rate (p ≥ .21). GRH60 did not differ from C in the oxidative status (p ≥ .19). Our study highlights that GA could prevent the negative effects of ghrelin during bovine IVM.

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The aim of this study was to evaluate the genotoxic and cytotoxic effects of amitraz (AMZ) on the primary culture of bovine cumulus cells (CC) and oocyte nuclear maturation. Cytotoxicity was evaluated by assessing mitochondrial activity with the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Genotoxicity was estimated using the alkaline single cell gel electrophoresis (SCGE) assay. Apoptosis was detected with the Annexin V-affinity assay. The in vitro maturation test was performed in bovine oocytes. To understand AMZ action, glutathione content, superoxide dismutase enzyme activity, and lipid peroxidation were evaluated in CC. Results showed that AMZ lethal concentration (LC 5024h) for bovine CC was 32.55 µg/mL (MTT assay). A 25 µg/mL induced late apoptosis and necrotic cells (p < 0.05); however, DNA damage was decreased at the same concentration (SCGE assay; p < 0.05). A decrease in metaphase II was observed at 25 µg/mL, and degenerate oocytes were observed at 15 and 25 µg/mL (p < 0.05). None of the oxidative stress parameters evaluated showed significant differences. This study contributes to a better understanding of AMZ in this model, suggesting its potential cytotoxicity and impact on bovine reproduction.

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In many species, alpha-lipoic acid (ALA) is essential for embryo development. There, therefore, was investigation of effects of ALA supplementation to culture media for in vitro development of cattle embryos. In Experiment I, there were assessments of embryo production and oxidative status of cattle embryos derived by in vitro maturation and fertilization (IVM/IVF)that were cultured until the blastocyst stage of development using different ALA concentrations (5, 25 and 100 µM), fetal bovine serum (FBS) and amino acids (aa) as well as 20 % oxygen (O2) in the culture atmosphere. In Experiment II, embryos were cultured without FBS, at different ALA concentrations (2.5, 5 and 7.5 µM) and in the presence or absence of aa when there was a 7 % O2 atmosphere. Embryo development rates and blastocyst quality were evaluated. With 20 % O2 concentration, treatment with 100 µM ALA resulted in lesser hatching rates and development to the blastocyst stage (P < 0.01), while with supplementation with 5 µM ALA there were lesser (P = 0.04) glutathione concentrations and greater protein contents of embryos (P < 0.01). Culturing in the 7 % O2 atmosphere, combined with supplementation with 2.5 µM ALA with FBS and aa resulted in a greater blastocyst cell number (P = 0.03) and lesser hatching rates (P = 0.04). Taken together, results indicate supplementation with the greater ALA concentrations resulted in impairment of embryo development, regardless of the O2 concentration imposed during the culture period, while the relatively lesser supplementation-concentrations with ALA led to improvements in embryo quality.

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Selenium (Se) is an essential trace element with important functions in animals and whose deficiency is associated with reproductive failures. The aim of this study was to investigate the effect of Se concentrations during in vitro maturation (IVM) of Bos taurus oocyte within the reference ranges for Se status in cattle. For this purpose, Aberdeen Angus cumulus-oocyte complexes (COCs) were matured in IVM medium supplemented with 0, 10, 50, and 100 ng/mL Se (control, deficient, marginal, and adequate, respectively). The results demonstrated that marginal and adequate Se concentrations added during IVM increased viability and non-apoptotic cumulus cells (CC). Moreover, the addition of Se to culture media decreased malondialdehyde level in COC with all studied concentrations and increased total glutathione content in CC and oocytes with 10 ng/mL Se. On the other hand, total antioxidant capacity of COC, nuclear maturation, and the developmental capacity of oocytes were not modified by Se supplementation. However, 10 ng/mL Se increased hatching rate. In conclusion, supplementation with 10 ng/mL Se during in vitro maturation of Bos primigenius taurus oocytes should be considered to improve embryo quality.

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The eicosapentaenoic acid (EPA) is an n-3 polyunsaturated fatty acid (PUFA) present in the lipid composition of bovine oocytes. Little is known about the importance of EPA in bovine oocyte maturation and embryo development in vitro. Although previous work suggest that n-3 PUFAs may inhibit oocyte maturation, the available data are inconsistent. In this study, we evaluated the effect of EPA (1, 10, 100 nM) during in vitro maturation (IVM) of bovine oocytes, alone and in combination with vitamin E (VE) or cysteamine (CYS). EPA treatment in IVM decreased oocyte lipid content and affected lipid droplets pattern (P < 0.05). EPA 100 nM reduced oocytes maturation rate (P < 0.05), without affecting cumulus expansion. At the concentrations tested, EPA did not modify embryo development. However, the addition of antioxidants during IVM reduced the levels of reactive oxygen species in the culture system by increasing intracellular glutathione content (P < 0.05). Besides, the combination of EPA with VE or CYS reduced the percentages of MI oocytes after 24 h of IVM (P < 0.05). EPA reduced oocyte lipid content without any detrimental for embryo development.

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We have developed a new method for the measurement of subcutaneous tumour volume which consists in taking photographs of mice in their home cages, to refine the standard method of measurement with calipers. We consider this new method to be non-aversive, as it may be more compatible with mice behavioural preferences and, therefore, improve their welfare. Photographs are captured when mice voluntarily go into an acrylic tube containing graph paper that is later used as a scale. Tumour volumes measured with the caliper and the non-aversive photographic method were compared to those obtained by water displacement volume and weight. Behavioural and physiological changes were evaluated to assess animal welfare. Significant differences were found between measurements obtained with the caliper and the non-aversive photographic method, v. the reference volume acquired by water displacement (P < 0.001). Nevertheless, there was good consistency for these measurements when tumours were measured repeatedly, with all Intra-Class Correlation Coefficients above 0.95. Mice on which the non-aversive photographic method was employed were significantly less reluctant to establish contact with the experimenter (P < 0.001) and behaved less anxiously in a modified-Novelty Suppressed Feeding test. Particularly, statistically significant differences were found in connection with the latency to eat an almond piece (P < 0.05), the frequency of grooming (P < 0.001) and the frequency of defecation (P < 0.001). Corticosterone concentration in faeces and blood glucose were determined and no significant changes were found. Therefore, we propose the non-aversive photographic method to measure subcutaneous tumours as a way to refine methodologies in the field of experimental oncology.

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The objectives of this study were to evaluate the effect of feeding an enriched diet with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) to finishing lambs born from ewes supplemented either with or without EPA and DHA during late gestation on productive performance, muscle fatty acid (FA), and hypothalamus mRNA concentration of metabolic genes and hormone receptors. Lambs born from dams fed during the last 50 d of gestation either with a control diet containing 0.39% Ca salts of palmitic fatty acid distillate (C) or Ca salts enriched with EPA and DHA (PFA) were used. After weaning lambs (n = 70) were blocked by weight (BW) and used in a 2 × 2 factorial into 2 finishing diets containing 1.5% of C or PFA. The 2 factors were the ewe diet and the finishing diet. Lambs (37.9 ± 0.4 kg) were weighed and blood sampled for glucose and NEFA measurements at days 1, 14, 28, and 42. Dry matter intake (DMI) was measured daily. At day 43, 14 females and 14 males were slaughtered, and hot carcass weight, body wall thickness, rib eye area, and FA composition of Longissumus thoracis muscle were evaluated. Female hypothalamuses were obtained and mRNA concentration of hormone receptors, neuropeptides, and their receptors was measured. Lambs born from PFA dams were heavier (P < 0.01). There was a time × finishing diet interaction for BW (P = 0.03), and lambs fed C had a greater BW. Lambs fed C had an increase in DMI (P < 0.01). There were no significant differences in plasma glucose and NEFA concentration (P > 0.1). Lambs born from PFA dams had a greater concentration of C22:0 (P < 0.03). Lambs fed C had higher concentrations of C18:1c15 (P < 0.01), C17:0 (P < 0.09), C18:0 (P < 0.09), and n6/n3 (P < 0.01). Lambs fed PFA had greater concentration (P < 0.05) of C16:1, C22:1, C20:5, C22:5, C22:6, total n3 FA, and total EPA and DHA. There was a significant dam × finishing diet interaction (P ≤ 0.08) on mRNA concentration for MCR3, CCK-R, Cort-R, and CART. Lambs, which had the same treatment as their dams, showed lower overall mRNA concentration than those with different treatments between them and their dams. Lambs born from PFA ewes had lower concentration of MCR4 mRNA (P = 0.09) than C. Agouti-related peptides mRNA concentration was lower in lambs fed PFA (P = 0.06) than C. In conclusion, changes on lamb performance, muscle fatty acid composition, and metabolic neuropeptides depend not only on the lamb diet, but also on the dam diet during late gestation.

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Gastric varices occur in one-third of patients with portal hypertension. Bleeding from gastric varices remains a significant cause of death. Currently the first-line of treatment for gastric varices is endoscopic obliteration with N-butyl-2-cyanoacrylate. Though relatively safe, this option has several well-known complications. We report the case of a 61-year-old male patient with cryptogenic cirrhosis, who presented with fever, tachycardia and hypoxemia after endoscopic obliteration with N-butyl-2-cyanoacrylate. Radiographic findings were consistent with pulmonary embolism of the sclerosing substance. The aim of this case report is to emphasize the clinical and radiological findings of this complication in order to distinguish it from other similar medical conditions and prevent a delay in diagnosis.

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Las várices gástricas están presentes en cerca de un tercio de los pacientes con hipertensión portal y el sangrado de las mismas representa una causa significativa de mortalidad. El tratamiento de primera línea es la obturación con N-butil-2-cianoacrilato, que si bien es seguro no está libre de complicaciones. Presentamos el caso de un paciente de 61 años de edad con antecedente de cirrosis criptogénica que se presentó a la consulta febril, taquicárdico e hipoxémico después de la obturación endoscópica con N-butil-2-cianoacrilato. Las imágenes mostraron embolia pulmonar bilateral del material obturante. El objetivo de este trabajo es destacar las manifestaciones clínicas y las imágenes de esta situación para ayudar a su diagnóstico precoz y diferenciarla de otras entidades que requieren un tratamiento específico.

Gastric varices occur in one-third of patients with portal hypertension. Bleeding from gastric varices remains a significant cause of death. Currently the first-line of treatment for gastric varices is endoscopic obliteration with N-butyl-2-cyanoacrylate. Though relatively safe, this option has several well-known complications. We report the case of a 61-year-old male patient with cryptogenic cirrhosis, who presented with fever, tachycardia and hypoxemia after endoscopic obliteration with N-butyl-2-cyanoacrylate. Radiographic findings were consistent with pulmonary embolism of the sclerosing substance. The aim of this case report is to emphasize the clinical and radiological findings of this complication in order to distinguish it from other similar medical conditions and prevent a delay in diagnosis.

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