Background:Heat shock during sperm capacitation affects the spermatozoa quality, resulting in increased early acrosome reaction and consequently decreasing their fertilizing capacity. Although the mechanisms involved in the regulation of sperm capacitation and acrosome reaction are not fully understood, it has been reported that Ca2+/calmodulin-dependent protein kinase II (CaMKII) is an important regulator of these processes. Thus, the present aimed to evaluate the effect of heat shock in the CaMKII signaling during the bovine sperm capacitation and acrosome. Methods:Bovine spermatozoa were in vitro capacitated for 4 hours. The acrosome reaction was induced by exposure to heparin and calcium ionophore A23187 for 1 hour. Heat shock was applied by incubating spermatozoa at 41 °C with 7% CO2, while the control group was maintained at 38.5 °C with 5% CO2. At the end of each treatment, the localization of total CaMKII and phosphorylated CaMKII (pCaMKII), as well as acrosomal membrane integrity, were evaluated by immunofluorescence. Results:It was observed that CaMKII and not phosphorylated CaMKII (pCaMKII) localization at the acrosome region was affected by sperm capacitation. In contrast, the localization of both, CaMKII and its phosphorylated form was affected by the acrosome reaction (p < 0.05). The acrosome membrane integrity, as well as the pCamKII localization in bovine spermatozoa, was affected by incubation time. This effect of incubation time was stronger in heated shock sperm, although it was observed only after 2 h of incubation. Heat shock also affected the acrosomal localization of pCaMKII in the acrosomal region of spermatozoa with intact acrosome. Discussion:Taken together, the data present here show that CaMKII and pCaMKII localization is dynamic during bovine sperm capacitation and acrosome reaction and that this pattern of localization is affected by heat shock, suggesting that failure in CaMKII signaling is probably involved in the early acrosome reaction observed in heated-shock spermatozoa.

Sperm function is susceptible to adverse environmental conditions. It has been demonstrated that in vivo and in vitro exposure of bovine sperm to elevated temperature reduces sperm motility and fertilizing potential. However, the cascade of functional, cellular, and molecular events triggered by elevated temperature in the mature sperm cell remains not fully understood. Therefore, the aim of this study was to determine the effect of heat shock on mature sperm cells. Frozen-thawed Holstein sperm were evaluated immediately after Percoll purification (0 h non-incubation control) or after incubation at 35, 38.5, and 41 degrees C for 4 h. Heat shock reduced sperm motility after 3-4 h at 41 degrees C while mitochondrial activity was reduced by 38.5 and 41 degrees C when compared to the control. Heat shock also increased sperm reactive oxygen species production and caspase activity. Heat-shocked sperm had lower fertilizing ability, which led to diminished cleavage and blastocyst rates. Preimplantation embryo developmental kinetics was also slowed and reduced by sperm heat shock. The microRNA (miR) profiling identified > 300 miRs in bovine sperm. Among these, three and seven miRs were exclusively identified in sperm cells exposed to 35 and 41 degrees C, respectively. Moreover, miR-181d was enriched in sperm cells exposed to higher temperatures. Hence, elevated temperature altered the physiology of mature sperm cells by perturbing cellular processes and the miR profile, which collectively led to lower fertilizing ability and preimplantation development.

European Journal of Clinical InvestigationVolume 52, Issue 3 e13749 EDITORIAL Bioenergetic remodeling in the pathophysiology and treatment of nonalcoholic fatty liver disease Anabela Marisa Azul, Anabela Marisa Azul orcid.org/0000-0003-3295-1284 CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, PortugalSearch for more papers by this authorMartin Winter, Martin Winter Micro-Biolytics GmbH, Esslingen am Neckar, Germany Lab Automation Network, Tübingen, GermanySearch for more papers by this authorDaniel Silva, Daniel Silva CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, PortugalSearch for more papers by this authorLiljana Georgievska, Liljana Georgievska orcid.org/0000-0002-7023-6356 CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, PortugalSearch for more papers by this authorPaulo J. Oliveira, Corresponding Author Paulo J. Oliveira pauloliv@cnc.uc.pt orcid.org/0000-0002-5201-9948 CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, Portugal Correspondence Paulo J. Oliveira, CNC—Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Lot 8A, Biocant Park, 3060-197 Cantanhede, Portugal. Email: pauloliv@cnc.uc.ptSearch for more papers by this author Anabela Marisa Azul, Anabela Marisa Azul orcid.org/0000-0003-3295-1284 CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, PortugalSearch for more papers by this authorMartin Winter, Martin Winter Micro-Biolytics GmbH, Esslingen am Neckar, Germany Lab Automation Network, Tübingen, GermanySearch for more papers by this authorDaniel Silva, Daniel Silva CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, PortugalSearch for more papers by this authorLiljana Georgievska, Liljana Georgievska orcid.org/0000-0002-7023-6356 CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, PortugalSearch for more papers by this authorPaulo J. Oliveira, Corresponding Author Paulo J. Oliveira pauloliv@cnc.uc.pt orcid.org/0000-0002-5201-9948 CNC-Center for Neuroscience and Cell Biology, Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, UC Biotech, Cantanhede, Portugal Correspondence Paulo J. Oliveira, CNC—Center for Neuroscience and Cell Biology, University of Coimbra, UC Biotech Building, Lot 8A, Biocant Park, 3060-197 Cantanhede, Portugal. Email: pauloliv@cnc.uc.ptSearch for more papers by this author First published: 13 February 2022 https://doi.org/10.1111/eci.13749Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Volume52, Issue3Special Issue: FOIE GRAS ‐ Bioenergetic Remodeling in the Pathophysiology and Treatment of Non‐Alcoholic Fatty Liver DiseaseMarch 2022e13749 RelatedInformation

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing neurodegenerative disease that affects motor neurons. This disease is associated with oxidative stress especially in mutant superoxide dismutase 1 (mutSOD1) patients. However, less is known for the most prevalent sporadic ALS due to a lack of disease models. Here, we studied oxidative the stress profiles in lymphoblasts from ALS patients with mutSOD1 or unknown (undSOD1) mutations.mutSOD1 and undSOD1 lymphoblasts, as well as sex/age-matched controls (3/group) were obtained from Coriell and divided in 46 years-old-men (C1), 46 years-old-women (C2) or 26/27 years-old-men (C3) cohorts. Growth curves were performed, and several parameters associated with redox homeostasis were evaluated, including SOD activity and expression, general oxidative stress levels, lipid peroxidation, response to oxidative stimulus, glutathione redox cycle, catalase expression, and activity, and Nrf2 transcripts. Pooled (all cohorts) and paired (intra-cohort) statistical analyses were performed, followed by clustering and principal component analyses (PCA).Although a high heterogeneity among lymphoblast redox profiles was found between cohorts, clustering analysis based on 7 parameters with high chi-square ranking (total SOD activity, oxidative stress levels, catalase transcripts, SOD1 protein levels, metabolic response to mM concentrations of tert-butyl hydroperoxide, glutathione reductase activity, and Nrf2 transcript levels) provided a perfect separation between samples from healthy controls and ALS (undSOD1 and mutSOD1), also visualized in the PCA analysis.Our results show distinct redox signatures in lymphoblasts from mutSOD1, undSOD1, and healthy controls that can be used as therapeutic targets for ALS drug development.Highlights ### Competing Interest StatementThe authors have declared no competing interest.* ALS : Amyotrophic Lateral Sclerosis ARE : Antioxidant Response Element BCA : Bicinchoninic Acid CSF : Cerebrospinal Fluid DTT : Dithiothreitol ECL : Enhanced Chemiluminescence reagent EDTA : Ethylenediaminetetraacetic Acid EGTA : Ethylene Glycol-bis (2-aminoethylether) N, N,’N, ’N-tetraacetic Acid fALS : Familial ALS GPx : Glutathione Peroxidase GR : Glutathione Reductase GSH : Reduced Glutathione GSSG : Oxidized Glutathione H2DCFDA : 2’,7’-Dichlorodihydrofluorescein Diacetate HPLC : High-Performance Liquid Chromatograph HRP : Horseradish Peroxidase MDA : Malondialdehyde mutSOD1 : Mutant SOD1 NAM : Nicotinamide Nrf2 : Nuclear Factor Erythroid-2-related Factor PBMCs : Peripheral Blood Mononuclear Cells PBS : Phosphate-Buffered Saline PCA : Principal Component Analysis PMSF : Phenylmethylsulfonyl Fluoride PVDF : Polyvinylidene Difluoride sALS : Sporadic ALS SDS : Sodium Dodecyl Sulfate SOD1 : Superoxide Dismutase 1 Ta : Annealing Temperature TBA : Thiobarbituric Acid t-BHP : tert-Butyl Hydroperoxide undSOD1 : Undetermined SOD1 Mutations vit E : Vitamin E

The exploration and beneficiation of mineral coal involve managing acid mine drainage (AMD), the acidic and metallic ion composition of which requires adequate treatment for disposal. In the present work, a passive pilot-scale AMD treatment system was developed using open channels of calcitic (CL-I and CL-II) and dolomitic (DL-I and DL-II) limestone beds and mixtures with natural zeolites (NZ) and functionalized zeolites (FZ). Investigated parameters include pH, electrical conductivity, total acidity, total alkalinity, and concentrations of aluminum, iron and manganese ions. DL-I, CL-II and mixtures of CL-II/NZ and CL-II/FZ increased pH levels from 3.3 to 7.9, 8.2, 7.9 and 7.6, respectively; increased total alkalinity levels from 0 mgCaCO3.L-1 to 20, 107, 42 and 34 mgCaCO3.L-1, respectively; and reduced total acidity levels by 95, 91, 90 and 90%, respectively. All beds promoted the removal of aluminum, iron and manganese ions, but the CL-II/FZ mixture was the most efficient due to both neutralization and a higher uptake of manganese ions (roughly 99%), which are pollutants that are typically difficult to remove. The study results reveal ways to transform passive treatment systems using limestone beds and unconventional materials such as zeolites, combine neutralization and adsorption mechanisms in the same operation, ensure a simple maintenance and operational system and improve the economic and environmental sustainability of related processes.

Alzheimer’s disease (AD) is the most common form of dementia worldwide, being characterized by the deposition of senile plaques, neurofibrillary tangles (enriched in the amyloid beta (Aβ) peptide and hyperphosphorylated tau (p-tau), respectively) and memory loss. Aging, type 2 diabetes (T2D) and female sex (especially after menopause) are risk factors for AD, but their crosslinking mechanisms remain unclear. Most clinical trials targeting AD neuropathology failed and it remains incurable. However, evidence suggests that effective anti-T2D drugs, such as the GLP-1 mimetic and neuroprotector liraglutide, can be also efficient against AD. Thus, we aimed to study the benefits of a peripheral liraglutide treatment in AD female mice. We used blood and brain cortical lysates from 10-month-old 3xTg-AD female mice, treated for 28 days with liraglutide (0.2 mg/kg, once/day) to evaluate parameters affected in AD (e.g., Aβ and p-tau, motor and cognitive function, glucose metabolism, inflammation and oxidative/nitrosative stress). Despite the limited signs of cognitive changes in mature female mice, liraglutide only reduced their cortical Aβ1–42 levels. Liraglutide partially attenuated brain estradiol and GLP-1 and activated PKA levels, oxidative/nitrosative stress and inflammation in these AD female mice. Our results support the earlier use of liraglutide as a potential preventive/therapeutic agent against the accumulation of the first neuropathological features of AD in females.

Nitric oxide (center dot NO) is a reactive nitrogen species (RSN) that, over the years, has been shown to be integrated with biological and physiological events, including reproductive processes. center dot NO can affect the functionality of spermatozoa through free radical scavenging, deactivating and inhibiting the production of superoxide anions (O-2(center dot-)). However, the role of center dot NO in mammalian spermatozoa physiology seems paradoxical. The aim of this study was to investigate the effects of center dot NO on motility, hyperactivation, membrane integrity, peroxidation, and capacitation in cryopreserved equine sperm. Ejaculates were collected and cryopreserved. After thawing, samples were centrifuged, suspended in an in vitro fertilization (IVF) medium and incubated with the following treatments: 1) C = control (IVF); 2) A = L-arginine (10 mM - In); 3) L = L-NAME (1 mM 1h); 4) M = methylene blue (100 mM- Re); 5) AL = L-arginine + L-NAME (In + Ih); 6) AM = L-arginine + methylene blue (In + Re). The samples were evaluated for spermatic kinetics by CASA and other analyses [plasma and acrosomal membranes used the propidium iodide (PI) and Pisum sativum agglutinin (PSA), detection of tyrosine residues phosphorylation in the membrane (F0426), nitric oxide (DAF-2/DA), lipid peroxidation (C11-BODIPY581/591)] by flow cytometry. The L-arginine treatments reduced MOT, PROG, RAP and LIN only at time 0 min compared to the control and L-NAME. These treatments (MT and MP, VAP, VSL, LIN, RAP) also reduced the sperm movement characteristics but only at the beginning of the incubation period. After this period of incubation, motility recovered. center dot NO removal by methylene blue almost completely inhibited sperm motility, but these treatments had the highest percentages of intact membranes. L-arginine treatments improved acrosome reactions and differed from M and AM. center dot NO production, tyrosine phosphorylation and lipid peroxidation did not differ among treatments, except for M and AM, where a reduction in these variables was detected. Therefore, equine sperm capacitation and the acrosome reaction are part of an oxidative process that involves the participation of ROS, and center dot NO plays an important role in the maintenance and regulation of motility, hyperactivation, induction of acrosome reaction and possibly in capacitation, which are indispensable processes for the fertility of equine sperm.

Follicular development and deviation processes during the postpartum period are not completely known in horses. Thus, we aimed to study the characteristics of follicular dynamics and ovarian vascular perfusion during the postpartum period in mares that demonstrated estrous behavior and had early (<10 days) or late (≥10 days) postpartum ovulation. Ten mares were scanned daily by transrectal ultrasonography from the first day postpartum (d1) to the sixteenth day after the first postpartum ovulation (D0 = ovulation). The animals were split in the early (n = 3) and late (n = 7) ovulation groups (average interval between parturition and ovulation: 8.0 ± 0.0 and 14.7 ± 1.2 days, respectively). For the follicular growth, no difference (P > 0.1) was detected between the groups when the data were normalized for the days preceding the first postpartum ovulation (from D-7 to D-1). However, when the data were normalized to days postpartum, the dominant follicle was larger (P < 0.05) in the early-ovulated group in all days during this period (d1 to d7). The number of follicles >25 mm diameter was greater (P < 0.05) in the early-ovulated group during the first 3 days postpartum, and the late-ovulated mares showed greater number of follicles with 20-25 mm during d4 to d7. For blood flow characteristics, no difference (P > 0.1) was detected between groups in vascular perfusion of the dominant follicle or in the ovarian pedicle ipsilateral to the largest follicle. Similarly, no difference (P > 0.1) was detected in progesterone concentrations, corpus luteum (CL) area and vascular perfusion of the CL. Pregnancy rate did not differ (P > 0.1) between the early (3/3; 100%) and late (5/7; 71.4%) groups. Therefore, the characteristics of the follicle growth on the preceding days of ovulation were similar between the early- and late-ovulated mares and were consistent with the follicular dynamics expected in non-pregnant and non-lactating mares. However, when the data were analyzed for the days relative to parturition, greater follicle development was present in mares that ovulate earlier during the postpartum period (<10 days). The results suggest that important events may occur before parturition, resulting in early follicle development, mainly in those mares that show estrus and ovulate within 10 days postpartum.

In horses, limited data is found regarding the vascular events during uterine involution at the puerperal period. Thus, we aimed to evaluate the morphological aspects (size of uterus and intrauterine fluid content) and the hemodynamics (endometrial and mesometrial vascular perfusion) of the uterus during its postpartum involution process. Ten mares were daily scanned by transrectal color Doppler ultrasonography from the first day postpartum (d1) to the 16th day after first postpartum ovulation (D0 = ovulation). The formerly gravid horn (GH) and formerly nongravid horn (NH) were individually evaluated. A reduction (P < .05) in the uterine diameter was observed during the first 7 days postpartum, and the rate of uterine involution decreased after this period. The involution was completed on d21 and d24 for the NH and GH, respectively. Presence of intrauterine fluid was present in large amounts between d1 and d2 postpartum, followed by a decrease (P < .05) between d4 and d7. No fluid was observed after d16 postpartum or after the third day postovulation (D3). During the early postpartum period, an increase (P < .05) in the endometrial and mesometrial vascularization was detected, respectively, between d1 and d4, and between d1 and d2. The vascular perfusion did not differ after d4 for endometrial tissue, whereas was reduced (P < .05) between d2 and d10 for mesometrium. After the first postpartum ovulation, an increase (P < .05) in vascular perfusion was observed from D0 to D5, followed by a decrease (P < .05) between D5 and D11 and an increase (P < .05) between D11 and D14. The novel vascular perfusion profile here described in the endometrium and mesometrium after ovulation is similar to the uterine vascular profile observed during estrous cycles and early pregnancy, indicating a fast return of the mare's uterus to cycling postpartum conditions.

O colesterol desempenha papeis importantes em varias funcoes espermaticas, incluindo efeitos sobre as propriedades da membrana. Um desses efeitos e o de estabilizar as membranas a baixas temperaturas. O objetivo do presente estudo foi avaliar o efeito da associacao do colesterol (Ciclodextrinas Carregadas com Colesterol - CLC) e das Lipoproteinas de Baixa Densidade (LDL) e das possiveis interacoes entre estes agentes crioprotetores e o tempo de equilibrio, durante o processo de criopreservacao, sobre a motilidade espermatica, integridade, estabilidade e na peroxidacao lipidica das membranas espermaticas pos-descongelamento. Foram utilizadas amostras de semen de 15 touros Nelore, andrologicamente normais, com idades de 2 a 6 anos, coletadas por vagina artificial e avaliadas segundo as normas do CBRA (1998). As amostras foram divididas em 4 aliquotas e diluidas a 32oC (30 x 106 sptz/mL) com os seguintes diluidores: 1) Tris-Gema (controle); 2) LDL (8%); 3) LDL + CLC (8% LDL + CLC); 4) L-CLC (0,8% LDL + CLC). O semen diluido foi resfriado ate a temperatura ambiente (25oC) e envasado em palhetas de 0,5 mL. Para a criopreservacao utilizaram-se tres aparelhos automatizados (TK-3000®), com as mesmas curvas de resfriamento (-0,25°C/min) e de congelamento (-20°C/min) para todos os tratamentos, variando apenas o tempo de equilibrio a 5oC: 0h (T0), 4h (T4), e 6h (T6) totalizando doze tratamentos. Apos o congelamento, as palhetas foram armazenadas em N2 liquido (-196 oC). As avaliacoes do semen apos descongelamento consistiram na analise computadorizada da motilidade espermatica (CASA) e nas analises pela citometria de fluxo quanto a integridade das membranas plasmatica e acrossomal (PI/FITCPSA/H33342); peroxidacao lipidica (PI/C11-BODIPY581/591/H33342) e capacitacao espermatica atraves da estabilidade da membrana plasmatica (Merocianina 540/Yo-Pro1/H33342). Os tratamentos sem tempo de equilibrio (T0) apresentaram os menores valores, tanto para as motilidades total (MOT) e progressiva (PROG), quanto para a integridade das membranas plasmatica e acrossomal (AIMPI), para todos os diluidores. O diluidor LDL (8%) apresentou a melhor atividade crioprotetora em todos os tempos, com maiores motilidade total e progressiva, melhores caracteristicas do movimento espermatico (alto VAP, VSL e VCL), maior porcentagem de celulas rapidas (Rapid), a maior integridade das membranas plasmatica e acrossomal, e a maior proporcao de celulas viaveis nao capacitadas (Meroc (-)). O diluidor a base de gema apresentou a melhor protecao contra a peroxidacao lipidica das membranas espermaticas. O diluidor L-CLC apresentou a pior atividade crioprotetora. A adicao de colesterol atraves do pre-tratamento do semen com CLC nao permitiu uma reducao do tempo de equilibrio. Alem disso, a adicao de CLC a diluidores a base de LDL purificado a 8%, reduziu a sobrevivencia espermatica em T0 e T6. O tempo de 4 h de equilibrio foi adequado para se obter uma alta sobrevivencia espermatica, independente do diluidor utilizado, porem, os diluidores Gema e LDL se beneficiaram de um maior tempo de equilibrio (6h). O melhor metodo de criopreservacao foi aquele que utilizou o diluidor LDL (8%) e 6h de equilibrio (LDL-T6). Concluiu-se que o tempo de equilibrio e necessario no processo de criopreservacao do semen bovino para preservacao da motilidade e da integridade das membranas espermaticas, independente do diluidor utilizado. Alem disso, conclui-se que a CLC sozinha ou associada a baixas concentracoes de LDL nao protegem adequadamente as membranas espermaticas dos efeitos lesivos da criopreservacao.

Bull heat stress increases scrotal and testicular temperature, reduces sperm quality, motility and fertilization ability. Sperm changes in motility are directly associated with organization of cytoskeletal proteins. Therefore, the objective of this study was to determine the effect of heat shock on bovine sperm motility and cytoskeleton organization. A pool of three Bos indicus semen straws was used for each replicate (N = 6 replicates). Semen straws were purified on Percoll gradient. Sperm sample (1x106 spermatozoa/ml) was subjected to motility evaluation and immunofluorescence for localization of microtubules and the molecular motor dynein immediately after Percoll gradient (0 h Control) and after SP-TALP incubation at Control (38.5°C) and Heat Shock (41°C) for 4 hours. Sperm motility was assessed every hour during the incubation period. Samples of the 0 hour control and after 4 hours incubation were processed with anti-α-tubulin mouse monoclonal IgG, anti-cytoplasmic dynein rabbit polyclonal IgG, Alexa Fluor 488® goat mouse IgG, Alexa Fluor 555® goat mouse IgG and Hoechst 33342 for sperm tubulin, dynein and DNA localization. Tubulin (N= 600 sperm/treatment) and dynein (N = 300 sperm/treatment) morphological localization and pixel fluorescence intensity was determined with Image J software version 1,49j. Non-parametric data were analyzed using the Wilcoxon test of the statistical package SAS. Zero hour control (not incubated) sperm motility was higher than all the other groups (73.12 ± 3.6%, P < 0.05). Sperm incubation at 38.5°C affected (P < 0.05) sperm motility at different incubation times (68.12 ± 3.6%, 53.75 ± 3.6%, 41.25 ± 3.6% and 27.12 ± 3.6% for 1, 2, 3 and 4 hours, respectively). Similarly, incubation of spermatozoa at 41°C (P < 0.05) reduced sperm motility over time (52.5 ± 3.6%, 43.75 ± 3.6%, 20.62 ± 3.6 % and 3.62 ± 3 6% for 1, 2, 3 and 4 hours, respectively). Heat shock of 41°C reduced (P < 0.05) sperm motility as compared to control 38.5°C at all incubation times. Immunofluorescence indicated that tubulin was localized at the sperm tail insertion and along the tail. Tubulin pixel fluorescence intensity increased (P < 0.01) with incubation and temperature (13.6 ± 0.29, 15.2 ± 0.28, 15.4 ± 0.28 arbitrary units (AU) for 0 hour control, 38.5°C and 41°C, respectively). The molecular motor dynein was localized along all the sperm cell. Similarly, dynein pixel fluorescence intensity increased (P < 0.0001) with incubation (4.2 ± 0.5, 26.8 ± 0.5, 26.2 ± 0.5 AU for 0 hour, 38.5°C and 41°C, respectively). In conclusion, incubation and elevated temperature reduced sperm motility and affected the pattern of cytoskeletal proteins organization.

The postpartum period is characterised by the rapid uterine involution process and return of ovarian activity (foal heat), resulting in a fertile oestrus in most of the mares. However, the follicular development and selection processes during this period are not completely known in horses. We aimed to study the characteristics of follicular growth and vascular perfusion in the ovary during the early postpartum period in mares that demonstrated oestrous behaviour and had early (<10 days) or late (≥10 days) ovulation. Ten mares were scanned daily from the first day postpartum (Day 1) until the day of the first postpartum ovulation (Day 0). The animals were split in the early (n = 3) and late (n = 7) ovulation groups (averaged interval between parturition and ovulation: 8.0 ± 0.0 and 14.7 ± 1.2 days, respectively). For ultrasound exams a Duplex B-mode and colour Doppler instrument (M5VET®, Mindray, Shenzhen, China) was used with a multifrequency linear probe. Data were analysed for the main effects of group, day, and their interaction using the PROC MIXED procedure of SAS software (version 9.3, SAS Institute Inc., Cary, NC, USA). For the follicular growth, no difference (P > 0.05) was detected between the groups when the data were analysed for the days relative to ovulation (from Day 7 to Day 1). However, the dominant follicle was larger (P < 0.05) in the early-ovulated group (37.2 ± 1.6 v. 21.9 ± 1.1) in all days during early postpartum (Day 1 to Day 7). The number of follicles with >25 mm diameter was also greater (P < 0.05) in the early-ovulated group (1.1 ± 0.1 v. 0.1 ± 0.1) during the first 3 days postpartum. In addition, the late-ovulated mares showed greater number of follicles with 20–25 mm during Day 4 to Day 7 (2.0 ± 0.2 v. 0.7 ± 0.1). For the blood flow characteristics, no difference (P > 0.05) was detected in the coloured signals of blood flows in the follicular wall of the dominant follicle or in the ovarian pedicle ipsilateral to the largest follicle. Therefore, the characteristics of the follicle growth on the preceding days of ovulation were similar between the early- and late-ovulated mares and consistent with the follicular dynamics expected in non-pregnant and non-lactating mares. However, when the data were analysed for the days relative to parturition, a greater follicle development was present in mares that ovulate earlier during the postpartum period (<10 days). In conclusion, the results suggest that important events may occur previous to the parturition, resulting in an early follicle development, mainly in those mares that show heat signs and ovulate within 10 days postpartum. Research was supported by FAPESP process number 2010/10692-9 and CNPq process number 135954/2011-8.

In horses, a rapid involution of the uterus occurs right after parturition, allowing the reestablishment of a favourable uterine environment for embryo development. However, limited evidence is found regarding the vascular events during puerperal period in mares. We aimed to evaluate the morphological (size of uterus and uterine fluid content) and haemodynamic (endometrial and mesometrial vascular perfusion) characteristics of the uterine involution process. Mares (n = 10) were evaluated by transrectal ultrasonography from the first day postpartum (d1) to the sixteenth day after first ovulation (D0 = ovulation). For ultrasound exams, a duplex B-mode and Doppler ultrasound instrument (M5 VET; Mindray Medical International Limited, China) equipped with a transrectal transducer was used. The previously pregnant (PH) and nonpregnant (NPH) horns were individually evaluated. Data were analysed for the main effects of horns (PH and NPH), day, and their interaction, using the PROC MIXED procedure of SAS software (9.3 version; SAS Institute Inc., Cary, NC, USA). Discrete variables were analysed by ANOVA. A reduction (P < 0.05) in the uterine diameter was observed during the first 7 days postpartum, but the rate of uterine involution (decrease in uterine size) decreased thereafter. The involution was complete around the d21 for the NPH and around d24 for the PH. Presence of uterine luminal fluid (mm) was increased between d1 (no fluid) and d2 (31.41 ± 2.88) postpartum, followed by a decrease between d4 (30.43 ± 4.52) and d7 (10.20 ± 1.76). No fluid was observed after d16 postpartum or after the third day postovulation (D3). For endometrial and mesometrial vascular perfusion, only a day effect (P < 0.05) was observed. An increase in the endometrial and mesometrial vascularization was detected, respectively, between d1 and d4, and between d1 and d2. Vascular perfusion did not differ after d4 for endometrial tissue, whereas it was reduced (P < 0.05) between d2 and d10 for mesometrium. For the vascular perfusion after ovulation, an increase (P < 0.05) from D0 to D5, followed by a decrease (P < 0.05) between D5 and D11 and an increase (P < 0.05) between D11 and D14 was observed in the endometrial and mesometrial tissues. The profile of the vascular perfusion in endometrium and mesometrium after first ovulation postpartum is similar to that observed during oestrous cycles and early pregnancy, indicating a return of the uterus to the pre-pregnant uterine characteristics in mares.Research was supported by FAPESP process no. 2010/10692-9 and CNPq process no. 135954/2011-8.

Dioctophyme renale is the largest roundworm known. The complex epidemiological chain this is indirect life cycle that involves different species. The first-stage larvae-containing eggs must be ingested by an aquatic oligochaete annelid intermediate host, and essential for the continuation of the cycle. It is located in the definitive host priority in the right kidney, but can be found in ectopic location, such as the abdominal cavity. This paper reports the occurrence of two cases in Sao Luis do Maranhao, Brazil. In the first case the animal had been sent to the Veterinary Hospital of the State University of Maranhao for mastectomy and the second case was a consultation in private practice Veterinary Saint Anthony for verification of a volume increase in the inguinal region left, thinking in both cases, the nematode D. renale. The present report contributes to the importance of observing during cavity surgery the anatomy of the viscera

titulo en ingles: Cycle erratic Dioctophyme renale: report of two cases titulo en español: Ciclo errático de Dioctophyme renale: relato de dos casosResumo: O Dioctophyme renale é o maior nematelminto conhecido. A complexa cadeia epidemiológica deste se dá pelo ciclo evolutivo indireto que envolve diferentes espécies. Os ovos contendo larvas de primeiro estágio devem ser ingeridos por um anelídeo oligoqueta aquático, hospedeiro intermediário e essencial para a continuação do ciclo. Localiza-se no hospedeiro definitivo prioritariamente no rim direito, mas pode ser encontrado em localização ectópica, como a cavidade abdominal. Este trabalho relata a ocorrência de dois casos em São Luís do Maranhão, Brasil. No primeiro caso o animal tinha sido enviado ao Hospital Veterinário da Universidade Estadual do Maranhão para mastectomia e o segundo caso foi a uma consulta na clínica particular Veterinária Santo Antônio para verificação de um aumento de volume na região inguinal esquerda, achando nos dois casos, o nematóide D. renale. O presente relato contribui na importância se observar durante as cirurgias de cavidades a anatomia das vísceras e a presença de neoformações, no sentido do clínico-cirurgião se antecipar ao aparecimento de quaisquer tipo de sintomatologia clínica provocada por esse nematóide.Palavras chave: ciclo ectópico, dioctofimose, nematelminto, parasita, tumor de mamaAbstract: Dioctophyme renale is the largest roundworm known. The complex epidemiological chain this is indirect life cycle that involves different species. The first-stage larvae-containing eggs must be ingested by an aquatic oligochaete annelid intermediate host, and essential for the continuation of the cycle. It is located in the definitive host priority in the right kidney, but can be found in ectopic location, such as the abdominal cavity. This paper reports the occurrence of two cases in São Luís do Maranhão, Brazil. In the first case the animal had been sent to the Veterinary Hospital of the State University of Maranhão for mastectomy and the second case was a consultation in private practice Veterinary Saint Anthony for verification of a volume increase in the inguinal region left, thinking in both cases, the nematode D. renale. The present report contributes to the importance of observing during cavity surgery the anatomy of the viscera and the presence of in the direction of clinical surgeon to anticipate the emergence of any kind of clinical symptoms caused by this nematode.key words: cycle ectopic, dioctophymosis, nematelminthe, parasite, breast tumor