Pharmacokinetics of intravenous and oral administration of enrofloxacin to the late-term pregnant and non-pregnant mares.

BACKGROUND: Enrofloxacin may be an alternative antimicrobial for unresponsive cases of severe bacterial infections in pregnant mares. As pregnancy may affect drug bioavailability, distribution, metabolism and excretion, dose adjustment might be necessary. OBJECTIVES: To determine the disposition of orally and intravenously administered enrofloxacin in pregnant and non-pregnant mares. STUDY DESIGN: Randomised cross-over experiment. METHODS: Six light-breed, healthy pregnant mares (260 days gestation) were given a single dose of either intravenous (5 mg/kg bwt) or oral compounded (7.5 mg/kg bwt) enrofloxacin, with the opposite dose administered after a 7-day washout. The protocol was repeated 45-60 days post-partum, 15-30 days after foals were weaned. Plasma samples were obtained via venepuncture at 0, 5, 10, 20, 30, 45, 60, 90 min, and 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72 h after enrofloxacin administration. Enrofloxacin and ciprofloxacin concentrations were measured by LC-MS/MS. Concentration versus time data were analysed based on non-compartmental pharmacokinetics. RESULTS: Enrofloxacin AUC0-∞ was significantly higher in pregnant mares than non-pregnant mares after PO administration and tended to be higher after i.v. administration. Ciprofloxacin maximum plasma concentration (Cmax ) and concentration at 24 h (C24h ) were higher, and half-life of the terminal phase (t½λz ) was longer in pregnant mares than non-pregnant mares after oral administration. Similarly, ciprofloxacin C24h was higher in pregnant mares with intravenous administration. Oral bioavailability did not differ based on pregnancy status. MAIN LIMITATIONS: Only six healthy light breed mares were assessed. Disease or horse breed may affect the endpoints evaluated. A lack of established enrofloxacin AUC/MIC targets for equine pathogens limits pharmacokinetic-pharmacodynamic conclusions. CONCLUSIONS: The oral form of enrofloxacin was well absorbed, and oral bioavailability was comparable to previous studies. While differences in enrofloxacin and ciprofloxacin pharmacokinetics were seen between pregnant and non-pregnant mares, the recommended drug dose and dose intervals are appropriate for MIC <0.25 µg/mL. Dosages may need to be adjusted for bacteria with a MIC >0.25 µg/mL.

Quality Improvement of Post-Thawed Stallion Epididymal Spermatozoa with Single Layer Centrifugation.

BACKGROUND: Epididymal sperm cryopreservation represents the ultimate option to preserve spermatozoa of valuable stallions. OBJECTIVE: The study aims to evalute whether single layer centrifugation (SLC) prior to cryopreservation or after post-thawing improves the quality of stallion epididymal sperm. MATERIALS AND METHODS: Epididymal sperms of stallions were harvested (N=20). Sperm samples were subjected to treatments: conventional centrifugation, SLC prior to cryopreservation (SLC-PC) or SLC post-thaw (SLC+). All samples were cryopreserved, thawed and evaluated. SLC+ were thawed, single layer cenrifuged and resuspended in freezing extender (SLC+F) or cooling extender (SLC+C). Total motility, progressive motility, morphology, mitochondrial functionality, membrane integrity and DNA integrity were evaluated. RESULTS: SLC-PC and SLC+F yielded higher total motility, while SLC+F yielded the highest progressive motility. Mitochondrial functionality was significantly higher in all SLC groups. Membrane integrity was higher in SLC-PC. The percentage of morphologically normal spermatozoa was higher in SLC-PC and SLC+F. CONCLUSION: SLC prior to cryopreservation or post-thaw improves the quality of stallion epididymal spermatozoa. When SLC is performed post-thaw, freezing extender is the best medium to resuspend the pelleted semen.

The effects of xanthan gum on equine sperm quality during cooling storage / Efeitos de xantam gum em qualidade de esperma equino durante armazenamento resfriado

This study was designed to evaluate the possible benefits of adding xanthan gum to a standard extender for equine through in vitro analyzes of sperm quality. Semen was collected four times from five different stallions (n= 20 samples) and subjected to cooled storage under different conditions: control (only standard extender) and three different concentrations of xanthan gum (0.01%, 0.12%, and 0.25%) supplemented to the extenders. Sperm parameters, such as motility, mitochondrial functionality, and membrane, acrosome, and DNA integrity were measured after 0h, 24h, 48h, and 72h of sperm storage at 5ºC. Our observations indicated that sperm motility declined with longer cooling period with the 0.25% xanthan gum supplementation group compared with the control group. Other parameters, such as mitochondrial functionality and membrane and acrosome integrity also declined for all treatments during storage; however, no differences were observed between xanthan gum and control groups. DNA integrity did not significantly change during the storage. In conclusion, the addition of xanthan gum to equine semen extender is not harmful to the sperm structure, despite reducing the sperm motility.(AU)

Esse estudo foi desenvolvido para avaliar os possíveis benefícios de acrescentar xanthan gum a um extensor padrão através de analises in vitro de qualidade de esperma. Semen foi coletado quatro vezes de cinco garanhões diferentes (n = 20 amostras) e submetido a armazenamen to resfriado em diferentes condições: controle (apenas extensor padrão) e três diferentes concentrações de xanthan gum (0,01%, 0,12% e 0,25%) suplementado aos extensores. Parâmetros dos espermatozoides, como mobilidade, funcionamento mitocondrial e integridade de membranas, acrossomos e DNA forma medidos após 0h, 24h, 48h e 72h de armazenamento a 5oC. Nossas observações indicaram que motilidade reduziu com armazenamento resfriado prolongado no grupo de 0,25% de suplementação de xanthan gum comparado ao grupo controle. Outros parâmetros, como funcionalidade mitocondrial e integridade de membrana e acrossomos também reduziu em todos os tratamentos durante o armazenamento, no entanto não foram detectadas diferenças significativas entre grupos tratados e grupo controle. Integridade de DNA não mudou significativamente durante armazenamento. Em conclusão, a adição de xanthan gum a extensor de sêmen equino não é danosa à estrutura do espermatozoide apesar de reduzir motilidade.(AU)

Effect of urine contamination on stallion semen freezing ability.

Urospermia is a common ejaculatory dysfunction of stallions. Current practice suggests that urine contaminated semen should not be used for cryopreservation. The aim of this study was to determine effects of urine contamination on semen freezing. Sixty-five ejaculates from eight stallions were divided into no urine (CONT), low (20% urine, LOW), and high (50% urine, HIGH) samples. Semen was extended with a commercial cooling extender, cushion-centrifuged, resuspended to 200 million/mL in a commercial egg-yolk based extender, and cryopreserved in liquid nitrogen. A subset of ejaculates (n = 20) were split in two after cushion-centrifugation, and one half of the ejaculate was submitted to a single-layer gradient centrifugation before cryopreservation. Sperm motility parameters were assessed pre- and post-freezing with an automated sperm analyzer. Semen pH, creatinine, and urea concentrations were assessed in raw samples, after urine contamination and after centrifugation and extension. Statistical analyses were performed with ANOVA and Tukey's posthoc. There were significant reductions in total and progressive sperm motilities (i.e., %TM and %PM, respectively) with increasing urine contamination pre-freezing (%TM 67 ±â€¯1.7, %PM 50 ±â€¯2.2, CONT), (%TM 60.3 ±â€¯1.7, % PM 42.5 ±â€¯2.1, LOW), and (%TM 41.3 ±â€¯2, %PM 21.3 ±â€¯1.5, HIGH). Post-thaw motilities for CONT (%TM 54 ±â€¯2.3, %PM 40.8 ±â€¯3.3) and LOW (%TM 51.7 ±â€¯1.8, %PM 36.2 ±â€¯2.1) were not different, but were higher than the HIGH (%TM 31.5 ±â€¯1.2, %PM 17.1 ±â€¯1.0) (p < 0.05). Post-thaw sperm viability was significantly lower in the HIGH (54.7 ±â€¯2.4) than in the CONT (63.8 ±â€¯2.3) or LOW (64.6 ±â€¯3.4) groups. Semen creatinine and urine levels were significantly higher with increasing urine contamination and were significantly decreased after centrifugation and resuspension in freezing extender. Pre-treatment semen pH was significantly lower than semen contaminated with low or high amounts of urine, and pH decreased significantly after centrifugation and resuspension. Gradient centrifugation did not improve %TM in the control group, but it did improve pre-freeze %TM and %PM in the low and high groups and improved significantly post freezing %TM and %PM in the high urine contaminated group. Semen contaminated with a small amount of urine may be suitable for freezing, whereas highly contaminated semen might not be usable. Although urine was mostly removed in this fashion, the initial exposure to high quantities was sufficient to decrease sperm motility pre- and post-freezing, whereas low urine contamination was not as detrimental.

Avaliação hematológica e hemogasométrica de potros nascidos de éguas com placentite ascendente / Hematological and hemogasometric evaluation of foals born from mares with ascending placentitis

A placentite é a principal causa de partos prematuros, aborto e nascimento de potros comprometidos, podendo causar hipóxia e septicemia. A hematologia e a gasometria venosa fornecem informações importantes para o monitoramento da saúde de potros nascidos de éguas com placentite. O objetivo deste trabalho foi descrever os valores hematológicos e hemogasométricos durante as primeiras 24h de vida em potros nascidos de éguas mestiças Crioulas submetidas à indução experimental de placentite, com diferentes graus de maturidade. Foram utilizados 16 potros nascidos de éguas submetidas à indução experimental de placentite, divididos em três grupos de acordo com o grau de maturidade: prematuro (n=8), dismaturo (n=4) e a termo (n=4). Foram realizadas coletas sanguíneas nos momentos 0h, 12h e 24h para realização de hemograma completo e gasometria venosa. No eritrograma, foi observada anemia normocítica normocrômica no grupo prematuro em relação ao grupo dismaturo nas 12h e 24h. O grupo prematuro apresentou menor contagem de leucócitos totais nas 24h em relação ao grupo a termo (P=0,01). Os valores de pH, cHCO3 e SO2 não diferiram entre os grupos, porém os animais prematuros apresentaram acidose respiratória (pH=7,28). A PCO2 nos prematuros foi maior na 0h (P=0,02). Nos três grupos, a PCO2 apresentou uma curva adaptativa com redução dos valores durante as 12h e 24h. Os potros prematuros mostraram menores valores de excesso de base (BE) no nascimento (P=0,02), confirmando o quadro de acidose respiratória. Concluiu-se que as respostas hematológicas e hemogasométricas diferem entre potros com diferentes graus de maturidade. A acidose observada no grupo prematuro ao nascimento, com estabilização e resposta compensatória durante as 12h e 24h, demonstra a necessidade de avaliação hemogasométrica sequencial em potros de risco, o que permite a identificação da resposta clínica ao processo e, assim, auxilia no estabelecimento do tratamento e prognóstico para esses potros.(AU)

The placentitis is a major cause of premature birth, abortion and compromised foal delivery, and may result in hypoxia and sepsis. The blood gas analysis and hematology can provide important information for monitoring the foals born from mares with placentitis, with different degrees of maturity. The aim of this study was to describe the hematological and blood gas values during the first 24 hours of life in foals born from crossbreed mares with experimentally induced placentitis, presenting different degrees of maturity. Sixteen foals, born from mares with experimentally induced ascending placentitis were assigned to three groups according to degree of maturity: premature (n=8), dysmature (n=4), and full-term foals (n=4). Blood samples were collected at birth (0), at 12h and 24h, and hematological evaluation and blood gas variables were measured. In the premature group normocytic normochromic anemia was observed compared to dysmature group at 12h and 24h. The premature group showed lower count of white blood cells at 24h relative to the full-term group (p=0.01). The pH, cHCO3 and SO2 values do not differ among the groups; however the premature group showed respiratory acidosis (pH=7,28). The PCO2 was higher at 0h in the premature foals (p=0.02). In all groups, the PCO2 presented an adaptive curve with reduction between 12h and 24h. The premature foals showed lower base excess (BE) values at birth (p=0.02), confirming the respiratory acidosis in this group. We conclude that the hematological and blood gas response differs between foals with different degrees of maturity. Acidosis in the premature foals at birth, with stabilization and compensation of pH value during the first 12-24h demonstrate the necessity of sequential blood gas analysis in risk foals. This may help identify the clinical response to the process and assist in the establishment of adequate treatment and prognosis for these foals.(AU)

Quercetin in equine frozen semen.

　　BACKGROUND: Supplementation of sperm diluents to reduce the damage caused by the freeze-thaw cycle is broadly used in equine semen cryopreservation. OBJECTIVE: The present study aimed at determining the most appropriate quercetin supplementation in equine freezing extender. MATERIALS AND METHODS: Quercetin at four different concentrations (0.25, 0.5, 0.75 or 1 mM) was added in the sperm freezing diluent before the freeze-thaw cycle. The spermatozoa population was analyzed by flow cytometry and a statistical analysis was conducted to detect significant differences between control and treated samples. RESULTS: The statistical analysis did not reveal any significant modification of seminal parameters. CONCLUSION: Within the concentrations tested, quercetin supplementation in equine freezing extender did not affect progressive motility, mitochondrial functionality, acrosome reaction, membrane integrity or DNA fragmentation index in post-thaw equine semen.

Proteinograma sérico de éguas com placentite ascendente e seus respectivos neonatos: dados preliminares / Serum protein concentrationin mares with ascending placentitis and their newborns: preliminary data

Este estudo teve por objetivo estabelecer o proteinograma sérico em éguas com placentite induzida e em seus respectivos neonatos. Foram coletadas amostras de sangue das éguas em oito momentos diferentes e dos potros em quatro momentos. Para obtenção da concentração das frações proteicas, utilizou-se eletroforese em gel de acrilaminada contendo dodecil sulfato de sódio (SDS-PAGE). No método utilizado, foram observadas 23 bandas proteicas, cujos pesos moleculares variaram de 16KDa a 245KDa, sendo possível a identificação das seguintes frações: 175KDa, 102KDa, 83KDa, 63KDa, 50KDa, 41KDa, 39KDa e 28KDa. De todas as bandas proteicas encontradas, somente as de 39KDa e 41KDa apresentaram alteração na cinética nos momentos avaliados. De acordo com a solução marcadora, pode-se sugerir que essas proteínas seriam alfa1-glicoproteína ácida (39KDa) e haptoglobina (41KDa). A concentração de imunoglobulinas nos potros apresentou aumento significativo a partir das 12 horas de nascimento. Não está elucidado se estes níveis refletem a persistência do processo inflamatório placentário ou se são alterações fisiológicas do periparto. Não foram observadas alterações na cinética das proteínas nos potros nas primeiras 48 horas.(AU)

The aim of this paper was to identify the serum acute phase protein concentration in mares with induced placentitis and their neonates. Blood samples were collected from the mares in 8 different moments, and from the foals, in 4 moments. To obtain the concentration of protein fractions acrilaminada gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) was used. In the used method 23 protein bands whose molecular weights ranged from 16kDa to 245kDa were observed, it is possible to identify the following fractions: 175kDa, 102kDa, 83kDa, 63kDa, 50kDa, 41kDa, 39kDa and 28kDa. Of all the protein bands found only the 39KDa and 41KDa have changes in the kinetics in the evaluated times. According to the marker solution, we would suggest that these proteins are alfa1-acid glycoprotein (39kDa) and haptoglobin (41kDa). The concentration of immunoglobulins in foals increased significantly from 12 hours of birth.(AU)

Maturidade de potros nascidos de éguas com placentite / Maturity of foals from the mares with placentitis

Objetivou-se descrever a maturidade neonatal através da resposta clínica, comportamental e hematológica de potros nascidos de éguas com placentite. Participaram do estudo seis potros nascidos de éguas submetidas à indução experimental de placentite ascendente através da infusão intracervical de Streptococcus equi subespécie zooepidemicus e tratadas com Sulfa-trimetoprim e Flunixin meglumine. A formação dos grupos neonatais foi realizada de acordo com o grau de viabilidade e sobrevivência até 60 horas: Grupo Não Sobreviventes (n=2); Grupo Debilitados (n=2); Grupo Saudáveis (n=2). Foi considerado o tempo de gestação, período de intervalo inoculação-parto, avaliação comportamental, clínica e hematológica. O Grupo dos potros Saudáveis apresentou maior tempo de gestação (320±2 dias) e maior intervalo inoculação-parto (20,5±2,5 dias). Os Grupos Não Sobreviventes e Debilitados apresentaram atraso para decúbito esternal e reflexo de sucção. Foi observada bradicardia e hipotermia com 48h de vida no Grupo Não Sobreviventes. Os potros do Grupo Não Sobreviventes e Saudáveis apresentaram leucopenia no nascimento com discretas variações até as 48h. Os potros nascidos de éguas com placentite ascendente e tratadas demonstraram evolução clínica e respostas neonatais distintas. Conclui-se que, quanto maior o tempo de manutenção da gestação após a injúria placentária, melhor será a maturação fetal, o que refletirá em viabilidade e melhor capacidade de resposta neonatal...

The aim of this study was to describe the neonatal maturity through clinical, behavioral and hematologic response of foals born from mares with placentitis. Were used six foals born from mares subjected to experimentally induced ascending placentitis through intracervical infusion of Streptococcus equi subspecies zooepidemicus and treated with trimethoprim sulfametoxazole and flunixin meglumine. The neonatal groups were performed according to the viability and survival rate up to 60 hours: No Survivors group (n = 2); Debilitated group (n = 2); Healthy group (n = 2). Gestational length, the period between inoculation and delivery, and behavioral, clinical and hematologic evaluations were considered. The Healthy group showed longer gestation length (320±2 days) and longer inoculation-delivery interval (20.5±2.5 days). No Survivors and Debilitated groups showed delay in sternal recumbency and sucking reflex. Bradycardia and hypothermia was observed at 48 hours of life in No Survivors Group. Foals from No Survivors and Healthy groups showed leukopenia at birth with slight variations until 48h. Foals born from mares with ascending placentitis and treated showed distinct clinical and neonatal responses. It is concluded that the longer the maintenance of gestation after placental injury, better is the fetal maturation, which reflects in better viability and ability to neonatal response...

Resposta clínica e metabólica de potros neonatos em relação aos achados histopatológicos da placenta na égua / Clinical and metabolic response of neonatal foals related to histopathology finds in mare placenta

Avaliaram-se as respostas clínica e metabólica de potros neonatos em relação aos achados histopatológicos da placenta na égua. Foram avaliados dois grupos de éguas da raça Puro Sangue Inglês - um grupo-problema (n=25) e um grupo-controle (n=25), de acordo com os achados da placenta. O exame dos potros constou de avaliação clínica geral, hematologia e bioquímica sérica. O exame histopatológico da placenta apresentou resultado compatível com a apresentação clínica do potro, sendo que a presença de lesões inflamatórias resultou na produção de potros debilitados. A presença de lesões degenerativas não comprometeu o estado clínico do neonato, mas pode ser responsável pela manifestação de distúrbios subclínicos, evidenciados pelo aumento das taxas de AST e GGT. A ureia pareceu ser um indicador de dano renal decorrente de insuficiência placentária em potros neonatos.

The placenta represents the major communication between the mare and the fetus during the gestational period, and this suggests that any disturbance in the placenta can be an indicator of gestational damage with risk to the fetus. The aim of this paper was to evaluate the clinical and metabolic responses of the newborn foals related with the findings from the histopathological examination of the placenta. This study was conducted in a farm located in Bagé-RS, Brazil, where were evaluated two groups of Throughbred mares for this case-control study: One Problem Group (N=25) and the Control Group (n=25), based on the placental findings. The foal's evaluation was based on general clinical examination, hematology and serum biochemistry. Results from the placenta histopathological exams were compatible with clinical presentation of the foals, with the presence of inflammatory lesions resulting in the production of debilitated foals. The presence of degenerative lesions in the placenta does not compromise the clinical features of the newborn, but they can be responsible for the manifestation of sub-clinical disturbances, evidenced by increased levels of AST and GGT. Urea seems to be an indicator of renal damage due to placental insufficiency in neonatal foals.

Osteodistrofia fibrosa em equinos criados em pastagem de Panicum maximum cultivar Aruana: relato de casos / Fibrous osteodystrophy in horses raised on Aruana (Panicum maximum) pasture: case reports

Relata-se a ocorrência de osteodistrofia fibrosa em 38 equinos criados em pastagens de Panicum maximum cultivar Aruana, da raça Mangalarga Marchador, provenientes de uma propriedade localizada no estado do Paraná. No exame clínico geral, observou-se aumento bilateral e simétrico dos ossos da face e, também, aumento de volume na porção distal do rádio. A análise radiológica das áreas afetadas demonstrou redução da densidade óssea, e, na bioquímica sanguínea, 24 (63 por cento) animais apresentaram níveis de fósforo elevados. O exame bromatológico revelou níveis elevados de oxalato na pastagem de Aruana. Conclui-se que a ingestão de Aruana, forragem com altos níveis de oxalato, foi responsável pelo aparecimento de lesões de osteodistrofia fibrosa em equinos.

Fibrous osteodystrophy occurrence is reported in 38 Mangalarga Marchador horses raised on Aruana (Panicum maximum) pasture from a breeding farm in Paraná state. Clinical examination showed bilateral and symmetric increase in bones of face and an increase of volume at the radius distal portion. The radiologic analysis revealed bone density reduction in affected areas. Blood biochemist demonstrated high phosphorus levels in 24 animals (63 percent). Analysis of the pasture was performed and high levels of oxalate were found in the Aruana samples. It was concluded that the ingestion of Aruana, a pasture with high oxalate levels, was responsible for the appearing of fibrous osteodystrophy lesions in horses.

Vitrification of the inner perivitelline layer of chicken eggs for use in the sperm-egg interaction assay.

The sperm-egg interaction assay is a good predictor of the fertilizing potential of rooster semen; the ability of chicken sperm to interact with the egg can be assessed by counting the number of holes in the inner perivitelline layer (IPVL) of a freshly laid egg. Although isolated IPVL can be stored for up to 24h, preservation of IPVL for prolonged intervals in liquid nitrogen would facilitate the sperm-egg interaction assay. The objective of this study was to adapt the technique of vitrifying swine oocytes for use with the IPVL. Our hypothesis was that vitrification would not alter the ability of the membrane to bind sperm; therefore, there would be no difference between vitrified and fresh IVPL in the number of hydrolysis holes made by sperm. Our hypothesis was supported; there were no differences in the mean+/-SEM number of holes made by the same sample of sperm in vitrified and in fresh membranes (146.0+/-17.7 holes/mm(2) IPVL and 159.5+/-17.7 holes/mm(2) IPVL, respectively, P>0.05; n=123 IVPLs tested). Furthermore, 80% of frozen-thawed membranes were recovered intact. Because vitrification did not significantly change the ability of membranes to bind sperm, vitrified membranes can be safely used for the sperm-egg interaction assay. Vitrified IVPL would ensure availability for sperm evaluation and facilitate wide distribution of IPVL, enabling assays to be conducted even in the absence of facilities or expertise to prepare membranes.

Equine monocytic Ehrlichiosis (Potomac horse fever) in horses in Uruguay and southern Brazil.

A disease named locally as churrío or churrido equino (i.e., equine scours) has occurred for at least 100 years in Uruguay and southern Brazil in farms along both shores of the Merín lake. This report describes cases of churrido equino and provides serologic, pathologic, and DNA-based evidence indicating that the disease is in fact equine monocytic ehrlichiosis (Potomac horse fever). Results of an epidemiological investigation conducted on an endemic farm are also presented. Clinical signs in 12 horses were fever, depression, diarrhea, dehydration, and sometimes colic and distal hind limb edema. Postmortem findings of 3 horses were of acute enterocolitis. Inclusion bodies containing ehrlichial organisms were found in the cytoplasm of macrophages of the large colon of 1 horse. Eleven of the 12 horses were serologically positive to Ehrlichia risticii (indirect fluorescent antibody assay) and, of 3 paired samples, 2 showed seroconversion. Ehrlichia risticii DNA was identified by a nested polymerase chain reaction in peripheral blood of an affected horse. A healthy horse inoculated with peripheral blood from an affected horse developed the disease and antibodies to E. risticii. The disease had a peak incidence in March (summer) and was statistically associated with a marshy ecosystem near the Merín lake, where large numbers of Pomacea spp. (Ampullariidae) snails were found. Incidence density was almost 8 times higher in nonnative horses than in native horses. It was concluded that the previous diarrheic disease of horses known in Uruguay and southern Brazil as churrido equino is equine monocytic ehrlichiosis.