A leukocyte cryopreservation technique for cytogenetic studies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Trânsito gastrintestinal e digestibilidade aparente de nutrientes em eqüinos alimentados com dietas contendo grãos secos ou silagem de grãos úmidos de triticale

Cinco éguas mestiças (idade e peso corporal médios de seis anos e 480 kg PV) foram distribuídas em delineamento experimental em quadrado latino para se e avaliar a utilização de dietas contendo grãos de triticale (secos ou ensilados) em substituição ao milho na alimentação de eqüinos, por meio do ensaio de avaliação do trânsito gastrintestinal e da digestibilidade. Os tratamentos consistiram de três níveis de grãos de triticale em substituição aos grãos de milho (0, 50 e 100%) e duas formas de conservação dos grãos de triticale (secos ou ensilados). As dietas foram isoprotéicas (12,5% PB), com ingestão diária de MS pelos animais de 2,0% PV (relação volumoso : concentrado de 50:50). Os coeficientes de digestibilidade aparente (CDa) da MS, MO, PB e FDN foram determinados indiretamente. Os parâmetros de trânsito gastrintestinal avaliados foram: k1 (taxa de passagem pelo intestino grosso), k2 (taxa de passagem pelo estômago), TT (tempo de trânsito), TMR (tempo médio de retenção) e TMRT (tempo médio de retenção total). Não houve diferença para os CDa da MS, MO, PB e FDN entre as dietas experimentais, observando-se valores médios de 64,31; 65,14; 74,13 e 57,25%, respectivamente. Considerando-se a cinética das fases sólida e líquida, notou-se efeito somente para k2 na fase sólida da digesta, cujo valor nas rações contendo 100% de triticale (seco ou ensilado) foi de 19,63%/h e na dieta controle (0% de triticale), de 23,72%/h. Observou-se efeito linear crescente para o TT na fase sólida da digesta, com a elevação dos níveis de substituição dos grãos de milho pelos grãos de triticale ensilados. Concluiu-se que os concentrados para eqüinos podem ser formulados com grãos de triticale secos ou ensilados em substituição total ao milho. A inclusão de grãos de triticale na alimentação desta espécie animal promoveu trânsito lento da fase sólida da digesta.

Valor nutritivo e estudo cinético do trato digestivo de dietas contendo grãos secos ou ensilados de sorgo de baixo e alto tanino para eqüinos

Quatro éguas sem raça definida (idade e peso corporal médios de seis anos e 400 kg) foram distribuídas em delineamento experimental em quadrado latino para se avaliar o valor nutritivo e o estudo cinético do trato digestivo de grãos secos ou ensilados de sorgo de baixo e alto conteúdos de tanino na alimentação de eqüinos. Os tratamentos consistiram de dietas contendo dois híbridos de grãos de sorgo (baixo e alto níveis de tanino) e dois métodos de conservação (secos e ensilados). As dietas foram isoprotéicas (12,4% PB), com ingestão diária de MS estabelecida em 1,5% PV (relação feno:concentrado de 50: 50). Os parâmetros de trânsito gastrintestinal avaliados foram: k1 (taxa de passagem pelo intestino grosso), k2 (taxa de passagem pelo estômago), TT (tempo de trânsito), TMR (tempo médio de retenção) e TMRT (tempo médio de retenção total). Os tratamentos não afetaram os coeficientes de digestibilidade aparente (CDa) da MS e do amido, cujos valores médios foram 54,04 e 98,91%, respectivamente. Verificou-se efeito benéfico da ensilagem dos grãos de sorgo de alto conteúdo de tanino sobre a digestibilidade da PB e FDN. A CDa da PB e FDN para a dieta contendo grãos secos de sorgo de alto teor de tanino foi de 49,76 e 32,20% e para as dietas com grãos de sorgo de baixo conteúdo de tanino (seco ou ensilado) e grãos ensilados de sorgo de alto teor de tanino foi de 65,63 e 43,32%, respectivamente. Obteve-se somente efeito do método de conservação dos grãos de sorgo (secos vs ensilados) sobre o TMR, em que o valor para as dietas com silagens de grãos ensilados e secos foi, respectivamente, de 40,08 e 37,9h. Concluiu-se que os grãos de sorgo secos de alto teor de tanino não devem ser usados como principal grão energético nos concentrados para eqüinos, por diminuírem a digestibilidade da proteína e fibra.

Parâmetros bioquímicos de equinos submetidos à simulação de prova de enduro recebendo dietas com adição de óleo de soja

Com o objetivo de verificar o efeito da adição de níveis crescentes de óleo no concentrado sobre a atividade plasmática das enzimas creatina quinase (CK), aspartato aminotransferase (AST) e lactato desidrogenase (LDH) como indicativo de metabolismo energético, foram fornecidas dietas experimentais compostas de cinco níveis de óleo (controle, 6, 12, 18 e 24% do concentrado). Utilizaram-se 20 equinos da raça Árabe, peso médio de 400 kg, submetidos a prova de enduro de 80 km em esteira rolante. O enduro foi dividido em quatro anéis de 20 km, com duração média de 1 hora e dez minutos. A adição de óleo e a distância percorrida tiveram efeitos sobre as variáveis AST, CK e LDH, que apresentaram as respectivas expressões: AST (7,045-0,2292x+0,007991x2+0,008517z- 0,0003282xz), CK (8,06-,07020x+0,05546x2-0,001262x3+0,01204z+0,0006207xz) e LDH (6,624-0,3522x+0,03448x2-0,0008382x3+0,02401z-0,0007489xz) . O óleo é uma importante e bem aproveitada fonte de energia para equinos em exercício, pois sua adição na dieta de animais submetidos a prova de enduro promoveu alteração metabólica que favorece a produção de energia. O metabolismo animal poupou suas reservas energéticas oriundas da glicose, favorecendo a utilização do óleo. A menor atividade plasmática das enzimas AST, CK e LDH com a adição de óleo nas dietas indica direcionamento do metabolismo energético para a β-oxidação. Como apresentam várias isoenzimas, as enzimas estudadas atuam amplamente no metabolismo energético, favorecendo a constante reposição de ATP ao longo do exercício.

Dynamics of follicle populations and gonadotropin concentrations in fillies age two to ten months

Follicle populations and concentrations of circulating gonadotropins were studied during age 2-10 months in 10 spring-born pony fillies. Blood sampling and ultrasound scanning were done every 4 days and daily for four 30 day periods. During 5-12 weeks, FSH concentrations were lower in 6 fillies with follicles greater than or equal to 6 mm (mean +/- s.e. 1.4 +/- 0.1 ng/ml) than in 4 fillies with follicles <6 mm (2.8 + 0.3 ng/ml). The diameters and numbers of follicles and gonadotropin concentrations increased progressively during age 2-4 months. A plateau in follicle activity and reduced levels of gonadotropins occurred during 5-7 months. During 8-10 months, follicles grew to >10 mm and gonadotropin concentrations increased. Waves of follicular growth were identified during the 30 day periods by significant increases in the diameter of the 10 largest follicles. The waves did not partition into dominant and subordinate follicles. Results indicated an initial postnatal period of negative ovarian feedback, temporally related changes in gonadotropins and follicles for months 3-10, and development of follicles in waves.

The pre-emptive effect of epidural ketamine on wound sensitivity in horses tested by using von Frey filaments

Objective To evaluate the pre-emptive analgesic effect of pre-incisional epidural ketamine.Study Design A blinded, randomized experimental study.Animals Sixteen mixed breed mares, 17.6 +/- 2.8 years old, weighing 352 +/- 32 kg.Methods In a pilot study, an incision was made on one lateral thigh using a lidocaine block and no further analgesics, and it was verified that the nociceptive threshold was lower on the incised side than nonincised side (p < 0.05), and that von Frey filaments evoked a pain response. The 16 animals were divided into group A (ketamine, n = 9) and B (saline, n = 7). An epidural catheter was inserted 24 hours before the trials, the thigh was shaved bilaterally, and the right side was blocked (incised side) using lidocaine. Twenty-five minutes later, ketamine (A) or saline (B) was administered epidurally. Five minutes later, a 10-cm. skin incision was made on the right side, and then sutured. Nociceptive threshold was determined with von Frey filaments at 1, 3, and 5 cm. around the incision at 15-minute intervals for 2 hours, then at 4, 6, and 8 hours. Behavioral alterations, heart and respiratory rates were recorded. Nociceptive thresholds from these points were averaged to obtain mean values at each time, converted to a logarithmic scale, and submitted to a nonparametric analysis (Mann-Whitney and one-way repeated measures anova test,p less than or equal to 0.05).Results After 8 hours, the global range score revealed reduced hyperalgesia (p < 0.01) around the incision in 92% (4.65-4.27) of evaluated intervals in group A (ketamine). There were no significant changes in behavior, heart and respiratory rates,Conclusions It was concluded that pre-emptive epidural ketamine reduced post-incisional pain in the horse, and that von Frey filaments were able to quantify cutaneous sensitivity after tissue damage.Clinical relevance Epidural ketamine injection can reduce post-incisional sensitivity in the horse.

Radiographic study of distal radial physeal closure in thoroughbred horses

Monthly radiography was performed to study distal radial physeal closure in ten male and ten female Throughbred horses. The height, thoracic circumference and metacarpus circumference were also measured, Distal radial physeal closure time was sooner in females than males, and took 701 +/- 37 and 748 +/- 55 days respectively.

Development and characterization of an equine behaviour chamber and the effects of amitraz and detomidine on spontaneous locomotor activity

This report describes the development of a behaviour chamber and the validation of the chamber to measure locomotor activity of a horse, Locomotor activity was detected by four Mini-beam sensors and recorded on a data logger every 5 min for 22 h. Horses were more active during daytime than in the evening, which was at least partially related to human activity in their surroundings. To validate the ability of the chambers to detect changes in activity, fentanyl citrate and xylazine HCl, agents well-characterized as a stimulant and a depressant, respectively, were administered to five horses. Fentanyl citrate (0.016 mg/kg) significantly increased locomotor activity which persisted for 30 min, Xylazine HCl (1 mg/kg) significantly reduced locomotor activity for 90 min. Amitraz produced a dose-dependent decrease in locomotor activity, lasting 75 min for the 0.05 mg/kg dose, 120 min for the 0.10 mg/kg dose, and 180 min for the 0.15 mg/kg dose, In a separate experiment, yohimbine administration immediately reversed the sedative effect of amitraz, This suggests there is a similarity in the mode of action of amitraz, xylazine and detomidine, as yohimbine acts primarily by blocking central alpha 2-adrenoceptors that are stimulated by agents like xylazine, There was also a significant decrease in locomotor activity following injection of detomidine (0.02, 0.04 and 0.08 mg/kg) for 1.5, 3.5 and 5.0 h, respectively, the locomotor chamber is a useful, sensitive and highly reproducible tool for measuring spontaneous locomotor activity in the horse, which allows investigators to determine an agent's average time of onset, duration and intensity of effect on movement.

Intra-articular pressure profiles of the cadaveric equine fetlock joint in motion

The study of the influence of motion and initial intra-articular pressure (IAP) on intra-articular pressure profiles in equine cadaver metatarsophalangeal (MTP) joints was undertaken as a prelude to in vivo studies, Eleven equine cadaver MTP joints were submitted to 2 motion frequencies of 5 and 10 cycles/min of flexion and extension, simulating the condition of lower and higher (double) rates of passive motion. These frequencies were applied and pressure profiles generated with initial normal intra-articular pressure (-5 mmHg) and subsequently 30 mmHg intra-articular pressure obtained by injection of previously harvested synovial fluid.The 4 trials performed were 1) normal IAP; 5 cyles/min; 2) normal IAP; 10 cycles/min; 3) IAP at 30 mmHg; 5 cycles/min and 4) IAP at 30 mmHg; 10 cycles/min. The range of joint motion applied (mean +/- s.e.) was 67.6 +/- 1.61 degrees with an excursion from 12.2 +/- 1.2 degrees in extension to 56.2 +/- 2.6 degrees in flexion, Mean pressure recorded in mmHg for the first and last min of each trial, respectively, were 1) -5.7 +/- 0.9 and -6.3 +/- 1.1; 2) -5.3 +/- 1.1 and -6.2 +/- 1.1; 3) 58.8 +/- 8.0 and 42.3 +/- 7.2; 4) 56.6 +/- 3.7 and 40.3 +/- 4.6. Statistical analyses showed a trend for difference between the values for the first and last minute in trial 3 (0.05>P<0.1) with P = 0.1 and significant difference (P = 0.02) between the mean IAP of the first and last min in trial 4. The loss of intra-articular pressure associated with time and motion was 10.5, 16.9, 28.1 and 28.9% for trials 1-4, respectively. As initial intraarticular pressure and motion increased, the percent loss of intra-articular pressure increased.The angle of lowest pressure was 12.2 +/- 1.2 (mean +/- s.e.) in extension in trials 1 and 2, In trials 3 and 4, the lowest pressures were obtained in flexion with the joints at 18.5 +/- 2.0 degrees (mean +/- s.e.). This demonstrated that the joint angle of least pressure changed as the initial intra-articular pressure changed and there would not be a single angle of least pressure for a given joint.The volume of synovial fluid recovered from the MTP joints in trial 3 compared to 4 (trials in which fluid was injected to attain IAP of 30 mmHg) was not significantly different, supporting a soft tissue compliance change as a cause for the significant loss of intra-articular pressure during the 15 min of trial 4.The pressure profiles generated correlate well with in vivo values and demonstrated consistent pressure profiles. Our conclusions are summarised as follows:1. Clinically normal equine MTP joints which were frozen and then later thawed were found to have mostly negative baseline intra-articular pressures, as would be expected in living subjects,2. Alternate pressure profiles of the dorsal and plantar pouch at baseline intra-articular pressure document the presence of pressure forces that would support 'back and forth' fluid movement between joint compartments. This should result in movement of joint fluid during motion, assisting in lubrication and nutrition of articular cartilage,3. If joint pressure was initially greater than normal (30 mmHg), as occurs in diseased equine MTP joints, joint motion further increased joint capsule relaxation (compliance) and, therefore, reduced intra-articular pressure.4. Peak intra-articular pressures reached extremely high values (often >100 mmHg) in flexion when initial pressure was 30 mmHg. Joint effusion pressures recorded for clinical MCP joints are frequently 30 mmHg. These IAP values are expected to produce intermittent synovial ischaemia in clinical cases during joint flexion.5, Additional in vivo studies are necessary to confirm our conclusions from this study and to identify the contributions of fluid absorption and the presence of ischaemia in a vascularised joint.

Aerobic training, but not creatine supplementation, alters the gluteus medius muscle

The aim of the present study was to investigate the effect of oral supplementation of creatine on the muscular responses to aerobic training. Twelve purebred Arabian horses were submitted to aerobic training for 90 d, with and without creatine supplementation, and evaluated with respect to BW and BCS and to the area and frequency of the different types of muscle fibers in the gluteus medius. Supplementation consisted of the daily administration of 75 g of creatine monohydrate mixed into the ration for the 90 d of training. Physical conditioning was conducted on a high-performance treadmill, and training intensity was stipulated by calculating the velocity at which blood lactate reaches 4 mmol/L, determined monthly for each animal. The individual intensity of physical force at 80% of aerobic threshold was established. Morphometry of glutens medius muscle fibers was performed on frozen sections processed for histochemical analysis of myosin adenosine triphosphatase and immunohistochemistry of slow-contracting myosin. The results demonstrated that the animals maintained a moderate BCS without alteration of BW during the course of training, providing evidence of equilibrium between food intake and caloric expenditure during the study period. The present study demonstrated that aerobic training for 90 d caused hypertrophy of fiber types I (P = 0.04), IIA (P = 0.04), and IIX (P = 0.01), as well as an increase in the relative area occupied by type I fibers (P = 0.02) at the expense of type IIX fibers (P = 0.03), resulting in modifications of the contractile and metabolic characteristics of the gluteus medius muscle. It was not possible to show any beneficial effect from creatine on the skeletal muscle characteristics examined.

Serum iron and plasma fibrinogen concentrations as indicators of systemic inflammatory diseases in horses

Background: Detection of systemic inflammation, which is important for proper diagnosis and prompt treatment, can be challenging.Hypothesis: Measurement of plasma iron concentration is a sensitive method for detecting systemic inflammation in horses compared with measurements of plasma Fibrinogen concentration, a traditional marker for inflammation in the horse.Animals: Ninety-seven horses hospitalized with diseases causing systemic inflammation, 22 horses with localized inflammation, and 12 clinically normal horses were included in this study.Methods: A retrospective study was made on hospitalized horses that had both plasma iron and fibrinogen concentrations measured on hospital admission.Results: Plasma iron concentration was lower in horses with systemic inflammation (64 +/- 45 mu g/dL) than the reference interval minimum (105 mu g/dL) and were significantly lower (P = .001) than the value in a group of horses with local inflammation (123 +/- 45 mu g/dL) and in healthy transported horses (143 +/- 29 mu g/dL). Low plasma iron and high fibrinogen concentrations were both sensitive indicators of systemic inflammation in horses with sensitivity of 90 and 82%, respectively. There was a similar correlation between either continued decreases in iron concentration (R-sp of 0.239) or increases in fibrinogen concentration (R-sp of 0.280) during hospitalization and a worse prognosis.Conclusions and Clinical Importance: Measurement of plasma iron concentration better reflected acute inflammation than did fibrinogen concentration.

A COMPARATIVE-STUDY OF 4 DIFFERENT STAINING METHODS FOR ESTIMATION OF LIVE YEAST FORM CELLS OF PARACOCCIDIOIDES-BRASILIENSIS

A comparative study of four different staining methods for estimation of live yeast form cells of Paracoccidioides brasiliensis was carried out. The staining methods used were fluorescent staining, vital dye exclusion tests with erythrosin B and by Janus green and lactophenol cotton blue staining. Colony forming units (cfu) of the yeast form of eight P. brasiliensis isolates on brain heart infusion agar (BHIA) supplemented with 4% horse serum plus 5% P. brasiliensis cell extract (BHIA + HS + EXT) were examined for reliability of staining in determining the number of live fungal units in eight different isolates. Cfu on BHIA + HS + EXT plates showed an excellent plating efficiency over 96% in all isolates tested. The percentage of the live cells indicated by fluorescent staining (FL) or vital dye exclusion test with erythrosin B (EB) or Janus green (JG-1) was lower than that of cfu. By contrast, the percentage due to modified dye exclusion test with Janus green (JG-2) and that due to lactophenol cotton blue staining (LPCB) showed a close correration to that of cfu. Our results indicate that the modified dye exclusion test with Janus green and lactophenol cotton blue staining are useful for estimating cell viability of yeast form cells of P. brasiliensis.

Thermography and ultrasonography in back pain diagnosis of equine athletes

The object of the current study was to evaluate the efficacy of thermography and ultrasonography in the diagnosis of thoracolumbar lesions in Quarter Horse athletes and associate the different types of lesions found with the athletic modality practiced. Twenty-four horses were admitted to the Surgery Service for Large Animals of the Veterinary and Animal Science Faculty, UNESP, Botucatu, Brazil, with complaints of back problems. All the horses were submitted for physical examinations to confirm the existence of thoracolumbar alterations and then for thermography and ultrasonography Thermography was used to map the lesioned areas of this region and ultrasonography for lesion characterization. The lesions found were supraspinous desmitis, interspinous desmitis, dorsal intervertebral osteoarthritis, and impingement of the spinous processes or kissing spines. The existence of a relation between the type of event practiced by the horse and the type of lesion found was determined. In horses that competed in the barrel race, a predominance of lesions in the thoracic caudal, thoracolumbar, and cranial lumbar regions occurred, with intervertebral osteoarthritis and interspinous desmitis being the most common. In cutting horses, most of the lesions were observed in the caudal lumbar region, whereas horses competing in reining showed a preferential location for lesions in the middle lumbar, with a predominance of supraspinous desmitis and myositis. Thermography associated with ultrasonography was shown to be efficient in the diagnosis of the thoracolumbar lesions of these horses.

Ultrastructural and immunocytochemical evaluation of the effects of extracorporeal shock wave treatment in the hind limbs of horses with experimentally induced suspensory ligament desmitis

Objective-To evaluate the effects of extracorporeal shock wave therapy (ESWT) on affected ligaments in the hind limbs of horses with experimentally induced suspensory ligament desmitis by use of ultrasonographic, ultrastructural, and immunocytochemical techniques.Animals-10 horses.Procedure-Suspensory ligament desmitis was induced in both hind limbs of each horse by use of 2 collagenase injections (administered 2 weeks apart) in each suspensory ligament. Two weeks after the second injection, the right hind limb of each horse was treated with ESWT (3 treatments at 3-week intervals)- the left hind limb was not treated (control limb). Periodically during the study, the healing process was monitored ultrasonographically and the proportions of ligaments affected with lesions were assessed. Four weeks after the last ESWT treatment, biopsy specimens were collected from all ligaments for ultrastructural evaluation and immunocytochemical analysis of transforming growth factor beta-1.Results-The difference in the proportion of the lesion-affected ligament between ESWT-treated and control limbs was significant (P < 0.05) from 3 weeks after the second ESWT treatment to the end of the study. Compared with control ligaments, ESWT-treated ligaments had more small, newly formed collagen fibrils and greater expression of transforming growth factor beta-1 4 weeks after the last ESWT treatment was administered.Conclusions and Clinical Relevance-Results have indicated that ESWT appears to facilitate the healing process in horses with experimentally induced hind limb suspensory ligament desmitis.