Transferrina como marcador de maturação pulmonar em cordeiros nascidos a termo ou prematuros / Transferrin as a marker of pulmonary maturation in lambs born at term or premature

O objetivo do estudo foi procurar proteínas de fase aguda que possam indicar sinais de maturação no neonato prematuro, por meio da quantificação sérica delas. Identificou-se a imunoglobulina A, a ceruloplasmina, a haptoglobina, a glicoproteína ácida, a transferrina, a albumina e as imunoglobulinas G de cadeias leve e pesada, pela comparação do perfil dos proteinogramas de cordeiros nascidos a termo com os prematuros submetidos a diferentes protocolos terapêuticos, a fim de estimular a atividade respiratória. Constituíram-se seis grupos: PN (n= 9): nascidos de parto normal; CN (n= 7): nascidos de cesariana em tempo normal de gestação; CP (n= 6): nascidos de cesariana prematura sem nenhum tipo de tratamento; DEX (n= 9): prematuros cujas mães receberam dexametasona pré-parto; SURF (n= 6): prematuros tratados com surfactante; e DEXSURF (n= 6): prematuros tratados com surfactante cujas mães receberam dexametasona pré-parto. As avaliações foram realizadas nos momentos imediatamente após o nascimento (M0), após 24 (M24) e após 48 horas (M48). As amostras foram processadas por meio de eletroforese em gel de poliacrilamida contendo dodecil sulfato de sódio (SDS-PAGE). A albumina, as imunoglobulinas e a proteína total dos cordeiros tiveram elevação após a ingestão de colostro. Maiores valores séricos de transferrina são referentes a maior período gestacional, podendo essa proteína ser utilizada como marcador de maturação neonatal.(AU)

The aim of this study was to search for acute phase proteins that could indicate signs of maturation in the premature neonate by quantifying them in serum. Immunoglobulin A, ceruloplasmin, haptoglobin, acid glycoprotein, tranferrin, albumin, light and heavy chain immunoglobulin G were quantified, comparing the profile of proteinograms from term to preterm lambs submitted to different protocols that stimulate respiratory activity. Six groups were used: PN (n= 9): born from normal birth; CN (n= 7): born from caesarean section at normal time of gestation; CP (n= 6): born from premature cesarean without any type of treatment; DEX (n= 9) preterm whose mothers received prepartum dexamethasone; SURF (n= 6) preterm treated with surfactant; DEXSURF (n= 6): preterm treated with surfactant whose mothers received prepartum dexamethasone. The evaluations were performed immediately after birth (M 0), after 24 and 48 hours (M 24 and M 48). Samples were processed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Albumin, immunoglobulins, and serum total protein of the lambs were elevated, after colostrum ingestion. Higher serum transferrin values refer to a longer gestational period, and this protein may be used as a marker of neonatal maturation.(AU)

Does sperm quality and DNA integrity differ in cryopreserved semen samples from young, adult, and aged Nellore bulls?

BACKGROUND: In humans, it is now well documented that rising paternal age is correlated with decreased sperm DNA integrity and embryonic developmental failures. On the other side of the coin, it is also reported that very young fathers such as teenagers carry an increased risk of adverse birth outcomes. These observations suggest that, at least in humans, there is an age window for optimal sperm DNA integrity. In bovine, little is known about sperm DNA quality in young bulls and how it evolves with age. This study aimed to fill in this gap as it may be of importance for the bovine industry to know when exactly a bull is an optimal performer for reproductive programs. METHODS: Forty Nellore bulls were divided into three age groups: 1.8 to 2 years - young bulls; 3.5 to 7 years - adult bulls; and 8 to 14.3 years - aged bulls. Three ejaculates were collected from each bull, cryopreserved and evaluated for various parameters including: computer-assisted sperm analysis (CASA), plasma membrane and acrosome integrity, mitochondrial potential, sperm nuclear protamination, DNA oxidative damage, and Sperm Chromatin Structure Assay (SCSA). RESULTS: We report here that young bulls presented superior values for motility, plasma and acrosomal membrane integrity, and high mitochondrial potential. However, they also presented higher values for sperm morphological abnormalities compared to adult and aged animal groups (p < 0.05). In addition, young bulls exhibited more defective protamination than older animals did. The oldest bulls showed more nuclear oxidative damage than the younger groups of bulls while both the young and aged groups were found more susceptible to DNA denaturation as revealed with the SCSA test (p < 0.05). CONCLUSION: These results indicate that young bulls spermatozoa best survived the freezing procedure, followed by adult and aged bulls. However, young and aged bulls were found to be more susceptible to DNA damage, respectively caused by protamine deficiency and oxidation. Therefore, although young bulls have correct semen parameters according to classical evaluation, our results indicate that they may show some structural nuclear immaturity.

CONTEXTE: Chez l'homme, de nombreuses données indiquent maintenant que l'avancée de l'âge du père est associée à une réduction de l'intégrité de l'ADN des spermatozoïdes et aux échecs de développement embryonnaire. D'un autre côté, il est aussi rapporté que les jeunes pères, tels les adolescents, sont porteurs d'un risque accru d'issue défavorable de la grossesse. Ces observations suggèrent que, au moins chez l'humain, il existe une tranche d'âge dans laquelle l'intégrité de l'ADN des spermatozoïdes est optimale. Chez les bovins, on dispose de peu de connaissances sur la qualité de l'ADN des spermatozoïdes des jeunes taureaux et sur son mode d'évolution avec l'âge. La présente étude a pour but de combler ce manque car il peut être important, pour l'industrie bovine, de savoir à quelle période précise un taureau est un reproducteur optimal pour les programmes de reproduction. MATÉRIEL ET MÉTHODES: Quarante taureaux Nellore ont été répartis en trois groupes d'âge: 1, 8 à 2 ans ­ jeunes taureaux; 3,5 à 7 ans ­ taureaux adultes; et 8 à 14,3 ans ­ taureaux âgés. Trois éjaculats ont été collectés par taureau, cryopréservés et évalués pour différents paramètres incluant l'analyse assistée du sperme par ordinateur (CASA), l'intégrité des membranes plasmique et acrosomique, le potentiel mitochondrial, la protamination du noyau, l'altération oxydative de l'ADN et l'évaluation de la structure de la chromatine du noyau du spermatozoïde (SCSA). RÉSULTATS: Nous rapportons ici que les jeunes taureaux présentent des valeurs supérieures de la mobilité des spermatozoïdes et de l'intégrité des membranes plasmique et acrosomique, ainsi qu'un potentiel mitochondrial élevé. Cependant, les jeunes taureaux présentent aussi des valeurs plus élevées d'anomalies morphologiques des spermatozoïdes que celles des groupes adulte et âgé (p < 0.05). De plus, les jeunes taureaux ont une protamination plus défectueuse que celle des taureaux plus âgés. Les taureaux les plus âgés présentent plus d'altérations oxydatives du noyau que les jeunes taureaux alors que les deux groupes -jeunes et âgés- sont plus susceptibles d'avoir une dénaturation de l'ADN nucléaire comme indiqué par le SCSA (p < 0.05). CONCLUSIONS: Ces résultats indiquent que les spermatozoïdes des jeunes taureaux survivent le mieux au processus de congélation, suivis par les adultes puis les âgés. Toutefois, les jeunes taureaux et les âgés sont plus susceptibles d'avoir une altération de l'ADN, causée respectivement par une protamination déficiente et une oxydation. Par conséquent, bien que les jeunes taureaux aient des paramètres spermatiques corrects à l'évaluation classique, nos résultats indiquent que leurs spermatozoïdes peuvent présenter un certain degré d'immaturité structurale nucléaire.

Parâmetros hematológicos e perfil bioquímico renal de cordeiros nascidos a termo e prematuros / Hematologic parameters and renal biochemical profile in full-term and premature lambs

O objetivo do presente estudo foi avaliar as variáveis hematológicas e o perfil bioquímico renal sérico de cordeiros nascidos a termo e prematuros do nascimento às 48 horas de vida, bem como verificar a influência da dexametasona sobre tais variáveis. Foram constituídos quatros grupos experimentais: PN (cordeiros nascidos de parto normal, n=15, média de 146 dias); PNDEX (cordeiros nascidos de parto normal cujas mães receberam 16mg de dexametasona aos 141 de gestação, n=8, média de 143 dias); PRE (cordeiros prematuros nascidos de cesarianas aos 138 dias de gestação, n=10) e PREDEX (cordeiros prematuros nascidos de cesarianas aos 138 dias de gestação cujas mães receberam 16mg de dexametasona dois dias antes, n=9). Os valores médios do volume globular e de hemoglobina diminuíram ao longo das 48 horas de observação, nos quatro grupos experimentais, porém dentro dos limites fisiológicos para a espécie. Houve variação da concentração plasmática de proteínas totais em todos os momentos, sendo os menores valores no grupo PRE. A contagem leucocitária foi mais alta no grupo PN apenas no M24h. Ao longo do período, apenas o grupo PN mostrou diferença entre o M24h e os demais momentos, e o grupo PRE apresentou os menores valores de neutrófilos no M0h, M15min e M60min. As concentrações séricas de creatinina foram mais altas no grupo PRE no M60min, M24h e M48h. Em todos os grupos, houve diminuição no M24h e M48h. Os parâmetros avaliados foram afetados pela prematuridade na espécie ovina e a dexametasona teve influência positiva sobre a taxa de sobrevivência dos animais prematuros.(AU)

The aim of the study was to evaluate hematologic parameters and renal biochemical profile of full-term and premature lambs from birth to 48 hours of life, and assess the effect of dexamethasone on such variables. Four experimental groups were formed: NDG (normal delivery group - lambs vaginally delivered, n=15, average of 146-day gestation); NDEXG (normal delivery with dexamethasone group - lambs vaginally delivered whose mothers received 16 mg of dexamethasone at 141 days of gestation, n=8, average of 143-day gestation); PRE (premature lambs born by cesarean section at 138 days of gestation, n=10) and PREDEX (premature lambs born by cesarean section at 138 days gestation, whose mothers received 16 mg of dexamethasone two days before, n=9). Mean values of cell volume and hemoglobin content decreased during the observation period of 48 hours in all groups, but within the physiologic limits for ovine species. There was significant variation in plasma concentration of total protein in all periods evaluated, with the lowest mean values in PRE group. The leukocyte count was significantly higher in PN group only in M24h. Throughout the observation period, only PN group showed differences between M24h and the other moments and PRE group showed the lowest values of neutrophils in M0h, M15min and M60min. Serum creatinine concentration was significantly higher in group PRE in M60min, M24h and M48h. In all groups, there was a decrease in M24 and M48. The evaluated parameters were affected by prematurity in sheep and dexamethasone had a positive effect on the survival rate of premature animals.(AU)

Sperm morphometry and chromatin condensation in Nelore bulls of different ages and their effects on IVF.

The aim of this study was to evaluate the chromatin packing and sperm head morphometry of cryopreserved semen of Nelore bulls (Bos taurus indicus) of different ages. Furthermore, the influence of the degree of chromatin compaction on in vitro embryo production (IVP) was investigated. Forty bulls were divided into three groups: young (1.8-2 years), adult (3.5-7 years), and senile (8-14.3 years). The ejaculates were frozen according to standards established by the Artificial Insemination Center located in the Southeast of Brazil. Toluidine blue staining was used for simultaneous evaluation of the sperm chromatin and sperm head morphometry. Chromomycin A3 (CMA3) was applied to analyze sperm protamination and IVP for embryonic development. Spermatozoa of young bulls presented higher values for area (A, pixels), perimeter (P, pixels), and width (W, pixels) compared to adults and senile (young: A = 1848.5 ± 119.79, P = 10.23 ± 0.29, and W = 1.95 ± 0.1; adults: A = 1672.9 ± 104.46, P = 9.86 ± 0.33, and W = 1.81 ± 0.06; senile: A = 1723.1 ± 124.41, P = 9.97 ± 0.33, and W = 1.83 ± 0.09; P < 0.0001) and showed higher protamination deficiency when analyzed by CMA3 (young: 1.57 ± 0.76; adults: 1.09 ± 0.63, and senile: 0.90 ± 0.59; P < 0.05). Likewise, variables of sperm head size (A, P, and W) and protamination assessed by CMA3 showed negative correlation with age and positive correlation with ellipticity, evaluated by toluidine blue method (P < 0.05). Sperm head area was larger in spermatozoa presenting chromatin instabilities than spermatozoa without chromatin alteration (P < 0.0001). There was no difference in IVP when using semen with larger or smaller portions of spermatozoa with chromatin instabilities, indicating that the proportion of sperm with abnormal chromatin compaction (4%-16.15%) did not interfere with early embryonic development. From our results, it can be concluded that sperm of young Nelore bulls have larger heads compared to adults and senile due to reduced protamine content when evaluated by CMA3 and higher proportion of major sperm defects assessed by differential interference contrast microscopy.

Impaired protamination and sperm DNA damage in a Nellore bull with high percentages of morphological sperm defects in comparison to normospermic bulls / Deficiências de protaminação e danos de DNA no sêmen de um touro Nelore com elevada porcentagem de patologias espermáticas em comparação a touros normospérmicos

The routine semen evaluation assessing sperm concentration, motility and morphology, does not identify subtle defects in sperm chromatin architecture. Bulls appear to have stable chromatin, with low levels of DNA fragmentation. However, the nature of fragmentation and its impact on fertility remain unclear and there are no detailed reports characterizing the DNA organization and damage in this species. The intensive genetic selection, the use of artificial insemination and in vitro embryo production associated to the cryopreservation process can contribute to the chromatin damage and highlights the importance of sperm DNA integrity for the success of these technologies. Frozen-thawed semen samples from three ejaculates from a Nellore bull showed high levels of morphological sperm abnormalities (55.8±5.1%), and were selected for complementary tests. Damage of acrosomal (76.9±8.9%) and plasma membranes (75.7±9.3%) as well as sperm DNA strand breaks (13.8±9.5%) and protamination deficiency (3.7±0.6%) were significantly higher compared to the values measured in the semen of five Nellore bulls with normospermia (24.3±3.3%; 24.5±6.1%; 0.6±0.5%; 0.4±0.6% for acrosome, plasma membrane, DNA breaks and protamine deficiency, respectively) (P<0.05). Motility and percentage of spermatozoa with low mitochondrial potential showed no differences between groups. This study shows how routine semen analyses (in this case morphology) may point to the length and complexity of sperm cell damage emphasizing the importance of sperm function testing.(AU)

O exame de rotina de sêmen, o qual avalia a concentração de espermatozoides, a motilidade e a morfologia, pode não identificar defeitos sutis na arquitetura da cromatina de espermatozoides. Os touros parecem ter cromatina estável com baixos níveis de fragmentação do DNA. No entanto, a natureza da fragmentação e o seu impacto sobre a fertilidade ainda não estão claros e não há relatos que caracterizam a organização do DNA e os danos nessa espécie com mais detalhes. A seleção genética intensiva e o uso da inseminação artificial e da produção in vitro de embriões, além do processo de criopreservação, podem contribuir para o dano da cromatina, e sabe-se a importância da integridade do DNA espermático para o sucesso dessas tecnologias. Amostras de sêmen de três ejaculados de um touro Nelore com altos níveis de alterações morfológicas (55,8±5,1%) foram selecionadas para realização de exames complementares. Os danos de acrossoma (76,9±8,9%) e das membranas plasmáticas (75,7±9,3%), bem como quebras de fita de DNA de espermatozoides (13,8±9,5) e deficiência de protamina (3,7±0,6) foram significativamente maiores em comparação aos valores avaliados no sêmen de cinco touros Nelore com normospermia (24,3±3,3%; 24,5±6,1%; 0,6±0,5%; 0,4±0,6% para acrossoma, membrana plasmática, quebras de DNA e deficiência de protamina, respectivamente) (p<0,05). Motilidade e porcentagem de espermatozoides com baixo potencial mitocondrial não diferiram estatisticamente. Essas avaliações mostram que análises de sêmen de rotina (neste caso, morfologia) podem apontar para a extensão e a complexidade dos danos na célula espermática, o que indica que a deficiência de protamina e os danos no DNA podem ocorrer simultaneamente a defeitos morfológicos. Tal ocorrência enfatiza a importância das análises de sêmen clássicas e dos testes complementares.(AU)

Molecular characterisation of Cryptosporidium spp. in lambs in the South Central region of the State of São Paulo / Caracterização molecular de Cryptosporidium spp. em cordeiros na região centro sul do Estado de São Paulo

Considering the proximity of sheep farmers to animals that are possibly diseased or releasing fecal oocysts into the environment and the marked pathogenicity in lambs, the aim of this study was to determine the occurrence and to molecularly characterize the infection by Cryptosporidium spp. in lambs in the South Central region of the state of São Paulo, Brazil. A total of 193 fecal samples were collected from sheep of several breeds, males and females, aged up to one year. Polymerase chain reaction (nested-PCR) was used to amplify DNA fragments from the subunit 18S rRNA gene and indicated 15% positivity; sequencing of amplified fragments was possible for 19 samples. Analysis of the obtained sequences showed that the identified species were Cryptosporidium xiaoi for 15 samples, constituting thus the first molecular characterization study of this Cryptosporidium species in Brazil. Cryptosporidium ubiquitum was identified for three samples and Cryptosporidium meleagridis for one sample; the latter two are considered zoonotic species.(AU)

Devido à proximidade de criadores de ovinos com animais possivelmente doentes e/ou eliminando oocistos fecais no ambiente e pela acentuada patogenicidade em cordeiros o objetivo foi, determinar a ocorrência e caracterizar molecularmente a infecção por Cryptosporidium spp. em cordeiros na região Centro Sul do Estado de São Paulo, Brasil. Num total de 193 amostras de fezes foram coletadas de ovinos de diversas raças, machos e fêmeas, com idade de até um ano. Por meio da reação em cadeia da polimerase (nested PCR) para a amplificação de fragmentos de DNA a partir do gene da subunidade 18S do rRNA houve positividade de 15% e o sequenciamento dos fragmentos amplificados foi possível em 19 amostras. A análise das sequências obtidas mostraram que as espécies identificadas nesses animais foram Cryptosporidium xiaoi em 15 amostras, sendo o primeiro estudo de caracterização molecular desta espécie de Cryptosporidium no Brasil. Cryptosporidium ubiquitum em três amostras, e Cryptosporidium meleagridis em uma amostra, sendo estas duas últimas consideradas espécies zoonóticas.(AU)

Avaliação do efeito trombogênico da perfusão regional intravenosa com gentamicina em equinos / Evaluation of intravenous regional perfusion thrombogenic effect with gentamicin in equine

A extremidade distal dos equinos é frequentemente afetada por processos sépticos de difícil tratamento. A perfusão regional intravenosa de antimicrobianos é uma opção de tratamento para esses processos, pois aumenta a concentração do fármaco no local mediante pequenas doses. Entretanto, apesar de ser muito utilizada recentemente, ainda apresenta várias questões. O objetivo deste trabalho foi avaliar as possíveis complicações da técnica, com o uso da gentamicina na dose de 2,2mg/kg na veia cefálica de equinos. Utilizaram-se 15 equinos hígidos divididos aleatoriamente em três grupos de cinco animais: grupo somente com torniquete (GT), grupo que recebeu 40mL de solução fisiológica (GSF) e grupo que recebeu gentamicina na dose de 2,2mg/kg, acrescida de solução fisiológica até completar o volume de 40mL (GSG). O membro perfundido foi avaliado por termografia e ultrassonografia duplex em cores ao longo do tempo. Os dados foram submetidos à análise pelos testes de Tukey e de Dunnett, e o nível de significância adotado foi de P<0,05. Todos os grupos apresentaram escore trombótico zero, e não houve diferença significativa entre os grupos quanto ao diâmetro médio da veia e da temperatura dos membros. A antibiose intravenosa regional com 2,2mg/kg de gentamicina, em dose única, não acarretou o desenvolvimento de trombose na veia cefálica...

The distal end of the horse is often affected by septic processes that are difficult to treat. The intravenous regional perfusion of antimicrobials is a treatment option for these processes, increasing the local concentration of the drug using small doses, and is currently widely used, but still presenting various issues. The aim of this study was to evaluate the possible complications of the technique, using gentamicin at a dose of 2.2mg/kg in the cephalic vein of horses. Fifteen healthy horses were assigned to three groups of 5 animals. Group 1, tourniquet group (TG), group 2, tourniquet and 40mL of physiologic saline solution (SPG) and group 3, tourniquet and 2.2mg/kg gentamicin completed to 40mL by the addition of physiologic saline solution (ASG). The perfused limb was evaluated by thermography and ultrasound Duplex color over time. The data were analyzed by Tukey's and Dunnett's method, and the accepted significance level was P<0.05. All groups showed thrombotic score zero and there was no significant difference between groups in the average diameter of the vein and member temperatures. The intravenous regional antibiosis with 2.2mg/kg of gentamicin as a single dose did not affect the development of thrombosis in the cephalic vein...

Heparin and penicillamine-hypotaurine-epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development.

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development.

Anestesia local por tumescência com lidocaína em cadelas submetidas a mastectomia / Tumescent local anesthesia with lidocaine in dogs undergoing mastectomy

O presente estudo investigou os benefícios da anestesia por tumescência com lidocaína em cadelas submetidas à mastectomia, visando ao conforto do paciente e à sua recuperação pós-operatória. Foram utilizados sete animais, de peso e raças variadas, que apresentavam neoplasia em região de cadeia mamária e que foram submetidos à cirurgia de mastectomia. Todos os animais receberam o mesmo protocolo anestésico, sendo utilizado como MPA a associação entre acepromazina e morfina, nas doses de 0,04mg/kg e 0,4mg/kg (IM), respectivamente. Após 15 minutos, foi alocado um cateter em veia cefálica e realizou-se a indução com propofol 4mg/kg e midazolam 0,2mg/kg, seguida de manutenção anestésica com isofluorano. Posteriormente à instrumentação, procedeu-se à técnica de anestesia por tumescência com solução gelada composta por ringer lactato, lidocaína 2% sem vasoconstritor e adrenalina, em um volume total de 15mL/kg. Em média, o tempo de duração do procedimento foi de 74±18 minutos. O pico plasmático de lidocaína deu-se entre 30 e 60 minutos após a infiltração da solução. O resgate analgésico foi realizado após sete horas, aproximadamente, da infiltração. Pode-se concluir que a anestesia por tumescência com lidocaína deve ser considerada como constituinte do protocolo anestésico e analgésico de cadelas a serem submetidas à cirurgia de mastectomia, proporcionando estabilidade de parâmetros, segurança e recuperação pós-operatória de boa qualidade.

The present study investigated the benefits of tumescent anesthesia with lidocaine in dogs undergoing mastectomy, seeking the patients' comfort and their postoperative recovery. Seven animals, with different weight and breed, who had cancer in the region of mammary chain underwent mastectomy surgery. All animals received the same anesthetic protocol being used as the association between acepromazine and morphine doses of 0.04mg.kg-1 and 0.4mg.kg-1 (IM), respectively. After 15 minutes a catheter was placed in the cephalic vein and induction with propofol 4mg.kg-1 and 0.2mg.kg-1 followed by maintenance with isoflurane anesthesia was done. After instrumentation, we proceeded to the tumescent anesthesia technique with ice-cold solution consisting of Ringer's lactate, lidocaine 2% without epinephrine and adrenaline in a total volume of 15mL.kg-1. The average duration of the procedure was 74±18 minutes. The plasmatic peak of lidocaine was between 30 and 60 minutes after infiltration. The rescue analgesic was performed after approximately seven hours of infiltration. It can be concluded that the tumescent anesthesia with lidocaine should be considered as a constituent of anesthetic and analgesic protocol in dogs undergoing mastectomy surgery providing parameter stability, safety and good quality postoperative recovery.

Efeito da utilização de diferentes diluidores para a produção in vitro de embriões bovinos / Effect of different bovine semen extenders on in vitro development of bovine embryos

Objetivou-se avaliar as características morfológica e funcional do sêmen bovino congelado comparando-se a eficácia de dois diferentes diluidores. O ejaculado de quatro touros foi dividido em duas partes iguais, uma submetida ao diluidor Tris e gema de ovo (A) e outra ao diluidor à base de lecitina de soja (Andromed®) (B). No experimento I, cinco palhetas dos diluidores A e B de cada touro foram descongeladas e avaliadas quanto à motilidade, vigor, concentração, morfologia espermática e teste de termor-resistência lento. Foram feitas, ainda, avaliação da integridade de membranas, por meio da associação das sondas iodeto de propídio, isotiocionato de fluoresceína - Pisum sativum e carbocianina catiônica lipofílica, e avaliação funcional da membrana plasmática com teste hiposmótico. A avaliação da integridade da cromatina foi realizada pelo método de coloração com laranja de acridina. No experimento II, o sêmen com os diferentes diluidores foi utilizado na fecundação in vitro, sendo observadas taxas de clivagem e desenvolvimento embrionário in vitro. Em relação aos resultados obtidos, apenas a porcentagem de espermatozoides no sêmen congelado foi discretamente maior com o diluidor A, concluindo-se que o diluidor composto por lecitina de soja pode substituir o composto por Tris e gema de ovo, respeitando-se as variações individuais de cada touro utilizado no presente experimento.

The goal of this study was to evaluate the protective effect of egg yolk compared with a soybean lecithin-based extender on morphologic and functional characteristics of frozen bovine semen. The ejaculate of four bulls was divided into two equal parts, one diluted with egg-yolk-Tris extender (A) and the other with a soy-lecithin based extender (Andromed®) (B). In experiment I, five straws of extender A and B from each bull were thawed and assessed regarding motility (subjective and computerized analysis), vigor, concentration, sperm morphology and slow thermoresistance (STR), evaluation of membrane integrity through association of propidium iodite probes (PI), fluorescein isotiocianate - Pisum sativum (FITC-PSA) and lipophilic cationic carbocyanine (JC-1) and functional evaluation of the plasmatic membrane through Hyposmotic Swelling Test (HOST). An evaluation of chromatin integrity was performed with the acridine orange staining procedure. In experiment II, the frozen semen with different extenders was used for in vitro fertilization (IVF), analyzing cleavage rates and in vitro embryo development. No statistical difference between extenders was identified, suggesting that the soy lecithin extender may substitute egg-yolk-Tris extender, considering the individual variations of each bull used in this experiment.

Detection of cross infections by Leishmania spp. and Trypanosoma spp. in dogs using indirect immunoenzyme assay, indirect fluorescent antibody test and polymerase chain reaction.

The aim of this study was to detect cross infections by Leishmania spp. and Trypanosoma spp. using enzyme-linked immunosorbent assay (ELISA), indirect fluorescent antibody test (IFAT) and polymerase chain reaction (PCR). Thus, 408 blood samples were collected from dogs domiciled in Araçatuba Municipality, São Paulo State, Brazil; the dogs were of both sexes, of several breeds and aged 6 months. For Leishmania spp., 14.95% (61 out of 408) of dogs were reactive using IFAT. Positivity was 20.10% (82 out of 408) using ELISA and 29.66% (121 out of 408) using PCR, with significant differences for the sex and age of these animals (p < 0.05). For Trypanosoma spp., antibody occurrence using ELISA was 10.54% (43 out of 408), while PCR indicated 2.45% (10 out of 408) positive dogs. Using IFAT, 10.29% (42 out of 408) of animals were considered positive and only sex showed a significant difference (p < 0.05). In this study, 10.54% (43 out of 408) of animals were seropositive according to ELISA for Trypanosoma spp., of which 79.07% (34 out of 43) showed positive results in the molecular diagnosis for Leishmania spp., while of the 10.29% (42 out of 408) positive dogs according to IFAT, 95.24 % (40 out of 42) had confirmed infection by this parasite. The obtained results demonstrate evidence of cross infections by both protozoa in the animals analysed in this study.

Efeito das células do cumulus e cisteamina durante o cultivo de maturação in vitro de oócitos bovinos sobre a maturação nuclear e aquisição da competência para desenvolvimento embrionário / Effects of cumulus cells and cysteamine during bovine oocyte in vitro maturation on meiosis progression and acquisition of developmental competence

Complexos cumulus-oócito (COC), oócitos desnudos (DO) e DO cocultivados com células do cumulus em suspensão (DO+CC) foram maturados in vitro (MIV) na presença ou ausência de cisteamina (50mM). Observou-se efeito benéfico da cisteamina durante o cultivo de MIV, pois a maturação nuclear no grupo COC cisteamina foi maior do que a do COC controle (P<0,05). No grupo sem a adição de cisteamina, foi observado que a ausência de CC durante o cultivo de MIV prejudicou a maturação nuclear em DO, em relação ao COC (P<0,05), todavia a cisteamina restaurou a capacidade de progressão da meiose em DO, tornando-os semelhantes aos COC (P>0,05). O acoplamento entre oócitos e CC durante MIV demonstrou ser essencial para aquisição da competência do oócito para suportar o desenvolvimento embrionário inicial, pois COC apresentaram maior porcentagem de blastocistos e eclosão quando comparados a DO e DO+CC (P<0,05). A inclusão de cisteamina no cultivo de MIV não restaurou a aquisição da competência em DO e DO+CC, que permaneceram semelhantes aos do grupo-controle (P>0,05). Conclui-se que a cisteamina no meio de MIV melhora as taxas de maturação nuclear em COC e restaura a capacidade de progressão da meiose em DO. Todavia, na concentração utilizada neste estudo, não promove efeito benéfico no desenvolvimento embrionário.

Cumulus-oocyte complexes (COC), denuded oocytes (DO) and DO co-cultured with cumulus cells in suspension (DO+CC) were in vitro matured (IVM) in the presence or absence of cysteamine (50mM). A beneficial effect of cysteamine was observed during IVM, because the nuclear maturation in the COC cysteamine group was higher than in COC control (P<0.05). In the control group, the absence of CC during IVM impaired nuclear maturation in DO when compared to COC (P<0.05), but cysteamine restored the ability of meiosis progression in DO, making them similar to COC (P>0.05). The coupling between oocytes and CC during IVM proved to be essential for the acquisition of oocyte competence to support early embryonic development, as COC had higher percentages of blastocyst and hatching when compared to DO and DO+DC (P<0.05). However, the inclusion of cysteamine in the IVM culture did not restore the acquisition of competence in DO and DO+DC, which remained similar to the control group (P>0.05). It is concluded that cysteamine in the IVM culture improves the nuclear maturation in COC and restores the progression ability of meiosis in DO. However, in the concentration used in this study, cysteamine does not promote a beneficial effect on embryo development.

Evaluation of TNF-α, IL-4, and IL-10 and parasite density in spleen and liver of L. (L.) chagasi naturally infected dogs.

Dogs are the main domestic reservoirs of L. (L.) chagasi. Once in the vertebrate host, the parasite can cause visceral leishmaniasis, which can also be transmitted to humans. Cytokines are key elements of the host immune response against Leishmania spp. To investigate whether tumor necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 are associated with pattern infection in dogs, these cytokines were quantified in the spleen and liver of dogs naturally infected with L. (L.) chagasi, with or without clinical manifestations, and their levels were correlated with the parasite load verified in these organs. A total of 40 adult dogs naturally infected with L. (L.) chagasi were assessed, together with 12 uninfected control dogs. Samples from spleen and liver were used to determine the cytokine levels by capture ELISA and for quantifying parasite load by real-time PCR. Statistical analysis was performed using the minimum Chi square method and group means were compared using the Tukey test. TNF-α, IL-4 and IL-10 levels in infected dogs were higher than in control groups; the liver was the main cytokine-producing organ during infection. The level of splenic TNF-α showed correlation with parasite load and may represent an important marker for infection process evolution, with the participation of IL-10. These results may contribute to a clearer understanding of the immune response in dogs infected with L. (L.) chagasi, which may lead to the development of prophylactic or preventive measures for these animals.

Levels of matrix metalloproteinase-2 and metalloproteinase-9 in the cerebrospinal fluid of dogs with visceral leishmaniasis.

Inflammation causes increases in the level of matrix metalloproteinases (MMPs), which, in central nervous system (CNS), are associated with neuroinflammation and disruption of blood-brain barrier. Analysis of cerebrospinal fluid (CSF) is pivotal for detecting diseases in CNS and, although a specific diagnosis may not be achieved, this analysis is helpful to confirm the diagnosis or to rule out relevant differential diagnoses. This study examined the levels of MMP-2 and MMP-9 in the CSF of dogs using gelatin zymography to verify possible alterations in these enzymes during natural systemic infection with Leishmania chagasi. Latent and active forms of MMP-2 were detected in some dogs of both groups, with high levels in the control group. In contrast, latent and active forms of MMP-9 were detected only in some animals with leishmaniasis. These results clearly demonstrate that MMP-9 is elevated in CSF of dogs with visceral leishmaniasis (VL). Although these results are preliminary, they suggest that MMP-9 might play a role in disruption of blood-brain barrier and/or blood-CSF barrier. While the presence of MMPs in CSF is not a condition exclusive to VL, their presence and persistence in CSF supports the hypothesis of an inflammatory state within CNS of dogs with VL.

Influência do butorfanol sobre os períodos de latência e de ação da ropivacaína pela via peridural na ovariossalpingo-histerectomia em cadelas / Influence of butorphanol on latency and duration of ropivacaine epidural anesthesia for ovariosalpingohysterectomy in bitches

Avaliaram-se os períodos de latência e de duração do efeito do butorfanol associado à ropivacaína aplicados pela via peridural e a possibilidade de uso dessa associação como protocolo anestésico para realização de ovariossalpingo-histerectomia (OSH) em cadelas. Utilizaram-se 16 cadelas pré-medicadas com acepromazina e midazolam compondo dois grupos: no grupo 1 (n=8) aplicou-se ropivacaína isolada (0,3mL/kg) e no grupo 2 (n=8), butorfanol (0,1mg/kg) e ropivacaína (até o volume de 0,3mL/kg), pela via peridural. Consideraram-se nove momentos (M): M0 - animal sem anestesia, M1 - 15 minutos após medicação pré-anestésica; M2 - 30 minutos após a aplicação peridural; M3, M4 e M5 - correspondentes ao início do procedimento e ao pinçamento dos pedículos esquerdo e direito, respectivamente; M6 - ligadura da cérvix uterina; e M7 e M8 - início da laparorrafia e fim da sutura de pele, respectivamente. Foram avaliados os períodos de latência e ação do bloqueio, além da viabilidade de realização do procedimento cirúrgico por meio do bloqueio efetuado. O que recebeu ropivacaína + butorfanol apresentou sedação pronunciada e permitiu a realização de OSH em 75 por cento dos animais, sem indução anestésica. Não houve diferença entre os grupos quanto aos períodos de latência e duração. A associação do butorfanol à ropivacaína proporcionou bloqueio anestésico compatível com a realização de OSH e período de latência curto, com duração de efeito suficiente para o procedimento cirúrgico.

The periods of latency and duration of butorphanol associated with ropivacaine used via epidural, and this combination as anesthetic protocol for carrying out ovariosalpingohysterectomy (OSH) in bitches were evaluated. Sixteen animals pre-medicated with acepromazine and midazolam were used composing two groups that received: 1 (n=8) ropivacaine (0.3mL/kg) and 2 (n=8) butorphanol (0.1mg/kg) and ropivacaine (up to the volume of 0.3ml/kg) via epidural. Nine moments were studied: M1 - 15 minutes after pre-anesthetic medication; M2 - 30 minutes after the epidural medication; M3, M4, and M5 - at the beginning of surgery and at clamping left and right pedicles, respectively; M6 - at ligation of the uterine cervix; and M7 and M8 - at laparorhaphy and end of skin suture, respectively. Periods of latency and blocking the action of random double-covert manner were evaluated as well as the feasibility of carrying out the surgery performed by the blockade. It was observed that the group receiving ropivacaine + butorphanol allowed the execution of OSH in 75 percent of animals without the need for anesthesia. There was no statistical difference between the periods of latency and duration. It was concluded that the combination of butorphanol to ropivacaine provides, in bitches, block compatible with the implementation of OSH with short period of latency and duration of effect sufficient for the surgical procedure.

Pharmacokinetics of tetracycline in plasma, synovial fluid and milk using single intravenous and single intravenous regional doses in dairy cattle with papillomatous digital dermatitis.

The purpose of this study was to compare the pharmacokinetics of tetracycline in plasma, synovial fluid, and milk following either a single systemic intravenous (i.v.) injection or a single i.v. regional antibiosis (IVRA) administration of tetracycline hydrochloride to dairy cattle with papillomatous digital dermatitis (PDD). To this end, plasma and synovial fluid tetracycline concentrations were compared with the minimal inhibitory concentration (MIC) values of the major bacteria, which are known to cause digital diseases and thus assess its efficacy in PDD. Residual tetracycline concentrations in milk from cows treated by both methods were also determined. Twelve Holstein cows with various stages of PDD were randomly assigned to two groups of six animals. Group 1 received a single systemic i.v. injection of 10 mg/kg of tetracycline hydrochloride. Group 2 received 1000 mg of tetracycline hydrochloride by IVRA of the affected limb. Blood, synovial fluid and milk samples were taken prior to tetracycline administration (time 0 control), and then at 22, 45 and 82 min, and 2, 3, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h following drug administration. Tetracycline concentrations were determined by high-performance liquid chromatography. Mean tetracycline plasma and milk concentrations in Group 1 were higher than Group 2. The opposite was observed for synovial fluid concentrations. Group 2 synovial fluid concentrations were higher than the MIC value over 24 h for the bacteria most frequently responsible for claw disease. Compared with i.v. administration, IVRA administration of tetracycline produced very high synovial fluid and low plasma and milk concentrations.

Effects of lidocaine infusion during experimental endotoxemia in horses.

BACKGROUND: The clinical efficacy of IV infusion of lidocaine for treatment of equine endotoxemia has not been studied. HYPOTHESIS: Lidocaine infusion after exposure to lipopolysaccharide (LPS) will inhibit the inflammatory response and have inhibitory effects on the hemodynamic and cytokine responses to endotoxemia. ANIMALS: Twelve horses. METHODS: Two equal groups (n=6): saline (GI) and lidocaine (GII). In all animals, endotoxin (500 ng/kg body weight [BW]) was injected intraperitoneally over 5 minutes. Twenty minutes later, animals received a bolus of GI or GII (1.3 mg/kg BW) over 5 minutes, followed by a 6-hour continuous rate infusion of GI or GII (0.05 mg/kg BW/min). Treatment efficacy was judged from change in arterial blood pressure, peripheral blood and peritoneal fluid (PF) variables (total and differential cell counts, enzyme activities, and cytokine concentrations), and clinical scores (CS) for behavioral evidence of abdominal pain or discomfort during the study. RESULTS: Compared with the control group, horses treated with lidocaine had significantly lower CS and serum and PF tumor necrosis factor-alpha (TNF-alpha) activity. At several time points in both groups, total and differential cell counts, glucose, total protein and fibrinogen concentrations, and alkaline phosphatase, creatine kinase, and TNF-alpha activities were significantly different from baseline values both in peripheral blood and in PF. CONCLUSIONS AND CLINICAL IMPORTANCE: Lidocaine significantly decreased severity of CS and inhibited TNF-alpha activity in PF.

Anestesia peribulbar com ropivacaína como alternativa ao bloqueio neuromuscular para facectomia em cães / Peribulbar anesthesia with ropivacaine as an alternative to neuromuscular blocking agents for cataract surgery in dogs

Desenvolveu-se uma técnica de bloqueio peribulbar comparando-a à técnica de anestesia oftálmica com bloqueio neuromuscular parcial em cães submetidos à facectomia extracapsular. Doze cães, de diferentes raças, foram alocados em dois grupos (G1 e G2) e anestesiados com acepromazina (0,05mg/kg, IV) e propofol (5mg/kg, IV) e mantidos com isofluorano sob ventilação espontânea. Os cães do G1 receberam o bloqueio peribulbar com ropivacaína 0,75 por cento, e os do G2 o bloqueio neuromuscular parcial com brometo de pancurônio (0,01mg/kg, IV). Utilizaram-se, como parâmetros comparativos, escores de posicionamento do globo ocular, controle do reflexo oculocardíaco e pressão intraocular (mmHg). Foi possível propor uma técnica eficaz para cães baseando-se na técnica de bloqueio peribulbar posterior realizada no homem. Em ambos os grupos, o globo ocular permaneceu centralizado. Não ocorreram alterações eletrocardiográficas atribuídas ao reflexo oculocardíaco. Houve redução significativa da pressão intraocular em G1 após o bloqueio (10,7±0,6 e 14,7±0,6). Conclui-se que o bloqueio peribulbar proporcionou condições cirúrgicas apropriadas para a realização da facectomia, com a vantagem de promover bloqueio sensitivo do olho.

A peribulbar block technique was developed and its clinical efficacy was compared with neuromuscular blockade in dogs undergoing cataract surgery. Twelve dogs of different breeds were randomly and equally allocated in two groups. After given acepromazine (0.05mg/kg, IV), anesthesia was induced with propofol (5mg/kg, IV) and maintained with isoflurane in oxygen during spontaneous breathing. A peribulbar block with 0.75 percent ropivacaine was performed in G1 dogs while partial neuromuscular blockade with pancuronium (0.01mg/kg IV) was provided in G2 dogs. Globe position scores, oculocardiac reflex, and intra-ocular pressure (mmHg) were evaluated at pre-defined intervals during surgery. Peribulbar blocks were successful performed according to posterior peribulbar block described in humans. In both groups, the globe was centralized and globe position scores did not differ between groups. The intra-ocular pressure was significantly lower in G1 after the block (10.7±0.6 vs 14.7±0.6). There were no electrocardiographycal changes attributed to the oculocardiac reflex. In conclusion, satisfactory surgical conditions were provided by the peribulbar block. This technique can be used as an alternative to the use of neuromuscular blocking agents in dogs undergoing cataract surgery, with the advantage of providing analgesia of the eye.

Qualidade espermática de sêmen de cães naturalmente infectados por leishmania sp / Semen quality of dogs naturally infected by leishmania sp

Avaliaram-se alterações espermáticas associadas à infecção por leishmaniose no sêmen de cães naturalmente infectados, utilizando-se, durante oito semanas consecutivas, ejaculados de seis cães soronegativos e seis cães soropositivos. As amostras foram colhidas uma vez por semana e avaliadas quanto ao volume, concentração, motilidade, vigor, morfologia espermática, integridade da cromatina, avaliação simultânea da integridade da membrana plasmática, acrossoma e potencial mitocondrial. Concomitantemente foram dosadas a proteína total do plasma seminal e sanguíneo. A leishmaniose visceral causou aumento dos defeitos maiores e menores nos espermatozoides dos animais acometidos pelo estágio moderado a severo da doença. Em estágios mais avançados da enfermidade, a integridade das membranas acrossomal e plasmática foi afetada negativamente. Não foi possível estabelecer um critério quanto à avaliação do potencial mitocondrial. A incidência de alterações morfológicas nos animais acometidos não promoveu aumento de injurias à cromatina. Todos os animais com leishmaniose apresentaram hiperproteinemia do sêmen.

The spermatic changes associated with the natural infection in dogs by Leishmania sp was evaluated during eight consecutive weeks, using ejaculates of six seronegative and six seropositive dogs. The samples were collected once a week and evaluated for volume, concentration, motility, vigor, sperm morphology, chromatin integrity, simultaneous evaluation of the plasmatic membrane integrity, acrosome, and mitochondrial potential. The total proteins of the seminal plasma and blood were measured. The visceral leishmaniasis caused increase of major and minor defects in spermatozoa of animals attacked by moderate to severe stages of the disease. In more advanced stages of the illness, the acrosomal and plasmatic membranes integrity was adversely affected. It was not possible to establish a pattern refering the evaluation of the mitochondrial potential. The incidence of morphological changes in the seropositive animals did not promote an increase of injuries to the chromatin. All animals with leishmaniasis presented hyperproteinemia of the semen.

Effect of antioxidants during bovine in vitro fertilization procedures on spermatozoa and embryo development.

Increased amounts of reactive oxygen species (ROS) during in vitro fertilization (IVF) may cause cytotoxic damage to gametes, whereas small amounts of ROS favour sperm capacitation. The aim of this study was to investigate the effect of antioxidants [50 microm beta-mercaptoethanol (beta-ME) and 50 microm cysteamine (Cyst)] or a pro-oxidant (5 mm buthionine sulfoximine) on the quality and penetrability of spermatozoa into bovine oocytes and on the subsequent embryo development and quality when added during IVF. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes, and mitochondrial function, was diminished (p < 0.05) after 4-h culture in the presence of antioxidants. Oocyte penetration rates were similar between treatments (p > 0.05), but antioxidants adversely affected the normal pronuclear formation rates (p < 0.05). The incidence of polyspermy was high for beta-ME (p < 0.05). No differences were observed in cleavage rates between treatments (p > 0.05). However, the developmental rate to the blastocyst stage was adversely affected by Cyst treatment (p < 0.05). The quality of embryos that reached the blastocyst stage, evaluated by total, inner cell mass (ICM) and trophectoderm cell numbers and ICM/total cell ratio was unaffected (p > 0.05) by treatments. The results indicate that ROS play a role in the fertilizing capacity in bovine spermatozoa, as well as in the interaction between the spermatozoa and the oocytes. It can be concluded that supplementation with antioxidants during IVF procedures impairs sperm quality, normal pronuclear formation and embryo development to the blastocyst stage.