Ethanol, Carnoy, and paraformaldehyde as fixative solutions for histological evaluation of preantral follicles in equine ovarian tissue.

The most adequate fixative solution for equine ovarian tissue is still to be determined as a tool to evaluate the improvement of methodological studies in assisted reproductive techniques and fertility preservation. This study aimed to evaluate a short-time ethanol 70% (ST-EtOH, 45 min) exposure as an alternative fixative compared with two classically fixatives [Carnoy's (CAR) solution and paraformaldehyde 4% (PFA)] at different fixation times (6 h, 12 h). The end points evaluated were morphology and classes of preantral follicles, follicular and stromal cell densities, and follicular and oocyte nuclear diameters in equine ovarian tissue. Ovaries (n = 6) from ovariectomized young mares were fragmented (3 × 3 × 1 mm; 20 fragments/ovary) and fixed in the tested treatments. Overall, a total of 11,661 preantral follicles were evaluated in 1444 histological slides. The ST-EtOH similarly preserved the preantral follicle morphometry and stromal cell density compared to the PFA fixative, regardless of the exposure time. Nonetheless, the CAR fixative solution had the greatest percentage of normal preantral follicles and the highest stromal cell density among all treatments. In conclusion, Carnoy's solution must be preferred compared with ST-EtOH and PFA fixatives for studies concerning the cellular morphology of equine ovarian tissue. Moreover, ST-EtOH fixative is a good alternative for equine ovarian tissue when a quick histological evaluation is required instead of more time-consuming and expensive techniques. Additional studies concerning the impact of different fixatives on the ultrastructure of cellular populations and their compatibility with IHC and molecular techniques in equine ovarian tissue are warranted.

Red cell distribution width values and red cell distribution width-to-platelet ratio in Thoroughbred foals in the first 24 hours of life.

OBJECTIVES: To report red cell distribution width (RDW) values, to calculate RDW-to-platelet ratio (RPR), and to investigate a possible correlation of RDW and RPR index values in neonatal foals classified as healthy or at risk based on clinical information from a population of foals up to 24 hours of life. DESIGN: Retrospective study conducted from records and CBCs of foals born between June and November from 2018 to 2020 foaling seasons. SETTING: Breeding farm. ANIMALS: Three hundred and nine neonatal full-term Thoroughbred foals. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Foals were evaluated by a veterinarian within 15 minutes after birth, and a blood sample was collected within 24 hours of life. Based on clinical information, 88 of 309 foals (28.4%) were considered at risk of perinatal disease, and 201 were healthy. Mean gestational age for the foals was 346.3 ± 9.7 days. RDW values did not differ between groups. Gestational length demonstrated to have a negative correlation with RDW (r = -0.156, P = 0.005) and mean corpuscular volume (r = -0.135, P = 0.01), indicating a link of these variables to foal maturity. RPR index was higher for at-risk (0.073 ± 0.018) than for healthy foals (0.068 ± 0.014, P = 0.01). CONCLUSION: RPR might be a promising early indicator of disease for the field triage of neonatal foals.

Climatic factors affecting gestational length in mares under subtropical climate.

The aim of this study was (i) to evaluate the relationship of climatic factors with gestational length (GL) and (ii) to evaluate the relationship of sire, foal gender and maternal factors with GL in mares. Retrospective data from 470 gestations of 202 respective mares were collected from a Criollo breeding farm in the southern hemisphere. GL was considered as the interval between ovulation and parturition. Climatic and environmental data (temperature, relative humidity, daily sunshine hours, precipitation, temperature humidity index - THI) were obtained daily and the mean values for foaling month and season were calculated. A multiple factor analysis of variance was performed to determine the relationship of the independent variables and interactions related to the mare, foal gender and stallion, and those related to the weather in each season of the year with GL as the dependent variable. The mean ± standard error (SE) of GL for the 470 gestations was 337 ± 0.4 days with a range of 311-363 days. Young-primiparous mares (340±0.9) had longer gestations (P < .001) than young-multiparous (336 ± 0.7) and mature-multiparous (334 ± 0.7) mares. Foal gender had no effect on GL. Gestational length was found to be affected by the stallion. Mares which experienced an autumn and winter gestational period with less daily sunshine hours had an increased GL (P < .05). In conclusion, environmental and climatic factors during pregnancy influenced the GL in mares, specifically, daily sunshine hours, precipitation and THI presented a negative correlation with GL. Young-primiparous Criollo mares presented longer gestational lengths than young or mature multiparous mares.

Serum cortisol and thyroid hormone concentrations and survival in foals born from mares with experimentally induced ascending placentitis.

BACKGROUND: There are few publications on occurrence of nonthyroidal illness syndrome in foals and on the prognostic value of cortisol and thyroid hormone (TH) concentrations in newborn foals. OBJECTIVES: To determine serum cortisol and TH concentrations (total and free thyroxine: T4 and F T4 ; total and free triiodothyronine: T3 and F T3 ) in foals born from mares with placentitis, to determine their association with survival, and their use as prognostic markers. ANIMALS: A cohort of 29 newborn foals comprising 5 Control, 14 Low-risk, and 10 Sick foals were evaluated over the first week of life. METHODS: In this prospective study foals born to mares with experimentally-induced placentitis were assigned to Low-risk or Sick groups while foals born to control mares were classified as Control based on clinical findings. Foals were also classified as Term (n = 13), Dysmature (n = 7), or Premature (n = 9), and survival rate was recorded. Serum cortisol and TH hormone concentrations were measured at 0, 12, 24, 48, and 168 hours of life. RESULTS: Sick non-surviving foals had lower (P < .05) T3 : cortisol ratio at 12 (3.68 ± 1.06 versus 18.58 ± 2.78), 24 (5.47 ± 2.34 versus 23.40 ± 3.82), and 48 (10.47 ± 6.29 versus 26.6 ± 2.90) hours of life when compared to Sick surviving foals and lower (P < .05) T4 : cortisol ratio at 12 (75.12 ± 21.71 versus 414.47 ± 58.47) and 24 hours (127.83 ± 55.21 versus 430.87 ± 80.31) after birth than Sick surviving foals. CONCLUSION AND CLINICAL IMPORTANCE: Placental infections can impair fetal thyroid function. Low T3 : cortisol and T4 : cortisol ratios seem to be good prognostic markers in newborn foals.

Single-Layer Colloid Centrifugation as a Method to Process Urine-Contaminated Stallion Semen After Freezing-Thawing.

Urospermia is a major ejaculatory dysfunction affecting stallions. It has been thought that urine-contaminated semen should not be cryopreserved; however, on select cases, urine contamination of semen cannot be avoided. A recent study suggested that urospermic semen can be cryopreserved after cushion centrifugation and extension. Thus, this study aimed to assess the use of single-layer colloid centrifugation (SLC) to process frozen-thawed urine-contaminated stallion semen. Raw ejaculates (n = 55) from eight stallions were split into three groups: no urine, low (20%), or high (50%) urine contamination. Semen was extended 1:1, cushion-centrifuged, and resuspended at 200 million sperm/mL in BotuCrio. Resuspended semen was loaded in 0.5 mL straws and cryopreserved in liquid nitrogen. Samples were thawed (37°C for 30 seconds) and processed by SLC (400 g/30 minutes). Percentages of total motility (TM) and progressive motility (PM) were assessed with computer-assisted semen analyzer. Sperm viability (%VIAB) and yield were assessed with a NucleoCounter before and after gradient centrifugation. Data were analyzed with two-way ANOVA and Tukey's test. The motility parameters TM before SLC (control: 35 ± 2; low: 33 ± 0.7; high: 22 ± 1.8) after SLC (control: 51 ± 3.6; low: 42 ± 2.2; high: 25 ± 2.8) and PM before SLC (control: 24 ± 1.8; low: 21 ± 1.14; high: 12 ± 1.5) and after SLC (control: 40.3 ± 3.2; low: 31 ± 3.9; high: 14 ± 2) significantly decreased with increasing urine contamination. Urine contamination marginally reduced (P < .05) sperm viability after cryopreservation before SLC (control: 45 ± 0.7; low: 27 ± 0.2; high: 27 ± 0.3) and after SLC (control: 54 ± 0.5; low: 49 ± 0.7; high: 38 ± 0.6). Recovery rates of sperm after centrifugation were not significantly different between groups. In conclusion, urine contamination affects sperm motility parameters in a dose-dependent manner. Post-thaw SLC selected sperm with higher motility and viability in control and low groups but only selected sperm with higher viability in the high group.

Cortisol, progesterone, 17αOHprogesterone, and pregnenolone in foals born from mare's hormone-treated for experimentally induced ascending placentitis.

This study aimed to evaluate steroid hormones in foals born from mares treated for ascending placentitis with different combinations of trimethoprim-sulfamethoxazole (TMS), flunixin meglumine (FM), long-acting altrenogest (ALT) and estradiol cypionate (ECP) for ten consecutive days, starting two days after experimental induction of placentitis with Streptococcus zooepidemicus. Fourty-six pregnant mares and respective foals were assigned as healthy group (Control, n = 8) or treated groups as follows: TMS+FM (n = 8), TMS+FM+ALT (n = 8), TMS+FM+ALT+ECP (n = 6), TMS+FM+ECP (n = 6) and no treatment (NO TREAT n = 10). At delivery, foals were classified as high-risk or low-risk based on clinical and hematologic findings, and survival rates were recorded during the first week of life for comparisons across groups. Cortisol, progesterone, 17αOHprogesterone, and pregnenolone concentrations were determined via immunoassays in 31 of the 46 foals immediately after foaling (0 h), at 12, 24, 48 h, and seven days post-partum (168h). At birth, serum cortisol concentrations were higher in Control and TMS+FM+ECP foals than in remaining groups (p < 0.05). Foals in TMS+FM+ALT and TMS+FM groups had higher 17αOHprogesterone concentrations at 24 h and 48 h, respectively (p < 0.05). Pregnenolone concentrations were higher in TMS+FM than TMS+FM+ALT+ECP foals at 7 days (p < 0.05). High-risk and non-surviving foals had decreased concentrations of cortisol at parturition, but increased concentrations of progesterone from 0 h to 48 h. Pregnenolone and 17αOHprogesterone concentrations were increased and pregnenolone after 12 h in high-risk and non-surviving foals (p < 0.05). In conclusion, adding ECP to the treatment of experimentally-induced placentitis appears to improve foal viability and endocrine response. Cortisol and progestogen profiles were abnormal in high-risk and non-surviving foals, and those treated with ALT or TMS+FM only.

Hematological and biochemical indicators of maturity in foals and their relation to the placental features / Indicadores hematológicos e bioquímicos de maturidade em potros e relação com suas características placentárias

Newborn's health is directly related to gestational conditions and placental efficiency. The aims of this study were: (1) To evaluate hematological and biochemical parameters of foals born from mares with placentitis at birth and at 24h of age, (2) to verify if placental pathology had any influence on neonatal maturity degree through hematological and biochemical response of those foals. According to placental findings (control and placentitis) and neonatal maturity degree (mature and immature), foals were divided into three groups: (1) Control group (n=22), foals born from mares with placentitis and classified as (2) Mature (n=26), and (3) Immature (n=10). The hematocrit and plasma concentration of fibrinogen, total plasma protein, white blood cells count, lactate, glucose, creatinine, urea, albumin, bilirubin, triglyceride, cholesterol, calcium, phosphorus, magnesium, aspartate aminotransferase (AST), creatine kinase (CK), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT) were measured. Placental features were significantly different between neonatal maturity degree (P=0.001). Mares that had acute placentitis foaled more immature neonates (n=8/10; 80%). Concentrations of fibrinogen (P=0.003), creatinine (P=0.021), total cholesterol (P=0.014), AST (P=0.001), GGT (P=0.002), total (P=0.001) and unconjugated bilirubin (P=0.010) were higher at birth in the Immature group, whereas albumin levels were lower (P=0.002). Foals born from mares with placentitis presented hyperlactatemia at 24h of age (P=0.002). Acute placentitis had an influence on the neonatal maturity, allowing an accelerated but incomplete fetal maturation. The monitoring of lactate, fibrinogen, creatinine, bilirubin, cholesterol, albumin, AST, and GGT levels, associated with clinical, physical, and behavior evaluation may contribute as indicators of neonatal maturity.(AU)

A saúde do neonato está diretamente relacionada às condições gestacionais e eficiência placentária. Os objetivos deste estudo foram: (1) avaliar parâmetros hematológicos e bioquímicos de potros nascidos de éguas com placentite ao nascimento e com 24h de vida e (2) verificar se a patologia placentária exerceu influência no grau de maturidade através da resposta hemato-bioquímica destes neonatos. De acordo com os resultados histopatológicos placentários (controle e placentite) e grau de maturidade neonatal (maturo e imaturo), os potros foram divididos em três grupos: grupo controle (n=22); e potros nascidos de éguas com placentite classificados como (2) maturos (n=26) e (3) imaturos (n=10). Foi avaliado hematócrito e concentrações sanguíneas de fibrinogênio, proteína plasmática total, leucócitos totais, lactato, glicose, creatinina, uréia, albumina, bilirrubinas, triglicerídeos, colesterol, cálcio, fósforo, magnésio, aspartato aminotransferase (AST), creatina quinase (CK), fosfatase alcalina (FA) e gama glutamiltranferase (GGT). As características placentárias foram significativamente diferentes entre os graus de maturidade neonatal (P=0.001). Éguas com placentite aguda produziram mais potros imaturos (n=8/10; 80%). No nascimento, os potros imaturos apresentaram maiores concentrações de fibrinogênio (P=0,003), creatinina (P=0,021), colesterol total (P=0,0014), AST (P=0,001), GGT (P=0,002), bilirrubina indireta (P=0,010) e total (P=0,001) e menor concentração de albumina (P=0,002). Os potros nascidos de éguas com placentite apresentaram hiperlactatemia com 24h de vida (P=0,002). A placentite aguda exerceu influência na maturidade neonatal, permitindo uma maturação fetal acelerada, porém, incompleta. Mensurações dos níveis sanguíneos de lactato, fibrinogênio, creatinina, colesterol total, AST, GGT, bilirrubinas e albumina, associado à avaliação clínica, física e comportamental, podem contribuir como indicadores de maturidade neonatal.(AU)

Dynamics of humoral immune response in pregnant mares and foals vaccinated with Theileria equi recombinant EMA-2 / Dinâmica da resposta imune humoral em éguas gestantes e potros vacinados com EMA-2 recombinante de Theileria equi

Theileria equi is an infectious hemoprotozoan agent of equine piroplasmosis, a disease that has severe economic and sanitary impact internationally. In addition to its common clinical features, piroplasmosis can cause gestational losses and neonatal damage, which makes neonates susceptible to this disease. The aim of this study was to evaluate the dynamics of humoral immune response to recombinant EMA-2 of T. equi in pregnant mares and foals, as well as the transfer of vaccine antibodies through the colostrum ingested by sucking foals. For vaccine production, the EMA-2 expression gene was cloned and expressed in the yeast species, Pichia pastoris. Thirty-six horses were used, of which 18 were pregnant mares and 18 were foals. The mares were divided into control and vaccinated groups, and the vaccinated group received three doses of rEMA-2 every 21 days starting at 300 days of gestation. Foals from vaccinated and control groups were evaluated until the sixth month of life. The production of antibodies by foals on the rEMA-2 vaccination schedule was also evaluated from the second month of life. Foals in the vaccinated group had received three doses of the vaccine every 21 days. The method used to evaluate serum and colostrum samples was indirect ELISA, and plates were sensitized with the rEMA-2 protein. At the end of the vaccination schedule, vaccinated mares showed a 2.3-fold increase in antibody levels when compared to baseline values. The colostrum of vaccinated mares presented antibody levels of 1.0432±0.33. Foals delivered by vaccinated mares presented levels of antibodies greater than those of foals delivered by control mares after their first time sucking (at about twelve hours after birth). Foals vaccinated in the second month of life showed an 8.3-fold increase in antibody levels when compared to baseline values. The vaccination schedule with rEMA-2 was able to stimulate humoral immunity in pregnant mares. Vaccine immunoglobins were concentrated in the colostrum of vaccinated mares and foals delivered by these mares showed an increase in serum levels of vaccine antibodies after the first-time sucking.(AU)

Theileria equi é um hemoprotozoário, agente da piroplasmose equina, doença de impacto sanitário e econômico internacional. Em éguas gestantes além da doença clínica, podem ocorrer abortos e danos ao neonato, caracterizando grande susceptibilidade à doença no período neonatal. O objetivo deste estudo foi avaliar a dinâmica da resposta imune humoral à EMA-2 recombinante de T. equi em éguas gestantes e potros, bem como a transferência de anticorpos vacinais no colostro. Foram utilizados 36 equinos, sendo 18 éguas gestantes e 18 potros. As éguas foram divididas em grupo controle e vacinado, que receberam rEMA-2 a partir dos 300 dias de gestação em três doses com intervalos de 21 dias. Para produção da vacina, o gene de expressão de EMA-2 foi clonado e a proteína expressa em Pichia pastoris. Os potros provenientes de éguas dos grupos vacinado e controle foram avaliados até o 6º mês de vida. Avaliou-se também a produção de anticorpos em potros submetidos ao esquema vacinal com rEMA-2 a partir do 2º mês de vida, que receberam três doses da vacina em intervalos de 21 dias. O método escolhido para a avaliação das amostras de soro e colostro foi ELISA indireto, com sensibilização pela proteína rEMA-2. Nas éguas gestantes vacinadas com rEMA-2 ocorreu o incremento de 2,3 vezes o valor basal ao final do esquema vacinal. O colostro de éguas vacinadas apresentou título médio de anticorpos de 1,0432±0,33, e potros provenientes de éguas vacinadas apresentaram média maior que os provenientes de éguas controle após a primeira mamada (12 horas). Os potros que passaram por esquema vacinal a partir do 2º mês de vida obtiveram incremento de 8,3 vezes o valor basal de anticorpos. O esquema vacinal com rEMA-2 foi capaz de estimular a imunidade humoral em éguas gestantes. Éguas gestantes vacinadas concentraram imunoglobulinas vacinais no colostro, e os potros provenientes destas obtiveram incremento nos níveis séricos de anticorpos vacinais após a primeira mamada.(AU)

Estradiol cypionate aided treatment for experimentally induced ascending placentitis in mares.

The overall goal of this study was to assess the efficacy of various therapeutic combinations of estradiol cypionate (ECP, a long-acting estrogen) and altrenogest (ALT, a long-acting progestin) in addition to basic treatment for placentitis with trimethoprim-sulfamethoxazole (TMS) and flunixin meglumine (FM). Specific outcomes measured in this experiment were (i) time from induction of bacterial placentitis to delivery, and foal parameters (high-risk, survival, and birth weight); and (ii) serum steroid concentrations (progesterone, 17α-hydroxyprogesterone, 17ß-estradiol, and cortisol) in response to treatment. Pregnant mares (∼300 days gestation, n = 46) were randomly assigned into healthy mares (control group, CONT, n = 8) and mares with experimentally induced ascending placentitis (n = 38). Placentitis was induced via intracervical inoculation of Streptococcus equi subspecies zooepidemicus. Thereafter, placentitis induced mares were randomly assigned into: (1) basic treatment, TMS+FM (n = 8); (2) basic treatment with ALT supplementation, TMS+FM+ALT (n = 8); (3) basic treatment with ECP supplementation, TMS+FM+ECP (n = 6); (4) basic treatment with ALT and ECP supplementation TMS+FM+ALT+ECP (n = 6); and (5) no treatment (INOC, n = 10). Treatments were started 48 h after bacterial inoculation and carried out for ten consecutive days. Blood samples were collected daily, and mares were assessed for signs of placentitis until the mare delivered, or for ten consecutive days after onset of treatment. Steroids were analyzed via RIA. Continuous data were analyzed by ANOVA, and categorical data analyzed by Fisher's exact test. Significance was set at p < 0.05. Foal survival at parturition and seven days post-delivery were similar across treated groups (66.7-100%), and to the CONT group. Similar to CONT group, mares in the TMS+FM+ECP group had no high-risk foals while mares in the other groups had higher incidences (50-75%) (p < 0.05). The inclusion of ECP in the treatments resulted in foals with body weight similar to CONT group (p > 0.05). There were no group effects or time by group interactions on concentrations of steroids assessed herein (p > 0.05). In conclusion, in addition to basic treatment TMS+FM, mares with experimentally induced ascending placentitis benefited from ECP supplementation. Conversely, ALT did not appear to make a difference in outcomes. The immunoassays used for measurements of steroid concentrations did not appear useful to assess treatment response.

Histological features of the placenta and their relation to the gross and data from Thoroughbred mares / Achados histológicos da placenta e sua relação com seus aspectos macroscópicas e dados de éguas Puro Sangue Inglês

The placenta is a transitory organ that originates from maternal and fetal tissues, the function of which is transporting nutrients from the mother to the fetus. The aim of this study was describe the histological features of placentas in healthy Thoroughbred mares at foaling and evaluate their relation with the gross placental and data of these mares. For this study 188 Thoroughbred mares were used. It was performed clinical observation for signs of placentitis during daily health checks and ultrasonic examination monthly to assess the fetus and placenta. All of the mares that exhibited clinical signs of placentitis were treated during gestation. The parturition was assisted, the placentas were grossly evaluated and samples were collected immediately after expulsion. The following data were considered for each mare: age, gestational age, number of parturition, time for placental expulsion, umbilical-cord length, placental weight and clinical signs of placentitis. Histological evaluation of the placentas revealed extensive cytoplasmic vacuolization of the epithelial areolar cells, presence of inflammatory infiltrates and hypoplasia-atrophy of the microcotyledons. Most of the gross placental findings were consistent with the histological results. In conclusion the mares with a vacuolated placental chorionic epithelium were older and had experienced a larger number of births. Great part of the mares with inflammatory infiltrates did not showed any clinical signs of placentitis during gestation.(AU)

A placenta é um órgão transitório originado do tecido fetal e materno, com função de transportar nutrientes da mãe para o feto. O objetivo deste estudo foi descrever os achados histológicos das placentas de éguas Puro Sangue Inglês (PSI) a termo e avaliar sua relação com a macroscopia da placenta e dados dessas éguas. O estudo utilizou 188 éguas PSI. Foram realizadas observações clinicas diárias para presença de sinais clínicos de placentite e ultrassonografia mensal para avaliar saúde do feto e placenta. As éguas que apresentaram sinais clínicos de placentite durante a gestação foram tratadas. Os partos foram assistidos, as placentas avaliadas macroscopicamente e coletadas imediatamente após sua expulsão. Como dados das éguas foram considerados: idade, tempo de gestação, número de partos, tempo de eliminação da placenta, comprimento de cordão umbilical, peso da placenta e sinais clínicos de placentite. A avaliação histológica das placentas demonstrou extensiva vacuolização citoplasmática das células do epitélio coriônico das regiões areolares, presença de infiltrados inflamatórios e hipoplasia-atrofia de microcotilédones. A maior parte dos achados macroscópicos da placenta foram condizentes com os resultados de histologia. Como conclusão, a vacuolização do epitélio coriônico foi característica de éguas mais velhas e com maior número de partos. Grande parte das éguas com infiltrados inflamatórios não demonstraram sinais clínicos de placentite.(AU)

Verificação da transmissão vertical de Neospora spp. em equinos / Verification of vertical transmission of Neospora spp. in horses.

O gênero protozoário Neospora é reconhecido como causador de desordens reprodutivas e abortos em bovinos. Entre os equinos pouco se sabe sobre os efeitos da infecção por estes protozoários. Atualmente é admitido que os efeitos da infecção por Neospora hughesi em equinos possam ocorrer no sistema nervoso central e, os efeitos provocados pela infecção por Neospora caninum recaiam sobre o sistema reprodutor de éguas. O presente trabalho verificou a presença de imunoglobulinas da classe G no soro sanguíneo de uma população de éguas de cria e, em seus respectivos potros antes da ingestão do colostro. Para execução deste trabalho foi empregada técnica de imunofluorescência indireta (RIFI), utilizando como antígeno taquizoítos de Neospora caninum, a diluição inicial dos soros das éguas foi de 1:50 e a diluição do soro dos potros empregada foi de 1:16. Foram assistidos 78 partos e todos os potros tiveram seu soro sanguíneo coletado imediatamente após o nascimento. A pesquisa de anticorpos contra Neospora spp. apontou que 50 (64%) éguas e 32 (41%) potros foram positivos. Das 50 éguas que apresentaram anticorpos contra Neospora spp. 24 geraram potros positivos. Entre as 28 éguas que não reagiram, oito deram a luz a potros positivos. De posse dos resultados encontrados podemos concluir que ocorreu a transmissão vertical de Neospora spp. nos equinos pesquisados...

The genre protozoan Neospora is recognized as causing reproductive disorders and miscarriages in cattle. Among the horses little is known about the effects of infection by these protozoa. It is currently accepted that the effects of infection by Neospora hughesi in horses may occur in the central nervous system, and effects of Neospora caninum infection occur in the reproductive system of mares. The present study examined the presence of class immunoglobulin G in blood serum of a population of brood mares and their foals before colostrum ingestion. For this assignment was employed indirect immunofluorescence assay (IFA) using as antigen tachyzoites of Neospora caninum, the initial dilution employed in sera of the mares was 1:50 and dilution in the serum of foals was 1:16. Were assisted 78 deliveries and all foals had their blood serum collected immediately after birth. The presence of antibodies against Neospora spp. found in mares was 50 (64%) and 32 (41%) foals were positive. Of the 50 mares that had antibodies to Neospora spp. 24 generated positive foals. Among the 28 mares unreacted eight gave birth to foals positive. Having the results we can conclude that vertical transmission occurred Neospora spp. researched in horses...

Comparação da composição bioquímica do líquido amniótico equino colhido em diferentes estágios gestacionais e no momento do parto / Comparison of the biochemical composition of equine amniotic fluid collected in different gestational phases and at delivery

A viabilidade e maturidade fetais podem ser estimadas através da avaliação dos fluídos fetais em muitas espécies, no entanto a composição bioquímica do líquido amniótico durante a gestação ainda não está bem definida para a espécie equina. O objetivo deste estudo foi estabelecer e comparar o perfil bioquímico do liquido amniótico em diferentes momentos da gestação e no momento do parto para um melhor entendimento da fisiologia da gestação de equinos. Foram avaliados valores encontrados para pH, osmolaridade, glicose, uréia, creatinina, gamma-GT, sódio, potássio, cloretos e proteína total no liquido amniótico colhido de 122 éguas comparando-se os resultados encontrados entre as colheitas em terço inicial (TI), médio (TM) e final (TF) da gestação e no momento do parto (MP). [...] Valores para gamma GT diferiram apenas entre os grupos TF e MP e mais estudos devem ser conduzidos sobre sua função no liquido amniótico das espécies domésticas. Os valores de sódio e cloretos não diferiram significativamente entre os grupos estudados e as concentrações de potássio diferiram apenas entre os grupos TF e MP refletindo a manutenção do equilíbrio eletrolítico do líquido amniótico equino durante a gestação. As concentrações de proteínas totais variaram de maneira heterogênea entre os grupos, porém todos estes demonstraram baixas concentrações. Concluímos que a composição bioquímica do liquido amniótico equino varia de acordo com o desenvolvimento fetal e pode ser utilizada como mensuração da viabilidade e maturidade do feto no futuro, no entanto devido ao pequeno número de estudos conduzidos e diferenças encontradas em seus resultados, outros estudos devem ser realizados para a melhor definição do perfil bioquímico do líquido amniótico equino durante as fases gestacionais e no momento do parto.

The viability and fetal maturity can be estimated by biochemical evaluation of the fetal fluids of several species; however the biochemical composition of amniotic fluid during pregnancy is not fully defined for equine. The aim of this study was to establish and compare the biochemical profile of amniotic fluid in different moments of pregnancy and at delivery, in order to better explain the peculiarities of the physiology of pregnancy in mares. The values founded for pH, osmolarity, glucose, urea, creatinine, gamma-GT, Sodium, potassium, chloride and total protein were evaluated in amniotic fluid collected from 122 mares comparing the results between the initial-third (IT), mid-third (MT) and latter-third (FT) of gestation and at delivery (D). [...] Concentrations of urea tended to be statistically different in at least one of the groups. A significant increase in creatinine concentrations was observed during the initial-third, medium-third and the final-third of pregnancy and the value found at delivery remained equal to final-third. Values for Gamma GT differed only between FT and D groups and more studies should be conducted about its role in the amniotic fluid of domestic species. For the sodium and chloride ions, they were not significantly different between the studied stages, and the potassium ion was significantly different only between FT and D reflecting the maintenance of electrolyte balance of the amniotic fluid during equine pregnancy. Total protein concentrations were different between groups, but all the groups showed low concentrations. We conclude that the values of the parameters studied varied according with the fetal development and can be used for the evaluation of fetal viability and maturity in the future, but the scarce number of studies in this area and the difference between the results found in literature demonstrate the need of more works to establish the biochemical profile of equine amniotic fluid during pregnancy and at delivery.

Isolamento, caracterização e diferenciação de células-tronco mesenquimais do líquido amniótico equino obtido em diferentes idades gestacionais / Isolation, characterization and differentiation of mesenchymal stem cells derived from equine amniotic fluid obtained from different gestacional ages

O interesse nas pesquisas com células-tronco derivadas de anexos fetais de diversas espécies cresceu exponencialmente nas últimas décadas em virtude de serem fontes de células-tronco adultas com potencial de diferenciação em diversas linhagens celulares que apresentam pouca ou nenhuma imunogenicidade, apresentando-se assim como alternativa de grande importância para a formação de bancos celulares. Apesar do crescente interesse, os estudos para espécie equina ainda são escassos. O objetivo deste trabalho foi isolar, caracterizar e diferenciar células-tronco mesenquimais (CTMs) derivadas do líquido amniótico equino obtidas do terço inicial, médio e final da gestação (LA-CTMs), comparando suas características. Foram colhidas 23 amostras de líquido amniótico as quais foram submetidas às análises morfológica, imunocitoquímica, imunofenotípica por citometria de fluxo e às diferenciações osteogênica, adipogênica e condrogênica in vitro. Todas as amostras demonstraram adesão ao plástico e morfologia fibroblastóide. No ensaio imunocitoquímico as células de todos os grupos foram imunomarcadas para CD44, PCNA e vimentina com ausência de marcação para citoqueratina e Oct-4. Na citometria de fluxo observou-se a expressão de CD44 e CD90 e ausência de expressão de CD34, sendo que os marcadores CD44 e CD90 mostraram padrão de expressão decrescente em relação ao desenvolvimento gestacional. As amostras obtidas de todas as fases da gestação foram capazes de diferenciação nas linhagens osteogênica, condrogênica e adipogênica. Portanto, as células obtidas do líquido amniótico apresentaram características morfológicas, imunofenotípicas e potencial de diferenciação típicos das CTMs, demonstrando que a colheita pode ser realizada em qualquer fase gestacional. No entanto, mais pesquisas devem ser realizadas principalmente quanto à expressão de marcadores de pluripotencialidade (como o Oct-4) e ao seu potencial de diferenciação em linhagens extra mesodermais já relatados na literatura.

The interest in stem cells derived from fetal annexes of many species has exponentially increased during the last decades, because they are adult stem cell sources with potential of differentiation in several cell lineages; which present little or no immunogenicity and are an alternative with great importance for storage cell banks. Despite the rising interest, studies for the equine species are still rare. The aim of this study was to isolate, characterize and differentiate mesenchymal stem cells derived from equine amniotic fluid obtained from initial, middle and late third of gestation (AF-MSCs), and compare their results. Twenty three samples from equine amniotic fluid were evaluated by morphological, immunocytochemical and immunophenotypical (Flow cytometer) assays and osteogenic, adipogenic and chondrogenic in vitro differentiation. All samples demonstrated plastic adhesion and fibroblastoid morphology. The immunocytochemical assay demonstrated cells from all the studied groups were positive for CD44, PCNA and vimentin and negative for cytokeratin and Oct-4. Flow cytometry demonstrated expression of CD44 and CD90 and no expression of CD34, where CD44 and CD90 markers presented decreasing pattern of expression in relation to the gestational development. All samples collected from all gestational phases were capable to differentiate in osteogenic, chondrogenic and adipogenic lineages. Thus, cells obtained from equine amniotic fluid presented morphological and immunophenotypical characteristics and potential of differentiation typical of MSCs showing that the collection can be performed at any stage of pregnancy. However, more studies should be performed about the expression of pluripotent markers as Oct-4 and the differentiation potential for extra mesodermal lineages prior demonstrated in the literature.

Campylobacter jejuni occurrence in chicken fecal samples from small properties in Pelotas, southern of Brazil

The aim of this study was to evaluate the occurrence of thermophilic Campylobacter species in broiler flocks from 26 small properties located near the campus of Federal University of Pelotas, RS, Brazil. A total of 404 chicken fecal samples were analyzed and after isolation, identification and biotyping was performed according to Lior´s scheme. Twenty one strains (5.2 percent) of Campylobacter jejuni biotype II were isolated from animals from seven properties (26.9 percent), with a variation from 3 percent to 34.6 percent among the positive properties. We also analyzed twelve C. jejuni isolates by polymerase chain reaction (PCR), using specific primers that generate a fragment of 402 pb. Our study allowed us to observe that chickens raised in non-industrial establishments without sanitary attention can harbour C. jejuni in their intestinal tract, and by doing so, they could be a serious health risk to humans.

O objetivo deste estudo foi avaliar a ocorrência de espécies termofílicas de Campylobacter em galinhas de 26 pequenas propriedades localizadas próximo ao campus da Universidade Federal de Pelotas, RS, Brasil. Um total de 404 amostras fecais de galinhas foram analisadas e após o isolamento, a identificação e a biotipificação foram realizadas de acordo com o esquema de Lior. Vinte e uma amostras (5,2 por cento) de Campylobacter jejuni biotipo II foram isoladas de animais de sete propriedades (26,9 por cento), com uma variação de 3,3 por cento a 34,6 por cento entre as propriedades positivas. Primers específicos, que geram um fragmento de 402pb, foram usados em PCR para analisar 12 isolados de C. jejuni. Concluímos que galinhas criadas em estabelecimentos não industriais e sem atenção sanitária, são portadoras de C. jejuni e, desta forma, podem ser consideradas um fator de risco para infecção humana.