Freezing and thawing in Antarctica: characterization of antifreeze protein (AFP) producing microorganisms isolated from King George Island, Antarctica.

Antarctic temperature variations and long periods of freezing shaped the evolution of microorganisms with unique survival mechanisms. These resilient organisms exhibit several adaptations for life in extreme cold. In such ecosystems, microorganisms endure the absence of liquid water and exhibit resistance to freezing by producing water-binding molecules such as antifreeze proteins (AFP). AFPs modify the ice structure, lower the freezing point, and inhibit recrystallization. The objective of this study was to select and identify microorganisms isolated from different Antarctic ecosystems based on their resistance to temperatures below 0 °C. Furthermore, the study sought to characterize these microorganisms regarding their potential antifreeze adaptive mechanisms. Samples of soil, moss, permafrost, and marine sediment were collected on King George Island, located in the South Shetland archipelago, Antarctica. Bacteria and yeasts were isolated and subjected to freezing-resistance and ice recrystallization inhibition (IR) tests. A total of 215 microorganisms were isolated, out of which 118 were molecularly identified through molecular analysis using the 16S rRNA and ITS regions. Furthermore, our study identified 24 freezing-resistant isolates, including two yeasts and 22 bacteria. A total of 131 protein extracts were subjected to the IR test, revealing 14 isolates positive for AFP production. Finally, four isolates showed both freeze-resistance and IR activity (Arthrobacter sp. BGS04, Pseudomonas sp. BGS05, Cryobacterium sp. P64, and Acinetobacter sp. M1_25C). This study emphasizes the diversity of Antarctic microorganisms with the ability to tolerate freezing conditions. These microorganisms warrant further investigation to conduct a comprehensive analysis of their antifreeze capabilities, with the goal of exploring their potential for future biotechnological applications.

Blind Source Separation in Persistent Atrial Fibrillation Electrocardiograms Using Block-Term Tensor Decomposition With Löwner Constraints.

The estimation of the atrial activity (AA) signal from electrocardiogram (ECG) recordings is an important step in the noninvasive analysis of atrial fibrillation (AF), the most common sustained arrhythmia encountered in clinical practice. This problem admits a blind source separation (BSS) formulation that has been recently posed as a tensor factorization, using the Hankel-based block term decomposition (BTD), which is particularly well-suited to the estimation of exponential models like AA during AF. However, persistent forms of AF are characterized by short R-R intervals and very disorganized (or weak) AA, making it difficult to model AA directly and perform its successful extraction through Hankel-BTD. To overcome this drawback, the present work proposes a tensor approach to estimate QRS complexes and subtract them from the ECG, resulting in a signal that, ideally, only contains the AA component. Such an approach tackles the problem of blind separation of rational functions, which models QRS complexes explicitly. The data tensor admitting a BTD is built from Löwner matrices generated from each lead of the observed ECG. To this end, this paper formulates a variant of the recently proposed constrained alternating group lasso (CAGL) algorithm that imposes Löwner structure on the decomposition blocks. This is done by performing an orthogonal projection, which we explicitly derive, at each iteration of CAGL. Results from experiments with synthetic data show the consistency of the proposed Löwner-constrained AGL (LCAGL) in extracting the desired sources. Experimental results obtained on a population of 20 patients suffering from persistent AF show that the proposed variant outperforms other tensor-based methods in terms of atrial signal estimation quality from ECG records as short as a single heartbeat.

Short communication: Does previous superovulation affect fertility in dairy heifers?

The aim of this study was to evaluate the potential negative effects of superovulation on subsequent fertility of dairy heifers. Holstein heifers (n = 1,783), 312 to 387 d of age, and 273 to 307 kg of body weight (BW) from 2 commercial farms were enrolled. These animals were first selected to be donors (446) or nondonors (CON, n = 1,327) according to their genomic values. Nondonor heifers (CON) were artificially inseminated (AI) according to standard procedures of each farm after reaching 320 kg of BW. Donor heifers were superovulated using a fixed FSH dose (180 mg NIH-FSH-P1; Folltropin-V, Vetoquinol Brasil, Mairiporã, SP, Brazil) and embryos were collected following standard procedures. Heifers that produced fewer than 8 viable embryos after first superovulation (SOV1, n = 337) were no longer used as donors, whereas the remaining heifers (SOV2, n = 109) were superovulated a second time within an interval of 48 to 54 d. Donor heifers (SOV1 and SOV2) were AI once they reached 320 kg of BW, at least 15 d after the last embryo collection. Data on age at first AI, at conception, and at parturition, as well as the number of services per conception, were analyzed by ANOVA, using the PROC MIXED procedure of SAS (SAS Institute Inc., Cary, NC) procedure. Binomial variables (pregnancy per AI, overall pregnancy rate, open heifers at 500 d age, and late pregnancy loss) were analyzed using the GLIMMIX procedure of SAS. The heifers selected to undergo superovulation twice (SOV2) yielded more total (12.6 ± 5.3 vs. 6.8 ± 4.6; respectively) and viable embryos (8.5 ± 3.8 vs. 3.9 ± 2.8; respectively) than those superovulated only once (SOV1). Age at first AI, conception, and at parturition was greater in SOV2, but not in SOV1 compared with nondonor controls. In addition, pregnancy per first AI, overall pregnancy rate, services per conception, open heifers at 500 d of age, and occurrence of pregnancy losses after 60 d of gestation were similar among CON, SOV1, and SOV2 heifers. In summary, a single superovulation performed before heifers reach a minimum weight for breeding did not affect age at conception, calving or other indicators of reproductive efficiency. On the other hand, heifers superovulated twice were first inseminated at a later age than their birth cohorts, but had similar reproductive performance.

Early resynchronization of non-pregnant beef cows based in corpus luteum blood flow evaluation 21 days after Timed-AI.

The study aimed to verify whether a hormone protocol started at Day 13 (D13) after Timed Artificial Insemination (TAI) influences the conception rate. Nelore cows (primiparous and multiparous) from two commercial beef farms (n = 1,431) were first TAI (D0). Timed AI was performed in lots (TAI Lots) ranging from 187 to 346 cows. On D13, regarding the TAI lot, cows were assigned for either receiving (Resynch group, n = 1,002) or not (Control group, a subset of approximately 30%, n = 429) another hormone protocol for resynchronization. The same hormone protocol was used for the first TAI and for the resynchronization, except for 1 mg instead of 2 mg of estradiol benzoate (EB) at the begging of the protocol. Eight days later (D21), the Resynch group was checked for corpus luteum blood flow by color Doppler ultrasonography, and in those detected as non-pregnant, the protocol was completed and a 2nd TAI was performed at D23. Pregnancy diagnosis was later (D30) performed by B-mode ultrasonography in the control group and confirmed in the presumptive pregnant cows from the 1st TAI of the Resynch group. The remaining cows were checked for pregnancy 30 days after the 2nd TAI (experimental Day 53). The statistical model to explain conception rate considered the effects of Group (Control or Resynch), Farm, Parity (primiparous or multiparous), Sire, Technician (who perform AI), TAI Lot and pertinent interactions (Group*Parity, Group*Farm and Group*TAI Lot). The statistical analyses of the model were performed using the Proc Glimmix (SAS virtual University Edition). The conception rate for the 1st TAI was similar (P > 0.4) between Control (50.3%, 216/429) and Resynch group (52.6%, 527/1002). The positive predictive diagnostic on D21 showed high relation with PD30 (90.7%, 527/581). In Resynch group, non-pregnant cows (n = 421, 1002 minus 581) were re-inseminated. The conception rate of the 2nd TAI (42.8%, 180/421) was affected (P < 0.002) by side effects of the Farm (48.5 vs. 33.1%) and Parity (51.2 vs. 40.3%, for multiparous vs. primiparous, p < 0.001). Nevertheless, after the 2 TAIs of the Resynch group, the cumulative conception rate was 70.5% (707/1002). In conclusion, the early resynchronization of cows with a low (1 mg) EB dose and progesterone device at D13 after TAI can be used as a strategy to reduce conception interval in beef cattle, and thus to increase the number of pregnant cows from artificial insemination after the breeding season.

Efficacy and limitations of different approaches to anticipate the diagnosis of pregnancy in cattle / Eficácia e limitações de diferentes abordagens para antecipar o diagnóstico de gestação em bovinos

The study evaluated sonographic and serologic exams performed for early (20 to 30d) diagnosis of pregnancy. One hundred-twenty (n= 120) bovine recipients were synchronized (estrous=D0) and timed embryo transferred (TET, D7) with fresh in vitro produced embryos. In the first trial (n= 46), diagnosis of pregnancy was performed on day 20 (D20) by detecting CL blood flow (BF) and by Pregnancy-Associated Glycoproteins (PAGs) serology. In the second trial (n= 30), pregnancy diagnosis was performed on D25 by ultrasound visualization of uterine contents and by PAGs serology. In the last trial, PAG's serology was performed on D30. Ultrasonographic detection of the uterine contents and embryo viability performed on D30 (DG30) was considered the gold standard. The PROC FREQ procedure was used to test the agreement between diagnostic methods. On D20, the Doppler ultrasonography of the CL had showed high sensitivity (100%), but only moderate specificity (53.3%). On the same day, serologic diagnostic had no agreement (k= -0.08, P< 0.46) with the gold standard, with very low sensitivity (6.3%). However, the sensitivity of the serologic exam increased dramatically (from 6.3 to 100%) from D20 to D25, and it contributed to detect false negatives from the ultrasound diagnosis, improving the overall accuracy from 90% to 96.7%.(AU)

O estudo foi planejado para correlacionar exames ultrassonográficos e sorológicos realizados para o diagnóstico precoce (20 a 30d) de gestação. Cento e vinte (n= 120) receptoras bovinas foram sincronizadas (estro=D0), e embriões frescos produzidos in vitro foram transferidos em tempo fixo (TETF, D7). No experimento 1 (n= 46), o diagnóstico de gestação foi realizado no D20, pela detecção do fluxo sanguíneo do CL e pela sorologia de glicoproteínas associadas à gestação (PAGs). No experimento 2 (n= 30), a detecção da gestação foi realizada por meio da visualização do conteúdo do útero e também pela sorologia para PAGs. No experimento 3, a sorologia para PAGs foi realizada no D30. Em todos os experimentos, a visualização ultrassonográfica da vesícula e da viabilidade embrionária, realizada no D30, foi considerada padrão-ouro. O procedimento PROC FREQ testou o nível de concordância dos métodos diagnósticos. No D20, o diagnóstico baseado na vascularização do CL mostrou alta sensibilidade (100%) e apenas moderada especificidade (53,3%). Nesse mesmo dia, o diagnóstico sorológico não apresentou concordância (k=-0,08, P<0,46) com o padrão-ouro, além de baixa sensibilidade (6,3%). No entanto, a sensibilidade do exame sorológico aumentou drasticamente (6,3 para 100%) do D20 para o D25, contribuindo para detectar falsos negativos diagnosticados pela ultrassonografia, melhorando a acurácia (90 para 96,7%).(AU)

Differential expression of LHCGR and its isoforms is associated to the variability in superovulation responses of Gir cattle.

The aim of this study was to evaluate the pattern of expression of LHCGR isoforms in Gir heifers characterized as good (10.3 ±â€¯1.2 ova/embryos per flush, n = 5) or poor responders (1.1 ±â€¯0.3 ova/embryos per flush, n = 5) to superovulation protocols. In both groups, an adapted ultrasound-guided follicular aspiration system was used to collect granulosa cells from 8 mm follicles formed either during a synchronized, non-stimulated follicular wave (no stimulation control, NS) or on the fourth day of a superovulation protocol (SOV) induced with 200 IU of pFSH. The recovered follicular fluid was centrifuged and granulosa cells were washed with NaCl 0.9% and kept in RNAlater®. RNA extraction was performed using a commercial RNeasy Micro Kit and eluted samples were quantified and reverse transcribed using the commercial Superscript III kit. cDNA samples were amplified by real-time PCR using a primer to target LH/hCG receptor gene - not selective for LHCGR isoforms (total LHCGR) - and four sets of isoforms selective primers (S1, S10, S10 + 11, and S11). Analyses were performed using the REST software and expression levels are shown as mean ± SEM. Under physiological conditions (NS), poor responders had a higher expression of total LHCGR (4.9 ± 1.7 fold-change, P < 0.01) as well as isoforms S10, S11 and S10 + 11, compared to good responders. In both phenotypes, superovulation down-regulated total LHCGR expression (-0.5 ± 0.2 and -0.9 ± 0.0 for good and poor responders, respectively; P < 0.05). However, in poor responders the exogenous FSH treatment up-regulated the S10 (2.4 ± 2.0; P < 0.05), S10 + 11 (3.8 ± 3.2; P < 0.01), and S1 isoforms (1.8 ± 1.3; P < 0.05), compared to good responders We conclude that down-regulation of total LHCGR, associated to up-regulation of their inactive isoforms, may have compromised follicle development and thus contributed to the low efficiency of superovulation in heifers with a poor responder phenotype.

Teofilina como agente capacitante do sêmen bovino / Theophylline as a capacitating agent of bovine semen

Objetivou-se avaliar a teofilina como agente capacitante substituto ou associado à heparina sobre a reação acrossômica dos espermatozoides e o desenvolvimento de embriões produzidos in vitro. O experimento foi realizado com quatro touros e três tratamentos, totalizando 12 grupos experimentais. O sêmen dos touros foi avaliado nos tratamentos descritos a seguir: tratamento 1 (HEP): heparina - 10µg/mL; tratamento 2 (TEO): teofilina - 5mM; tratamento 3 (HEP + TEO): heparina (10µg/mL) + teofilina (5mM), por zero, seis, 12 e 18 horas, corados com trypan blue/Giemsa para avaliação da reação acrossômica. Para a produção dos embriões, os agentes capacitantes foram adicionados aos meios de fertilização. Na análise espermática, a taxa de reação acrossômica verdadeira foi maior (P<0,05) no tempo zero hora, enquanto para espermatozoides mortos, as maiores taxas (P<0,05) foram nos tempos de 12h (84,46±5,82) e 18h (86,75±4,19). A taxa de embriões produzidos (37,97±13) e a taxa de eclosão (33,50±14) foram maiores (P<0,05) para o tratamento HEP. Não foi observada diferença (P>0,05) entre touros na análise de reação acrossômica nem na PIVE. A utilização da teofilina foi tão eficiente quanto a da heparina na indução da reação acrossômica, no entanto resultou em menores taxas de produção embrionária.(AU)

The sperm capacitating process should take special attention during in vitro embryo production (IVEP) once that affects the success of embryo production. The study aimed to evaluate theophylline as substitute capacitating agent or in combination with heparin on the sperm acrosome reaction and development of embryos produced in vitro. The experiment was carried out using 4 bulls and 3 treatments, establishing 12 experimental groups. Each bull was evaluated in the following treatments: Treatment 1 (HEP): Heparin - 10mg/mL; Treatment 2 (THEO): Theophylline - 5mM; Treatment 3 (HEP + THEO), Heparin (10mg/mL) + Theophylline (5mM). The semen of bulls was incubated in each treatment for 0, 6, 12 and 18h, stained with Trypan blue / Giemsa and analyzed by electron microscopy for assessment of acrosome reaction. Using sperm of same bulls, capacitating agents were added to the fertilization media, for IVEP. In sperm analysis, the true acrosome reaction rate was higher (P<0.05) in time 0h, while sperm dead rates were highest (P<0.05) at 12h (84.46±5, 82), and 18h (86.75±4.19). The produced embryos rate (37.97±13) and hatching rate (33.50±14) were larger (P<0.05) for HEP treatment. There was no difference (P>0.05) between bulls in acrosome reaction analysis neither for IVEP. The use of theophylline was as effective as heparin in the induction of the acrosome reaction, although it resulted in lower embryo production rates.(AU)

Establishment and cryptic transmission of Zika virus in Brazil and the Americas.

Transmission of Zika virus (ZIKV) in the Americas was first confirmed in May 2015 in northeast Brazil. Brazil has had the highest number of reported ZIKV cases worldwide (more than 200,000 by 24 December 2016) and the most cases associated with microcephaly and other birth defects (2,366 confirmed by 31 December 2016). Since the initial detection of ZIKV in Brazil, more than 45 countries in the Americas have reported local ZIKV transmission, with 24 of these reporting severe ZIKV-associated disease. However, the origin and epidemic history of ZIKV in Brazil and the Americas remain poorly understood, despite the value of this information for interpreting observed trends in reported microcephaly. Here we address this issue by generating 54 complete or partial ZIKV genomes, mostly from Brazil, and reporting data generated by a mobile genomics laboratory that travelled across northeast Brazil in 2016. One sequence represents the earliest confirmed ZIKV infection in Brazil. Analyses of viral genomes with ecological and epidemiological data yield an estimate that ZIKV was present in northeast Brazil by February 2014 and is likely to have disseminated from there, nationally and internationally, before the first detection of ZIKV in the Americas. Estimated dates for the international spread of ZIKV from Brazil indicate the duration of pre-detection cryptic transmission in recipient regions. The role of northeast Brazil in the establishment of ZIKV in the Americas is further supported by geographic analysis of ZIKV transmission potential and by estimates of the basic reproduction number of the virus.

Efficacy of induction of luteolysis in superovulated cows is dependent on time of prostaglandin F2alpha analog treatment: effects on plasma progesterone and luteinizing hormone profiles.

The objectives were to (1) evaluate the effectiveness of induction of luteolysis in superovulated (SOV) cows at two distinct time points after embryo flushing; and (2) compare the pattern of LH release after treatment with PGF in cows with single vs. multiple ovulations. In the first experiment, Holstein cows were SOV with 400 IU of FSH following standard procedures. Uterine flushing for embryo recovery was performed 7 days after artificial insemination (Day 0), and cows were randomly allocated into two groups to receive PGF (0.5-mg sodium cloprostenol, intramascular) either immediately after flushing (Day 7 group, N = 19) or 4 days later (Day 11 group, N = 20). Time of luteolysis was determined on the basis of plasma progesterone (P4) concentrations. There was no difference (P > 0.05) in plasma P4 before treatment between Day 7 and Day 11 groups. A decline in plasma P4 was observed 48 hours after PGF treatment in both the groups (P < 0.0001). In Day 11 cows, P4 continued to decrease thereafter, whereas Day 7 animals had no further reduction in plasma P4. Luteolysis (P4 < 1 ng/mL) occurred in all Day 11 cows. In the Day 7 group, however, luteolysis failure was observed for 11 of 19 cows (57.9%). In cows without luteolysis, plasma P4 increased after the initial PGF-induced decline. The second experiment compared luteolysis in (SOV, N = 6) vs. non-SOV (control, N = 8) cows. Both groups received a single PGF treatment on Day 11 after estrus, and luteolysis was monitored daily by ovarian ultrasonography and plasma P4 measurements. In addition, plasma LH was measured in blood samples taken every 20 minutes for 1 hour during five consecutive days after treatment. A similar percentage of reduction in P4 was observed in both groups 24 hours after treatment; however, SOV cows only reached plasma P4 values similar (P > 0.05) to controls 96 hours after treatment. There was no difference in initial LH values between SOV and controls (P > 0.05). The slower decrease in plasma P4 in the SOV group prevented an increase in LH for up to 96 hours after luteolysis induction, whereas LH values increased (P < 0.05) in controls 24 hours after treatment. In conclusion, (1) luteolysis may fail or be incomplete when PGF treatment is given on the day of uterine flushing (Day 7) in SOV cows; (2) induction of luteolysis 4 days later (Day 11) is effective, but the initial high-plasma P4 concentrations result in a slower slope of P4 decline to basal levels, and consequently, delayed increase in LH pulses.

Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae).

Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis.

Oocyte development and fecundity type of the Brazilian Snapper Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae) / Desenvolvimento ovocitário e tipo de fecundidade da baúna de fogo Lutjanus alexandrei Moura & Lindeman, 2007 (Perciformes: Lutjanidae)

Abstract Lutjanid species exhibit multiple spawning behaviour during an extended spawning season in warm months, asynchronous oocyte development and indeterminate fecundity. Although early studies have contributed to knowledge of the reproductive cycle of many species within the group, they have not considered aspects about the number of cortical alveoli oocyte stage throughout maturity phases along spawning season. The latter aspect is also considered very important to confirm indeterminate fecundity hypothesis. In the present study, were analyzed 154 Brazilian snapper Lutjanus alexandrei female gonads obtained from artisanal fisheries in Pernambuco State (Brazil) between October 2010 and March 2011. Were measured oocyte size frequency distribution for maturity phases (developing, spawning capable and actively spawning), and oocyte development stage (unyolked oocytes, cortical alveoli, primary, secondary and tertiary vitellogenic oocytes and hydrated oocytes), and also the oocyte stage frequency during spawning season. The frequency of cortical alveoli oocyte stage was constantly found in the spawning period (>37%), showing slight variation throughout maturity phases. The absence of gap in the oocyte size frequency distribution between primary and secondary oocyte growth stages during spawning season is a strong indicator of continuous oocyte recruitment from reserve stocks. In addition, co-occurrence of tertiary vitellogenic oocytes, hydrated oocytes, post-ovulatory follicles and yellow-brown bodies in the histological sections of ovaries reinforce indeterminate fecundity hypothesis.

Resumo Os lutjanídeos apresentam um comportamento de desovas parceladas ao longo de uma extensa estação reprodutiva nos meses mais quentes do ano, desenvolvimento assincrônico dos ovócitos e padrão de regulação de fecundidade indeterminado. Embora muitos estudos anteriores tenham contribuído no conhecimento do ciclo reprodutivo das espécies do grupo, não foram considerados os aspectos relacionados ao percentual dos ovócitos em estágio de cortical alveoli entre as fases de maturidade sexual durante a estação reprodutiva. Este último aspecto, considerado importante para confirmação da hipótese de fecundidade do tipo indeterminada. Para isso, no presente trabalho foram analisadas 154 gônadas de fêmeas da baúna de fogo Lutjanus alexandrei obtidas da pesca artesanal em Pernambuco entre o período de outubro de 2010 a março de 2011. Foram mensurados os diâmetros dos ovócitos nas fases de maturidade sexual (desenvolvimento, capaz de desovar e ativamente desovando), e para todos os estágios ovocitários (ovócitos pre-vitelogênicos, cortical alveolar, vitelogênese primária, secundária, terciária e ovócitos hidratados), e avaliada a frequência de ocorrência ao longo do período de desova. O percentual de ovócitos em estágio cortical alveoli permaneceu constante durante o período de reprodutivo (> 37%), apresentando pouca variação entre as fases de maturidade sexual. A ausência de um intervalo na frequência de diâmetro dos ovócitos em crescimento primário e secundário entre todas as fases de maturidade sexual é um forte indicador de recrutamento contínuo de ovócitos do estoque de reserva. Além disso, a presença conjunta de ovócitos em vitelogênese terciária, ovócitos hidratados, folículos pós-ovulatórios e corpos amarelados nos cortes histológicos reforçam a hipótese de fecundidade do tipo indeterminada.

Corpus luteum blood flow evaluation on Day 21 to improve the management of embryo recipient herds.

The aim of the present study was to use blood flow evaluation of the CL at 14 days after embryo transfer to detect nonpregnant animals and optimize the management of bovine recipients. The estrous cycle was synchronized in 165 recipients, and the day of expected ovulation was considered to be Day 0. Embryo transfer was performed 7 days later, on Day 7. On Day 21, pregnancy was diagnosed on the basis of blood flow evaluation of the CL (DG21-predictive diagnostic). To validate this methodology, visual scores for blood flow were compared to objective data extracted from CL ultrasound images recorded in the Doppler mode. The size was also evaluated using recorded images of the CL in the B mode. Blood samples were also collected for further analysis of the progesterone (P4) concentration. The diagnosis of pregnancy was confirmed at 35 days after estrus (DG35-definitive diagnostic). The DG21 showed that 55.2% (90 of 163) of the animals were presumptively pregnant, and this value was higher (P < 0.04) than that obtained at DG35 (43.6%, 71 of 163). The predictive diagnostic achieved moderate specificity (79.3%) for the detection of pregnancy, but most importantly, high sensitivity (100%) for the detection of nonpregnant recipients. The overall accuracy of the diagnosis was 88.3%. The P4 concentrations were different (P < 0.02) and correlated with each visual score assigned for the CL size. Visual scores for CL blood flow were also efficient (P < 0.0001) to distinguish animals with different levels of P4; however, P4 concentrations were higher for scores 1 and 2 (high and regular blood flow, respectively) than those for score 3 (low blood flow). This technique showed high sensitivity and facilitated the early detection of nonpregnant animals. The DG21 would allow about 79.3% of nonpregnant animals to be resynchronized 9 to 14 days earlier, when compared to conventional management based on pregnancy diagnosis at Days 30 to 35.

Perfil de progesterona em novilhas com diferente atividade luteal e tratadas com implantes vaginais / Serum progesterone in heifers with different luteal activity treated with vaginal devices

O dispositivo de liberação de progesterona (DLP) é muito importante em protocolos de Inseminação Artificial em Tempo Fixo (IATF). Representa cerca de 43% dos custos e é objeto de estudos sobre a eficiência da sua reutilização. No entanto, perfis de liberação de progesterona (P4) em animais com diferentes concentrações endógenas desse esteroide não são claramente descritos. Este estudo teve como objetivo avaliar a concentração sérica de P4 em fêmeas com diferentes situações de atividade luteal, tratadas com DLP novo (1g de P4) por 8 dias. Trinta novilhas mestiças cíclicas foram divididas em três grupos: em G1 e G2, o DLP foi inserido (D0) sete dias após a ovulação induzida. Adicionalmente, 0,15mg de D-cloprostenol foi administrado três dias depois para promover a luteólise em G2. Para G3, o corpo lúteo foi lisado antes da inserção do DLP para que a P4 exógena fosse a única fonte desse hormônio. O sangue foi coletado no D0, D3, D5 e D8, e a P4 avaliada por RIA. Médias de P4 foram comparadas entre os grupos em cada dia e dentro do grupo, entre os dias, utilizando o teste Tukey. Antes da inserção do implante (D0), os níveis de P4 foram, nos grupos, semelhantes em G1 e G2, e superiores a G3 (5,3±3,1a e 5,3±1,4avs 0,6±0.3bng/mL, respectivamente-P<0,05). No D3, ocorreu o mesmo perfil (5,7±2,6a e 5,4±2,0a e 3,6±0.8bng/mL, respectivamente para G1 e G2 vs G3, P<0,05). Trinta e seis horas (D5) após a PGF, a P4 no G2 caiu para níveis semelhantes aos do grupo G3 e ambos diferiram (P<0,05) de G1 (3,3±1,6b vs 2,4±0,9b e 2,1±0.7bng/mL). Essa diferença se manteve (P<0,05) em D8 (3,1±1,3a, 1,8±0,8b e 1,6±0.6b ng/mL). O aumento da P4 após a inserção (D3 - D0) foi maior (P<0,05) em G3 que em G1 e G2 (2,8±0,9a vs 0,4±1,8b e 0,2±1.4bng/mL). Os animais com maior P4 endógena levam a menor liberação de P4 exógena a partir do DLP. Portanto, os níveis remanescentes de P4 no DLP após o uso dependem da concentração endógena de P4 do animal e possíveis alterações durante a permanência.(AU)

The progesterone (P4) device is a very important step in the ovulation control in Timed Artificial Insemination (TAI) protocols. It represents about 43% of the hormone costs, thus it has been the subject of several studies on efficiency of the reused device as an alternative to reduce TAI costs. However, to our knowledge, profiles for P4 release in animals with different endogenous concentrations of P4 are not clearly described. This study aimed to evaluate serum concentration of P4 in females with different ovarian conditions - related to luteal activity - and treated with a new intravaginal device (1g of P4) for 8 days. Thirty normally cyclic crossbred heifers were divided into three groups: for G1 and G2, P4 device was inserted (D0) seven days after ovulation (7 day old CL). Additional PGF (0.15 mg of D-cloprostenol) was given three days later to promote luteolysis in the G2 group. For G3, CL was killed before P4 insertion and the exogenous progesterone was the only source of this hormone. Blood samples were collected on D0, D3, D5 and D8 and P4 concentration was measured by radioimmunoassay (RIA). Means for P4 concentration were compared among groups in each day and within the group among days using the Tukey test. Before P4 device insertion (D0), P4 levels were higher (P<0.05) in G1 and G2 when compared to G3 (5.3±3.1 and 5.3±1.4 vs. 0.6±0.3ng/mL, respectively). Three days later (D3), the same pattern was observed (5.7±2.6 and 5.4±2.0 and 3.6±0.8ng/mL, respectively for G1 and G2 vs. G3, P<0.05). Thirty-six hours (D5) after PGF injection (G2), P4 in G2 dropped to levels similar to the G3 group and both differed (P<0.05) from G1 (3.3±1.6 vs. 2.4±0.9 and 2.1±0.7ng/mL, G1 vs. G2 and G3, respectively). There were no differences (P>0.05) among groups on D8 (3.1±1.3, 1.8±0.8 and 1.6±0.6ng/mL, respectively, for G1, G2 and G3). Progesterone increase after P4 insertion (D3 - D0) was higher (P<0.05) in G3 compared to G1 and G2 (2.8±0.9 vs. 0.4±1.8 and 0.2±1.4ng/mL, respectively). The interpretation was that animals with higher endogenous P4 promote less release of the exogenous P4 from the device. Therefore, the remaining P4 levels from used progesterone devices depend on the physiological condition of the animal at the time of insertion and possible changes during the treatment.(AU)

Effectiveness of Different Mechanical Methods on Dentin Caries Removal: Micro-CT and Digital Image Evaluation.

PURPOSE: To determine the caries removal effectiveness (CRE) and minimal invasive potential (MIP) of caries excavation methods using digital imaging and microtomography analyses. METHODS: Twelve human molars with occlusal caries lesions in dentin were randomly divided into three groups (carbide bur, exacavator, and polymer bur). They were sectioned mesiodistally, and standardized digital and computed microtomography x-ray (micro-CT) images were taken from each section before and after caries excavation. On each image, initial carious dentin (IC), prepared cavity (PC), and residual caries (RC) were defined according to visual criteria using ImageJ software. CRE was determined based on the RC/IC ratio, whereas MIP was determined by the PC/IC ratio. Data were analyzed using one-way analysis of variance and Student t-test or with Kruskal-Wallis and Student-Newman-Keuls test. The level of significance was set at 0.05. RESULTS: For both digital image and micro-CT analysis, the carbide bur showed higher CRE values than the excavator (p=0.0063 and p=0.0263, respectively) and the polymer bur (p=0.0028 and p=0.0005, respectively). The latter two presented similar results (p>0.05). Regarding MIP, for the digital image analysis, the polymer bur was different from the carbide bur (p=0.0030) but was not different from that of the excavator (p=0.1240). For micro-CT analysis, the MIP values of all the groups were significantly different, and the polymer bur was the most conservative method (p<0.05). CONCLUSIONS: The carbide bur was the most effective method for caries removal but was not completely conservative. The polymer bur and excavator presented low invasive potential but were not able to remove all of the carious dentin.

Redução da resposta superovulatória de fêmeas bovinas superestimuladas com FSH em doses split / Reduced superovulatory response of bovine females overstimulated with FSH in split doses

Objetivou-se avaliar a superovulação (SOV) de vacas zebuínas, utilizando protocolo convencional ou protocolo com número menor de aplicações e similar dosagem (dose split). Utilizaram-se 16 fêmeas (total 32 SOV), com idade entre 17-42 meses e escore de condição corporal 2,5-4 (escala de 1-5), em delineamento tipo cross-over. No início do tratamento (D0), os animais receberam um dispositivo de progesterona e 2mg de benzoato de estradiol. As fêmeas do grupo convencional receberam 250UI de FSH/LH divididas em oito doses decrescentes administradas em intervalos de 12h (FSH/LH no D4, D5, D6 e D7 no período da manhã e tarde, nas respectivas dosagens: 50,0 UI; 37,5 UI; 25,0 UI; 12,5 UI). No D7 pela manhã, as fêmeas foram tratadas com 150μg de D+cloprostenol, e a remoção da progesterona foi realizada no D7 à tarde. As fêmeas do grupo split também receberam 250 UI de FSH/LH. No D4 de manhã, administraram-se 62,5 UI de FSH/LH via IM e 125 UI por via SC. Quarenta e oito horas após (D6) administraram-se 62,5 UI via SC e na manhã do D7 foi removida a progesterona e aplicaram-se 150μg de D+cloprostenol. As fêmeas de ambos os grupos receberam 50μg de análogo de GnRH no D8 pela manhã e foram inseminadas 12 e 24 horas após. No D15 realizou-se a colheita dos embriões em ambos os tratamentos. Avaliou-se a resposta superovulatória pela contagem do número de folículos e CLs de cada ovário, com auxílio de ultrassom. Todas as variáveis foram submetidas ao teste T de Student para amostras pareadas. Houve diferença (P<0,05) na quantidade de folículos acima de 8mm no D8 (9,06±4,54 e 5,50±4,59); número de CLs no dia da colheita (8,12± 3,26 e 4,69±3,46), número de embriões totais (6,69±3,05 e 3,37±2,50) e de embriões viáveis (5,25±2,29 e 2,37±1,78) nas vacas do grupo convencional em relação às do split, respectivamente. Conclui-se que o protocolo split tem pior resposta superovulatória e de produção in vivo de embriões, em vacas zebuínas, quando comparado ao protocolo convencional...

The aim of this study was to evaluate the superovulation (SOV) response of zebu cows, using conventional protocol or other protocol with similar dose but smaller number of applications (split dose). 16 females (32 SOV), aged 17-42 months, and body condition score of 2.5-4 (1-5 scale) were used in randomized cross-over. At the start of treatment (D0), the animals received progesterone device and 2mg of estradiol benzoate. The females from the conventional group received 250 IU of FSH/LH divided into eight decreasing doses administered at intervals of 12 hours (FSH/LH in D4, D5, D6 and D7, with their respective strengths: 50.0 IU, 37.5 IU, 25.0 IU, 12.5 IU). In D7, the females were treated with 150μg of D+cloprostenol, and the removal of progesterone device was held in the afternoon. The females from the split group also received 250 IU of FSH/LH. In the morning D4 was administered in 62.5 IU FSH/LH IM and 125 IU subcutaneously. Forty eight hours later (D6) 62.5 IU was administered subcutaneously in the morning and on D7, the progesterone device was removed and 150 μg of D+cloprostenol was applied. The females in both groups were given 50 μg of GnRH in the morning and on D8 were inseminated after 12 and 24 hours. On D15 the embryo collection was performed in both treatments. The evaluation of superovulatory response was done by counting the number of follicles and corpus luteum (CL) in each ovary, with the aid of ultrasound. The variables were assessed by Student's t test for paired samples...

Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

Florfenicol associado ou não ao cloprostenol no tratamento de retenção de placenta em vacas leiteiras / Florfenicol and sodium cloprostenol for the treatment of retained placenta in dairy cows

The aim of this study was to compare the reproductive efficiency in dairy cows with retained placenta (RP), treated with Florfenicol associated or not with Cloprostenol. One hundred and eight cows with RP from dairy herds in southern Minas Gerais state and northern São Paulo state, were included for this purpose. The experiment was designed in factorial 2X2 . The four treatments encompassed two levels of each factor : 1 ) Florfenicol - 4 0mg/kg ( IM) divided in two injections or 40mg / kg ( SC) in one injection after calving; 2) Cloprostenol (0.530mg) - two injections associated with florfenicol treatment or without cloprostenol. The analysis of discrete variables used the Kruskal - Wallis (fou r groups) or Wilcoxon (two groups) statistic to compare groups. The Student "T" test was used to compare means of continuous variables. The overall occurrence of RP was 20.7%. The florfenicol groups had no effects on postpartum uterine health or subsequent reproductive efficiency of the cows. The cloprostenol injections of RP hastened the uterine involution and decreased the occurrence of uterine infection, and increased the percentage of animals with corpus luteum between 25 and 40 days after parturition. The intervals from parturition to first artificial insemination (AI) and to conception, as well as the number of AI per conception were reduced in the cloprostenol treatment.

Efficiency of the spermatogenesis in zebu bulls (Bos taurus indicus).

The objective of the research was to evaluate the efficiency of the spermatogenesis through the morphology of the testicular parenchyma in bulls of different zebu breeds. We used testicular fragments from bull of the breeds Nelore (n = 10), Polled Nelore (n = 6), Gyr (n = 5), Guzerat (n = 5) and Tabapuã (n = 5). The tissue was perfused with Karnovsky solution, included in glycol methacrylate and stained with toluidine blue-sodium borate 1%. Animals of the Nelore breed presented higher population of primary spermatocyte in pre-leptotene/leptotene (38.30) and in pachytene (38.14) and round spermatids (113.30), higher yield of spermatogonia mitosis (21.2) and higher daily spermatic production per gram of testicular parenchyma (32.8 × 10(6) ) than those from breeds Gyr, Guzerat and Tabapuã and higher general yield of spermatogenesis (62.4) than breeds Gyr and Tabapuã. There was no significant difference in any of the evaluated parameters between breeds Nelore and Polled Nelore. The rate of Sertoli cells did not vary between the studied breeds. Apparently, the genetic selection applied to the breeds has been improving the yield in the spermatogenic process by decreasing cellular loss, although it did not increase the support capacity of the Sertoli cells.

Investigation of complexity of the instruction manuals for electrical coffeepots.

BACKGROUND: Electrical coffeepots are commonly used in professional and residential environments. Their instruction manuals are related to issues that involve the user's safety and ability to operate the machine correctly. To provide the best product performance to the user, one must indicate or inform the correct usability, to turn the interaction easier. OBJECTIVE: This research proposes to investigate the instruction manuals of the electrical coffeepots. METHODS: Four coffee pot instruction manuals were analyzed in relation to the complexity through the heuristic evaluation. For that, eight experts of the Graphic Design were chosen to answer twenty four questions with the aim of analyzing: images; texts; layout development; information and warnings. RESULTS: This study shows the results of the 04 (four) items analyzed: a) images; b) texts; c) layout development; d) information and warnings, together with the suggestions of improvements for each manual. CONCLUSIONS: It is believed that the methodological procedures for the application of the heuristic evaluation have facilitated the diagnosis of fragilities and barriers that the users find during the interaction with electrical coffeepot manuals.

Color Doppler flow imaging for the early detection of nonpregnant cattle at 20 days after timed artificial insemination.

The objective was to determine the accuracy of a pregnancy test for predicting nonpregnant cattle based on the evaluation of corpus luteum (CL) blood flow at 20 d (CLBF-d20) after timed artificial insemination (TAI). Crossbred Holstein-Gir dairy heifers (n=209) and lactating cows (n=317) were synchronized for TAI using the following protocol: intravaginal implant (1.0 g of progesterone) and 2mg of estradiol benzoate i.m. on d -10, implant removal and 0.526 mg of sodium cloprostenol i.m. on d -2, 1mg of estradiol benzoate i.m. on d -1, and TAI on d 0. On d 20, animals underwent grayscale ultrasonography (US) to locate the CL and color flow Doppler to evaluate CLBF-d20 using a portable ultrasound equipped with a 7.5-MHz rectal transducer. Based only on a visual, subjective CLBF evaluation, the animals were classified as pregnant or not pregnant. On d 30 to 35, blinded from results of the previous diagnosis, the same operator performed a final pregnancy diagnosis using US to visualize the fetal heartbeat (gold standard; US-d30). A second evaluator also analyzed the CLBF-d20 in the same animals by watching 7-s recorded videos. Blood samples were collected from a subset of 171 females to determine, by RIA, plasma progesterone (P4) concentrations, which indicate CL function. The final pregnancy outcome (US-d30) was retrospectively compared with the CLBF-d20 diagnoses and then classified either as correct or incorrect. The number of true positive, true negative, false positive, and false negative decisions were inserted into a 2 × 2 decision matrix. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of the CLBF-d20 test were calculated using specific equations. Binomial variables (pregnancy rate and proportions) were analyzed using Fisher's exact test for the effect of parity and to compare between evaluators and tests (CLBF-d20 vs. plasma P4). The kappa values were calculated to quantify the agreement between CLBF-d20 and the gold standard (US-d30) and between evaluators. The performance parameters of CLBF-d20 test were as follows: sensitivity=99.0%, specificity=53.7%, positive predictive value=65.1%, negative predictive value=98.5%, and accuracy=74.8%. False negatives represented only 0.4% of the exams. No differences existed in these parameters between evaluators (no. 1 vs. no. 2) and tests (CLBF-d20 vs. plasma P4). Moreover, a high level of agreement was observed between evaluators (0.91). In conclusion, visual evaluation of CLBF-d20 represents a quick, reliable, and consistent diagnostic test that enables the early detection of nonpregnant cattle.