Efeito de diferentes diluentes na qualidade do sêmen refrigerado de equinos.

O avanço das biotecnologias e o constante uso do sêmen refrigerado na espécie equina proporcionou a otimização da inseminação artificial (IA), intensificando desse modo, a utilização de produtos, como os diluentes comerciais, que possibilitam melhorias na manutenção e na qualidade seminal durante o processo de resfriamento. O presente trabalho teve como objetivo avaliar características seminais de motilidade e vigor, em diferentes diluentes comercias (a base de leite desnatado, de caseína, e de leite com adição de colesterol), e em momentos distintos, sendo eles 12, 24 e 36 horas. De acordo com os resultados obtidos para análises seminais de motilidade e vigor, não se observou diferença estatística para os meios testados. Concluindo-se que em todos os meios houve uma boa manutenção da longevidade da célula espermática, sendo assim, indicados para o emprego no resfriamento do sêmen equino

In vitro Metabolism of Grandisin, a Lignan with Anti-chagasic Activity

Tetrahydrofuran lignans represent a well-known group of phenolic compounds capable of acting as antiparasitic agents. In the search for new medicines for the treatment of Chagas disease, one promising compound is grandisin which has shown significant activity on trypomastigote forms of Trypanosoma cruzi. In this work, the in vitro metabolism of grandisin was studied in the pig cecum model and by biomimetic phase I reactions, aiming at an ensuing a preclinical pharmacokinetic investigation. Although grandisin exhibited no metabolization by the pig microbiota, one putative metabolite was formed in a biomimetic model using Jacobsen catalyst. The putative metabolite was tested against T. cruzi revealing loss of activity in comparison to grandisin.FINEPFINEPFAPESPFAPESPCAPESCAPESCNPqCNP

Reproductive parameters in bulls: seminal plasma proteomics

Na espécie bovina existem animais que acumulam espermatozoides senescentes na cauda do epidídimo, devido a abstinência sexual, esses animais caracterizam-se por ejacularem grande volume de sêmen com baixa motilidade espermática e que com coletas consecutivas tem o aumento gradual dessa característica. Essa condição se apresenta principalmente em touros criados sob manejo extensivo, e o uso desses animais pode diminuir a eficiência reprodutiva durante a estação de monta (BARTH 2007). O plasma seminal é uma mistura bioquimicamente complexa e que tem importância significativa na regulação da função espermática. Diante disso, o objetivo desse estudo foi identificar o perfil proteômico em ejaculados de touros aprovados e em repouso sexual imediatamente após a avaliação do primeiro ejaculado. Foram utilizados seis touros em repouso sexual, e dois animais como controle. Três grupos foram formados, sendo o grupo A constituído por amostras do sêmen da primeira coleta dos touros em repouso sexual; grupo B: amostra de sêmen da última coleta dos touros em repouso sexual; e grupo C: amostra de sêmen dos touros controle. Todos os touros foram submetidos à avaliação andrológica pelo método de eletroejaculação e foram avaliados os aspectos físicos e morfológicos dos ejaculado por meio de microscopia óptica convencional e de contraste de fase. Depois das análises, os ejaculados foram centrifugados e os sobrenadantes (plasma seminal) foram usados para estudo proteômico. Depois da centrifugação, as proteínas do plasma seminal foram quantificadas pelo método de Bradford e analisadas por meio de eletroforese monodimensional e espectrometria de massas (LC-MS/MS). As proteínas foram identificadas pelo MASCOT e validadas estatisticamente usando o software SCAFFOLD Q+. As proteínas foram caracterizadas de acordo a suas classes e processos biológicos pelo STRAP usando os termos de ontologia gênica depositados no banco de dados UniprotKB. As proteínas diferencialmente abundantes foram quantificadas pelo método emPAI considerando unicamente aquelas proteínas com foldchange superior a 1,5 vezes. Os dados referentes à motilidade espermática foram submetidos à transformação angular (Y^'=arcsen√Y). As variáveis quantitativas foram analisadas por ANOVA. As variáveis qualitativas foram analisadas pelo teste de Kruskal-Wallis. O nível de significância adotado foi α = 5%. Foram encontratros 1,67% de touros em repouso sexual (7/419). Houve diferença (p<0,05) em relação à motilidade, vigor e turbilhonamento espermático entre os grupos, porém não houve diferença das características morfológicas. Na análise proteômica foram identificadas 93 proteínas no plasma seminal. A maioria delas envolvidas no processo celular (36,22%), regulação biológica (35,20%) e interação celular (19,12%). Das proteínas identificadas 6 foram diferencialmente expressas entre o grupo A e B (ALBU, NPC2, Z13, SPAD1, CCL2, F1N1Z8), e 4 (Q4R0H2, SPAD1, NPC2 e Z13) foram diferencialmente expressas entre B e C. Em relação às funções moleculares das proteínas, as principais funções foram de ligação (49,50 %) e atividade catalítica (35,34%). Palavra-chave: Andrologia. Cruzamento. SêmenIn bovine species there are animals that accumulate senescent sperm in the tail of the epididymis, due to sexual abstinence, these animals are characterized by ejaculating large volume of semen with low sperm motility and that with consecutive collections has the gradual increase of this characteristic. This condition occurs mainly in bulls reared under extensive management, and the use of these animals may decrease reproductive efficiency during the breeding season. Seminal plasma is a biochemically complex mixture that is of significant importance in regulating sperm function. Therefore, the objective of this study was to identify the proteomic profile in ejaculates of approved bulls and in sexual rest immediately after the evaluation of the first ejaculate. Six bulls were used in sexual rest, and two animals as control. Three groups were formed, and group A consisted of semen samples from the first collection of the resting bulls; group B: semen sample from the last collection of bulls at sexual rest; and group C: semen sample from control bulls. All bulls were submitted to andrological evaluation by the electroejaculation method and the physical and morphological aspects of the ejaculate were evaluated by conventional optical microscopy and phase contrast. After the analyzes, the ejaculates were centrifuged and the supernatants (seminal plasma) were used for proteomic study. After centrifugation, seminal plasma proteins were quantified by the Bradford method and analyzed by one-dimensional electrophoresis and mass spectrometry (LC-MS / MS). The proteins were identified by MASCOT and statistically validated using SCAFFOLD Q + software. Proteins were characterized according to their classes and biological processes by STRAP using the gene ontology terms deposited in the UniprotKB database. Differentially abundant proteins were quantified by the emPAI method considering only those proteins with foldchange greater than 1.5 times. The sperm motility data were submitted to angular transformation (Y ^ '= arcsen√Y). Quantitative variables were analyzed by ANOVA. Qualitative variables were analyzed by the Kruskal-Wallis test. The significance level adopted was α = 5%. A total of 1.67% (7/419) of sexually resting bulls were found. There was a difference (p <0.05) in relation to motility, vigor and sperm swirling between the groups, but there was no difference in morphological characteristics. In proteomic analysis 93 proteins were identified in seminal plasma. Most of them involved in the cellular process (36.22%), biological regulation (35.20%) and cellular interaction (19.12%). Of the identified proteins 6 were differentially expressed between group A and B (ALBU, NPC2, Z13, SPAD1, CCL2, F1N1Z8), and 4 (Q4R0H2, SPAD1, NPC2 and Z13) were differentially expressed between B and C. The main molecular functions of proteins were binding (49.50%) and catalytic activity (35.34%). Keywords: Andrology. Crossbred. Seme

In vitro evaluation of cryopreserved bovine semen and its correlation with in vivo fertility

Um dos problemas mais importantes da indústria da inseminação artificial é a determinação do potencial de fertilidade do sêmen, pois, as metodologias convencionais utilizadas para avaliar a qualidade de sêmen não têm demonstrado boa correlação com fertilidade dos espermatozoides. Resultados de recentes estudos com sêmen congelado-descongelados quando submetidos a uma filtragem antes das avaliações espermáticas tem melhor correlação entre as características dos espermatozoides e a fertilidade in vivo. Além disso, a utilização de diferentes sondas fluorescentes nas análises do sêmen permitem avaliação ampla e individual dos espermatozoides, além de sua qualidade funcional, bioquímica e estrutural. Desde modo, o presente estudo teve como objetivo empregar diferentes testes complementares de avaliação espermática pré e pós-filtragem em solução coloidal de sêmen criopreservado e verificar sua correlação com a fertilidade in vivo. Foram utilizadas duas amostras (palhetas da mesma partida) de sêmen congelado de 18 touros doadores de sêmen, sendo 11 reprodutores da raça Nelore, cinco Composto Montana Tropical, um Senepol, e um Red Angus, escolhidos de acordo com os animais utilizados em programa de Inseminação Artificial em Tempo Fixo (IATF) imposto em um rebanho comercial. Os dados obtidos foram divididos de acordo com a fertilidade obtida após as inseminações, sendo fertilidade satisfatória (≥ 55 % de taxa de prenhez) e fertilidade insatisfatória (˂ 55 % de taxa de prenhez) Todas as fêmeas foram sincronizadas por uso de protocolos de sincronização e inseminação artificial em tempo fixo (IATF). Os testes empregados para análise do sêmen foram motilidade e vigor espermático, análise quanto à integridade de membrana plasmática e acrossomal, produção de peróxido de hidrogênio intracelular, e organização da bicamada, por meio de sondas fluorescentes (IP, FITC-PSA, M540) empregando a Citômetria de fluxo. Houve efeito de touro sobre a taxa de prenhez (P<0,05) com máximo de 76 % (19/25) e mínimo de 15,6 % (5/32) sendo a média de 54,6 % (723/1325) de prenhez com uma inseminação. Quanto aos grupos de fertilidade houve diferença (P<0,05) entre as médias, sendo 62 % de prenhez para o grupo FERTILIDADE SATISFATÓRIA e 40,6 % para o grupo FERTILIDADE INSATISFATÓRIA. Na avaliação convencional do sêmen, o grupo de fertilidade satisfatória apresentou maior média de motilidade espermática progressiva retilínea (60,49%) que o grupo de fertilidade insatisfatória. Nas avaliações realizadas por citometria de fluxo, o grupo de fertilidade insatisfatória obteve maiores médias percentuais para populações celulares com membrana plasmática lesionada e membrana acrossomal intacta (7,18 %), para células com alto grau de peroxidação e lesão de membrana plasmática (25,5 %) e menor média percentual de populações celulares com membrana plasmática íntegra e estável (19,5 %) que diferiram (p<0,05) do grupo e fertilidade satisfatória. A centrifugação do sêmen em solução coloidal foi eficiente em reter as células com lesões na membrana plasmática e células com lesões na membrana acrossomal (16,8; 31,9 e 7,0 %); células com alta peroxidação intracelular (4,17; 64,3 e 15,0 %). Concluiu-se que as populações de espermatozóides com lesão na membrana acrossomal representam o maior percentual das células espermáticas em sêmen submetido ao processo de congelamento e relacionam-se com à alta taxa de fertilidade obtida em programa de inseminação artificial em tempo fixo (IATF); populações de espermatozóides com lesão de membrana plasmática e alta taxa de peroxidação intracelular estão relacionadas à baixa taxa de fertilidade em programas de inseminação artificial em tempo fixo (IATF); a seleção de espermatozoides com membranas acrossomal e espermática intactas é possível pela centrifugação associado ao uso de soluções coloidais (Androcoll®) e a avaliação da motilidade espermática progressiva retilina está relacionada a alta taxa de fertilidade em programa de inseminação artificial em tempo fixo.One of the most important problems of the artificial insemination industry is the determination of semen fertility potential for the conventional methods used to assess the quality of semen have not shown good correlation with fertility of sperm. Results of recent studies with frozen-thawed semen when subjected to filtering before the spermatic reviews have better correlation between the characteristics of sperm and fertility in vivo. In addition, the use of different fluorescent probes in semen analysis allow for a wide and individual assessment of sperm, as well as its functional, biochemical and structural. In this way, the present study aims to employ different complementary tests pre sperm and post-filtering in colloidal solution and check its correlation with fertility in vivo semen. Two samples were used (vanes in the same match) frozen semen of 18 donor bulls semen, 11 breeding Nelore five Composite Montana Tropical, one Senepol, and Red Angus, chosen according to the animals used in program Fixed-Time Artificial insemination (FTAI). It was previously established that the data obtained would be divided into fertility group, being satisfactory (≥ 55% pregnancy rate) and unsatisfactory (˂ 55% pregnancy rate) All females were synchronized by using synchronization protocols and fixed-time artificial insemination (FTAI). For data analysis of semen were motility and sperm vigor, analysis of the integrity of plasma and acrosomal membrane of intracellular hydrogen peroxide production, and organization of the bilayer by means of fluorescent probes (IP FITC-PSA, M540) in flow cytometer. There was bull effect on the pregnancy rate (P<0.05) with a maximum of 76% (19/25) and a minimum of 15.6% (5/32) with a mean of 54.6% (723/1325 ) of pregnancy. As for fertility groups difference (P <0.05) between the average, 62% of pregnancy for SATISFACTORY group and 40.6% for UNSATISFACTORY group. In conventional semen evaluation, the satisfactory group showed higher mean rectilinear motility (60.49%) than the poor group. In the assessments performed by flow cytometry, the UNSATISFACTORY group had higher average percentage for cell populations with damaged plasma membrane and intact acrosome membrane (7.18%; IP + PSA-) for cells with a high degree of peroxidation and plasma membrane injury (25.5%; DCFDA + PI +) and lower mean percentage of cell populations with full and stable plasma membrane (19.5% M540-6DCFDA +) that differed (p <0.05) SATISFACTORY group. The centrifugation of semen in colloidal solution was efficient in retaining the cells with lesions in the plasma membrane and cell injuries in the acrosome membrane (16.8% IP + PSA-; 31.9%, IP + PSA +, 7.0% IP-PSA +); cells with high intracellular peroxidation (4.17% DCFDA-PI +; 64.3% DCFDA + PI + 15.0%, + DCFDA PI). It was concluded that the populations of sperm with injury acrosome membrane represent the highest percentage of sperm cells in semen subjected to the freezing process and are related to the high fertility rate obtained in fixed-time artificial insemination (FTAI); sperm populations of plasma membrane damage and high intracellular peroxidation rate represent the highest percentages of sperm populations of cryopreserved semen, and are related to low fertility rate in fixed-time artificial insemination (FTAI); the selection of sperm with intact acrosome and sperm membranes is possible by centrifugation associated with the use of colloidal solutions (Androcoll®) and evaluation of sperm motility by light microscopy is related to high fertility rate in fixed-time artificial insemination (FTAI) program

Relationship of testicular biometry with semen variables in breeding soundness evaluation of Nellore bulls

This study aimed to evaluate the correlation between testicular biometry and semen variables, as well as, to relate testicular variables to the probability of selecting Nellore bulls with desirable sperm morphology when conducting breeding soundness evaluations (BSE). A total of 2055 BSEs from 506 bulls comprised the dataset. Biometric variables evaluated were: scrotal circumference, testicular volume, width, length, ratio and eccentricity; and semen variables were sperm motility, major sperm defects, minor sperm defects and normal sperm. Data of testicular biometry were correlated with data for semen variables using the Pearson’s correlation assessment. Effects of testicular variables in selecting for sperm morphology of bulls in the BSE were evaluated by logistic regression. Scrotal circumference, testicular volume, length and width were positively correlated to sperm motility (0.18 to 0.19) and normal sperm (0.24 to 0.27) and negatively correlated with values for major defects (−0.24 to −0.27), but for testicular ratio and eccentricity there were coefficients near zero for all semen traits. Testicular ratio and eccentricity were not suitable for predicting the probability of selecting a bull based on semen variables using the BSE, but scrotal circumference, testicular volume, length and width were highly significant (P <  0.0001) with moderate values of area under ROC (Receiver Operating Characteristics) curve (0.608 to 0.620)

Differential abundances of four forms of binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability

The Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process

Supplementary Tables.

List of proteins identified in the seminal plasma of Nellore bulls and their functional classification by KOG.</p

Aspects of sexual precocity and morphometry of uterus, placenta and embryos/fetuses in Piau breed and Commercial line gilts

In view of the importance of the genetic material of local breeds in the swine industry and the lack of information about reproductive performance of Piau females, two experiments were conducted to evaluate puberty and sexual maturity as well as the morphometry of embryos/fetuses, placenta and uterus during the first 90 days of gestation in Piau breed and Commercial line gilts. In experiment I, 37 Piau and 25 commercial line gilts were used. From the 120 days of age, detection of estrus was performed using mature boars from the first to third estrus of each gilt. Data regarding to age, body weight and estrus duration were recorded. After third estrus, females were slaughtered and ovaries were collected to determine ovulation rate. In experiment II, 36 Piau and 18 commercial line gilts were distributed into three groups according to the mating: Commercial, commercial line females x commercial line male; cross-mated, Piau females x commercial line male; and Piau, Piau females x Piau male. Gilts were slaughtered at 7, 15, 30, 45, 60 and 90 days of pregnancy. Piau females reached puberty and sexual maturity at the same age as commercial line females, but with lower weight; moreover, Piau group showed negative correlations of birth weight with puberty (−0.27) and sexual maturity (−0.29). Commercial gilts presented higher ovulation rate, weight and length of uterus, and length and thoracic circumference of fetuses. Nevertheless, number of fetuses was similar in all groups at 90 days of gestation suggesting that Piau females present higher survival rates of the conceptuses. The results showed differences between the genetic groups related to fetal and placental development, gestational losses, number of ovulations and uterine development. In addition, an intermediate status of fetal weight was observed in Piau/Commercial line crossbred conceptuses; thus, the selection of Piau females on reproductive traits to be mated with commercial line males would be an alternative to contribute to improvement of intramuscular fat content

Can scrotal circumference-based selection discard bulls with good productive and reproductive potential?

<div><p>Nonlinear mixed models were used to describe longitudinal scrotal circumference (SC) measurements of Nellore bulls. Models comparisons were based on Akaike’s information criterion, Bayesian information criterion, error sum of squares, adjusted R² and percentage of convergence. Sequentially, the best model was used to compare the SC growth curve in bulls divergently classified according to SC at 18–21 months of age. For this, bulls were classified into five groups: SC < 28cm; 28cm ≤ SC < 30cm, 30cm ≤ SC < 32cm, 32cm ≤ SC < 34cm and SC ≥ 34cm. Michaelis-Menten model showed the best fit according to the mentioned criteria. In this model, <i>β</i>_<i>1</i> is the asymptotic SC value and <i>β</i>_<i>2</i> represents the time to half-final growth and may be related to sexual precocity. Parameters of the individual estimated growth curves were used to create a new dataset to evaluate the effect of the classification, farms, and year of birth on <i>β</i>_<i>1</i> and <i>β</i>_<i>2</i> parameters. Bulls of the largest SC group presented a larger predicted SC along all analyzed periods; nevertheless, smaller SC group showed predicted SC similar to intermediate SC groups (28cm ≤ SC < 32cm), around 1200 days of age. In this context, bulls classified as improper for reproduction at 18–21 months old can reach a similar condition to those considered as good condition. In terms of classification at 18–21 months, asymptotic SC was similar among groups, farms and years; however, <i>β</i>_<i>2</i> differed among groups indicating that differences in growth curves are related to sexual precocity. In summary, it seems that selection based on SC at too early ages may lead to discard bulls with suitable reproductive potential.</p></div