Glutathione and IGF-1 in bovine seminal cryopreservation: oxidative stress response and pregnancy rate / [Glutationa e IGF-1 na criopreservação seminal bovina: resposta do estresse oxidativo e taxa de gestação]

The objective of this study was to evaluate the rate of conception, metabolic, and structural conditions of cryopreserved bovine sperm cells, plus extender with IGF-1 and glutathione (GSH). 12 ejaculations of Nelore bulls were used, submitted to treatments: control, gSH (2mM/mL), IGF-1 (100ng/mL) and gSH (1mM/mL) + IGF-1 (50ng/mL). After cryopreservation and thawing the semen passed the fast thermo resistance test (TTR), plasma membrane and acrosomal integrity (PIAI), mitochondrial membrane potential (AP), oxidative stress, and conception rate. Tukey test was used for the statistical analysis of the parametric variables and the Friedman test for nonparametric. The gestation percentage was compared by the Chi-square test. There was no statistical difference (P<0.05) between treatments for the TTRr variable. Otherwise in the oxidative stress evaluated with the CellROX probe was noted that the IGF-1 showed the highest number of reactive cells (P<0.05). The PIAI, AP and gestation rate showed no difference among treatments (P>0.05), with an average of conceptions of 36.58%. It is concluded that IGF-1, gSH and their association did not cause changes in sperm motility, mitochondrial potential, plasma and acrosomal membrane integrity. IGF-1 increased oxidative stress, however, there was no difference in the gestation rate among the treatments.(AU)

Objetivou-se avaliar a taxa de concepção, as condições metabólicas e estrutural das células espermáticas bovinas criopreservadas, acrescidas de diluidores com fator de crescimento semelhante à insulina do tipo 1(IGF-1) e glutationa (GSH). Foram utilizados 12 ejaculados de touros da raça Nelore, submetidos aos tratamentos: controle, gSH (2mM/mL), IGF-1 (100ng/mL) e gSH (1mM/mL) + IGF-1 (50ng/mL). Após a criopreservação e descongelação, o sêmen passou pelos testes de termorresistência rápida (TTRr), integridade de membrana plasmática e acrossomal (PIAI), alto potencial mitocondrial (AP), estresse oxidativo e taxa de concepção. Utilizou-se o teste de Tukey para as análises estatísticas das variáveis paramétricas e o teste de Friedman para as não paramétricas, com significância de 5%. A percentagem de gestação foi comparada pelo teste do qui-quadrado. Não hove diferença estatística (P<0,05) entre os tratamentos para a variável TTRr. Já no estresse oxidativo avaliado com a sonda CellROX, observou-se que o IGF-1 apresentou maior quantidade de células reativas (P<0,05), 36,38± 24,10. A PIAI, o AP e a taxa de gestação não apresentaram diferença entre tratamentos (P>0,05), com média de concepções de 36,58%. Conclui-se que o IGF-1, a gSH e a sua associação não causaram mudanças na motilidade espermática, no potencial mitocondrial, na integridade da membrana plasmática e acrossomal. O IGF-1 aumentou o estresse oxidativo, porém sem diferença na taxa de gestação entre os tratamentos.(AU)

Insulina e IGF-1 no meio extensor de criopreservação seminal bovina / Insulin and IGF-I in extender medium for bovine semen cryopreservation

Objetivou-se avaliar a condição metabólica e estrutural das células espermáticas bovinas após congelação, com adição prévia de IGF-I e insulina no meio diluidor seminal. Os ejaculados de seis touros Nelore foram submetidos a quatro tratamentos: controle; insulina (100µUI/mL); IGF-I (150ng/mL) e insulina + IGF-I (50µUI/mL e 75ng/mL, respectivamente). Após a congelação, realizaram-se os testes de termorresistência rápida, coloração pelo corante azul de tripan e Giemsa, além da análise computadorizada da motilidade espermática, da integridade das membranas plasmática e acrossomal, e da peça intermediária por meio de sondas fluorescentes. O teste de termorresistência rápida apresentou efeito dentro do tempo de cada tratamento, mas não entre os tratamentos. Na análise computadorizada da motilidade espermática, foram observados movimento, motilidade e velocidade espermáticos; não houve efeitos dos tratamentos sobre qualquer uma dessas variáveis. Respostas iguais foram obtidas com as sondas fluorescentes e o corante azul de tripan/Giemsa. A adição de insulina e IGF-I, de forma isolada ou combinada, ao meio diluidor para congelação de sêmen não produziu efeitos sobre as condições metabólica e estrutural das células espermáticas.(AU)

This study aimed to evaluate the metabolic and structural condition of the spermatic bovine cells after the freezing, with addition, previously, of IGF-I and Insulin in the seminal thinner medium. The semen of 6 Nellore bulls were submitted to four treatments: Control, Insulin (100µUI/mL); IGF-I (150ng/mL) and Insulin + IGF-I (50µUI/mL and 75ng/mL, respectively). After freezing, rapid resistance tests, Tripan and Giemsa Blue staining, and computerized analysis of sperm motility and integrity of the plasma and acrosomal membranes and the intermediate part were performed by fluorescent probes. The term rapid resistance test had effect within the time of each treatment, but not between treatments. In the computer analysis of sperm motility, sperm movement, motility and velocity no effects of treatments were observed on any of these variables. The same results were obtained with the fluorescent probes and the Blue dye Trypan / Giemsa. The addition of Insulin and IGF-I, alone or in combination, to the semen freezing dilution medium had no effect on the metabolic and structural condition of sperm cells.(AU)

Antioxidant effect on viability of boar semen cooled to 15°C / Influência de antioxidantes na viabilidade do sêmen suíno resfriado a 15°C

This study aimed to evaluate the addition of Vitamin C, reduced Glutathione and trolox on sperm characteristics of pork refrigerated semen. Six pigs were collected through the technique of gloved hand (10 ejaculates/animals). The semen was diluted in MR-A®. After the previous evaluations, the treatments were added: Control group: diluent only; Vitamin C Group: 200µM/mL Vitamin C; Trolox Group: 200µM/mL Trolox; Glutathione group: 2.5mM/ml Reduced glutathione. The semen was stored in thermal boxes and placed inside the refrigerator at 15oC and evaluated at D0, 12, 48, 72 hours. After 30 hours of incubation, each treatment was divided into two equal fractions and the same concentration of antioxidants was added in one of the parts. The results show that reduced glutathione supplementation preserves sperm motility after 24 hours but also has a higher percentage of acrosome intact in the presence of this antioxidant. There was no effect of adding a second dose of the antioxidants. In conclusion, the addition of reduced Glutathione to the swine semen diluent is a promising alternative for better preservation of sperm characteristics and the addition of the second dose of antioxidants during storage is detrimental to semen.(AU)

Este estudo tem como objetivo avaliar a adição da vitamina C, da glutationa reduzida e do trolox sobre características espermáticas do sêmen refrigerado de suínos. Seis cachaços foram coletados pela técnica de mão enluvada (10 coletas/animal). O sêmen foi diluído em MR-A®. Após as avaliações prévias, os tratamentos foram adicionados: grupo controle: apenas diluidor; grupo vitamina C: 200µM/mL de vitamina C; grupo trolox: 200µM/mL de trolox; grupo glutationa: 2.5mM/mL de glutationa reduzida. O sêmen foi armazenado em caixas térmicas e alocado dentro do refrigerador a 15oC e avaliado nos tempos zero, 12, 48 e 72 horas . Após 30 horas de incubação, cada tratamento foi dividido em duas frações iguais e adicionou-se a mesma concentração de antioxidantes em uma das partes. Os resultados demonstram que a suplementação de glutationa reduzida preserva a motilidade espermática após 24 horas, bem como tem maior percentual de acrossoma intacto na presença desse antioxidante. Não houve efeito ao se adicionar uma segunda dose dos antioxidantes. Em conclusão, o acréscimo da glutationa reduzida ao diluidor de sêmen suíno é uma alternativa promissora para melhor preservação das características espermáticas, e a adição da segunda dose dos antioxidantes durante o armazenamento é prejudicial ao sêmen.(AU)

Avaliação clínica e bioquímica de cães submetidos a dois métodos de hidroterapia / Clinical and biochemical evaluation of dogs submitted to two methods of hydrotherapy

Avaliou-se a resposta dos parâmetros fisiológicos de frequência cardíaca (FC) e respiratória (FR), temperatura retal (TR) e a atividade sérica das enzimas creatina quinase (CK) e lactato desidrogenase (LDH) em cães adultos hígidos, machos e fêmeas, sob duas modalidades de hidroterapia. Dez cães foram distribuídos em dois grupos de cinco animais, denominados de grupo I (caminhada em esteira aquática) e grupo II (natação) e submetidos a duas sessões por semana com duração inicial de 10 minutos, acrescida de cinco minutos por sessão até 30 minutos, durante nove semanas. As avaliações foram realizadas antes, imediatamente depois e quatro horas após o exercício em todas as sessões para as variáveis de FC, FR e TR e nos dias um, sete, 28 e 60 para CK e LDH. Não houve diferença (P>0,05) quando comparados os valores médios dos parâmetros fisiológicos de frequência cardíaca e respiratória e de temperatura retal. Os níveis de CK e LDH não se elevaram após a hidroterapia nos diferentes tempos e dias avaliados. Dessa forma, foi possível concluir que as sessões de natação e de caminhada em esteira aquática não causaram alterações nos parâmetros fisiológicos de frequência cardíaca e respiratória, temperatura retal e nos níveis sanguíneos de creatina quinase (CK) e lactato desidrogenase (LDH) no protocolo proposto.(AU)

The physiological parameters of heart rate (HR), respiratory rate (RR), rectal temperature (TR), and serum activity of creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes were determined in healthy dogs, male and female, under two hydrotherapy modalities. Ten dogs were distributed in two groups of five animals, named Group I (underwater treadmill walk) and Group II (swimming), submitted to two sessions per week with initial duration of 10 minutes, plus 5 minutes per session up to 30 minutes, during 9 weeks. The evaluations were performed before, immediately after and 4 hours after exercise in all sessions for the HR, RR and TR variables and on days one, seven, 28 and 60 for CK and LDH. There was no difference (P> 0.05) when comparing the mean values of physiological parameters of heart and respiratory rate and rectal temperature. CK and LDH levels did not rise after hydrotherapy at different times and days evaluated. So, it was possible to conclude that swimming sessions and underwater treadmill walking did not cause alterations in the physiological parameters of heart and respiratory rate, rectal temperature, and the blood levels of creatine kinase (CK) and lactate dehydrogenase (LDH) in the proposed protocol.(AU)

Histopathological hoof laminar changes in horses with pituitary pars Intermedia adenoma: cases report / Alterações histopatológicas do tecido laminar do casco em cavalos com adenoma da pars intermedia da hipófise: relato de casos

Laminitis in horses is often associated with endocrine disorders, especially the pituitary pars intermedia dysfunction (PPID) in older animals. Morphologic exams of the laminar tissue of the hoof were performed in two horses with suspected PPID, with no clinical signs of laminitis. Changes compatible with laminitis of endocrine origin were observed, such as rounding of the nuclei of the basal cells, thinning and stretching of the secondary epidermal laminae and tissue proliferation. PPID horses with no clinical signs of laminitis may be affected by lesions of the laminar tissue of the hoof that compromise the integrity of the dermal-epidermal junction and may develop clinical symptoms of the disease. It has been suggested that the development stage of endocrine laminitis is longer, but further studies should be conducted to confirm it.

A laminite em cavalos está frequentemente associada a distúrbios endócrinos, como a disfunção da pars intermedia da pituitária (PPID) em animais mais velhos. Exames morfológicos do tecido laminar do casco foram realizados em dois cavalos com suspeita de PPID após o óbito, os quais não apresentaram sinais clínicos de laminite. Alterações compatíveis com a laminite de origem endócrina foram observadas, como arredondamento da núcleo das células basais, alongamento e afilamento das lâminas epidérmicas secundárias e proliferação tecidual. Cavalos com PPID sem sinais clínicos de laminite podem estar acometidos por lesões do tecido laminar do casco que comprometam a integridade das interdigitações dérmico-epidérmicas e podem desenvolver a sintomatologia clínica da doença. Sugere-se que o período de desenvolvimento da laminite endócrina seja mais longo, porém estudos adicionais devem ser realizados para confirmar essa hipótese.

Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor.

Sera from 112 mares from 5 horse-breeding farms was examined for the presence of antibodies to Neospora caninum and Toxoplasma gondii by an indirect fluorescent antibody test (IFAT), as well as from dogs and cattle present on these properties for the presence of antibodies to N. caninum. Among the 112 mares, 35 had a history of reproductive problems in the last breeding season and 77 had no reproductive problems. The rates of seroprevalence of N. caninum in mares with and without a history of reproductive problems were 25.71% and 6.49% and from T. gondii 2.85% and 1.29%, respectively. In dogs and cattle, the rates of seroprevalence of N. caninum were 10.52% and 15.55%, respectively. A positive correlation was found between the presence of antibodies against N. caninum (p=0.010) in mares and the occurrence of reproductive problems using the Fisher's exact test. Significantly higher seroprevalence for N. caninum in mares was observed on the farm that had seropositive dogs (p=0.018). Cattle on this farm were also seropositive. No significant differences in seropositivity were found on farms where dogs were seronegative, or absent. This result suggests, for the first time, the presence of seropositive dogs as a risk factor for N. caninum in mares and the necessity for further investigation of the epidemiology of this parasite in horse-breeding farms with reproductive problems and the presence of cattle and dogs. This is the first report on the occurrence of antibodies against N. caninum in horses from the state of Santa Catarina, Brazil.

Use of gastric balloon manometry for estimation of intra-abdominal pressure in horses.

REASONS FOR PERFORMING STUDY: Standing laparoscopic procedures, facilitated by abdominal insufflation with carbon dioxide, are being employed to an increasingly greater extent in horses. However, a sustained increase in abdominal pressure may be life-threatening. A practical method for intra-abdominal pressure (IAP) assessment is imperative. Although indirect methods for estimating IAP have been extensively studied in man, little work has been performed in veterinary medicine. OBJECTIVES: To investigate the utility of gastric manometry for purposes of evaluating IAP in horses. METHODS: Gastric pressure (P(ga) ) was estimated by balloon manometry in 8 healthy, mature horses, before and during a 30 min passive pneumoperitoneum induced by right paralumbar puncture. The balloon manometer was positioned within the gastric lumen and inflated using 2 separate volumes of air: 10 and 50 ml. P(ga) Gastric pressure was determined at baseline (0) and 5, 15 and 30 min after induction of passive pneumoperitoneum. Intra-abdominal pressure was measured directly by right paralumbar puncture using an 8 gauge needle at baseline and immediately following establishment of passive pneumoperitoneum. RESULTS: Baseline IAP values were negative and increased (P≤0.05) during development of passive pneumoperitoneum. However, recorded P(ga) measurements for both inflation volumes were positive before (baseline) and during the course of the passive pneumoperitoneum. Measured P(ga) values did not correlate with IAP at any time. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results suggest that the indirect method used in human patients for estimating IAP by P(ga) is not applicable for horses.

Effect of passive pneumoperitoneum on oesophageal pressure, cardiovascular parameters and blood gas analysis in horses.

REASONS FOR PERFORMING STUDY: Standing surgical procedures are being employed to an ever-greater extent in horses. Pneumoperitoneum during abdominal surgery might adversely affect the work of breathing. OBJECTIVES: To determine whether development of pneumoperitoneum during abdominal surgery adversely influences the work of breathing. METHODS: Eight healthy mature horses were equipped with carotid artery and thoracic vena cava catheters and an intraluminal manometry system. The following measurements were obtained before and at +5, +10, +15 and +30 min following establishment of pneumoperitoneum by paralumbar puncture using an 8 gauge needle: vital signs, oesophageal pressure, gastric pressure, arterial and central venous blood pressures, and arterial and mixed venous blood gas analyses. RESULTS: Significant changes in oesophageal pressure, central venous pressure and results of arterial and mixed venous blood gas analysis were not detected. Arterial diastolic and mean pressures and rectal temperature increased slightly (P ≤ 0.05). CONCLUSIONS: Passive pneumoperitoneum did not adversely affect breathing mechanics or haemodynamic variables under experimental conditions. Changes in arterial pressure could have occurred as a response to the passive pneumoperitoneum or be related to handling stress. Subtle variations in rectal temperature were not clinically relevant and likely resulted from stress associated with restraint. POTENTIAL RELEVANCE: It is unlikely that mature horses will develop signs of respiratory difficulty as a result of the development of passive pneumoperitoneum during standing laparoscopy.

Morphological abnormalities and apoptosis in lamellar tissue of equines after intestinal obstruction and treatment with hydrocortisone / Alterações morfológicas e apoptose em tecido laminar de equinos após obstrução intestinal e tratamento com hidrocortisona

Four experimental groups of equines were used in order to study morphological abnormalities and apoptosis in lamellar tissue. Group Cg (control) was composed of animals without any surgical procedure; group Ig (instrumented), animals that underwent enterotomy; group Tg (treated), animals that were subjected to intestinal obstruction and were treated with hydrocortisone; and group Ug (untreated), animals that were subjected to intestinal obstruction without treatment. The lamellar tissue was analyzed regarding the presence of tissue abnormalities and apoptosis. No morphological abnormalities were observed in animals of surgical groups, and no difference in apoptosis was observed between groups. It was concluded that intestinal obstruction allowed laminitis to develop, probably by systemic activation, and that the maneuvers performed in the enterotomy aggravated the process. Hydrocortisone did not aggravate the lesions of the lamellar tissue.

Foram utilizados quatro grupos de equinos para estudar alterações morfológicas e apoptose no tecido lamelar. O grupo CG (controle) foi composto por animais sem o procedimento cirúrgico; o grupo Ig (instrumentado), por animais submetidos à enterotomia; o grupo Tg (tratado), por animais submetidos à obstrução intestinal e tratados com hidrocortisona; e o grupo Ug (não tratado), por animais submetidos à obstrução intestinal, sem tratamento. O tecido laminar foi analisado quanto à presença de alterações morfológicas e de apoptose. Foram observadas alterações morfológicas nos equinos submetidos a procedimento cirúrgico, mas não houve diferença entre os grupos com relação às células apoptóticas. Conclui-se que a obstrução intestinal permite o desenvolvimento da laminite provavelmente por ativação sistêmica e que as manobras realizadas na enterotomia podem ser consideradas como agravantes no processo. A hidrocortisona não agravou as lesões do tecido laminar.