RESUMO

Uterine inflammation negatively affects reproductive performance and is an important cause of infertility and subfertility in dairy cows. Several studies have investigated the use of gene expression in endometrial samples collected by biopsy or cytology to evaluate the inflammatory response of the cow uterus. This study aimed to compare the expression of the CCL5, CXCL8, IL6, and IL1B genes in the bovine endometrium according to the site of sample collection [caruncular (C) or intercaruncular (IC)], the collection method (biopsy or cytology), and the category of inflammation based on endometrial cytology (zero, medium, or high) in subclinical endometritis. The reproductive tracts of dairy cows were collected from a slaughterhouse, and punch-biopsy samples of endometrial tissues were obtained from both regions (C and IC). Endometrial cells from these regions were collected with the cytobrush technique and then used for the analysis of mRNA expression by quantitative PCR. After counting polymorphonuclear cells (PMN) by endometrial cytology, 20 uteri with an ovary at stage I (d 1-4 of estrous cycle) were categorized into 3 groups. Uteri with 0% PMN (n = 10) were assigned to group zero, uteri with 5 to 15% PMN (n = 5) to group medium (12.2 ± 1.6% PMN), and uteri with >15% PMN (n = 5) to group high (53.8 ± 32.9% PMN). All data were analyzed with 2-way ANOVA with Bonferroni multiple comparison post test. The results from gene transcripts demonstrated that the region (C or IC) of the endometrial biopsy had no influence on any of the degrees of inflammatory reaction observed. However, gene expression was more elevated in the endometrium of cows with greater inflammation compared with those without inflammation (CCL5, CXCL8, IL6, IL1B) and those with medium inflammation (CCL5, IL6). Expression of the genes evaluated did not differ between the endometrium without inflammation and with medium inflammation. However, in the high inflammation group, all genes were comparatively more expressed in samples collected by cytology relative to those derived from biopsies for both anatomical regions. In conclusion, gene expression did not differ between the C and IC tissue. Samples collected from animals with greater inflammation had greater gene expression than those with zero or medium inflammation. In addition, cytology samples had greater gene expression than biopsy samples in the high inflammation group.

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This study evaluated the prevalence of cervicitis and endometritis and their interrelations in crossbred dairy cows that were more than 60 days in milk (DIM), and the efficiency of the cytology technique (cytobrush) and histology as diagnostic methods for these diseases. The reproductive tracts (n=149) derived from cows were collected from a slaughterhouse and grossly evaluated to determine uterine involution and the phase of the estrous cycle. Subsequently, cervical and uterine cytological evaluations were done to characterize the inflammatory response as cervicitis (with a neutrophilic count greater than 5%) or endometritis (with more than 6% neutrophils). Additionally, randomly obtained samples from the cervix and uterus were collected for histological evaluation. Cytological evaluation revealed that the frequency of cervicitis was 6% (9/149), and endometritis was diagnosed in 8.1% (12/149) of the samples; both inflammatory reactions were diagnosed 2.1% (3/149) in three of these. Histological evaluation revealed that of the cows diagnosed with cervicitis by cytology, 66.6% (6/9) had no inflammatory reaction in the cervical mucosa, and 33.3% (3/9) had mild inflammation in this region. Histological evaluation of the uterine horn revealed that of the 12 cows diagnosed with endometritis by cytology, 50% (6/12) of these had a histological diagnosis of mild and moderate inflammatory responses, 8.3% (1/12) of these demonstrated a severe inflammatory response, while no inflammatory reaction was identified in 41.7% (5/12) of the endometrial biopsies evaluated. In conclusion, the endometrial and cervical inflammatory response diagnosed by the cytobrush technique persisted in 12.1% of the cows. Comparatively, the frequency of the inflammatory responses at the cervix and uterus was reduced by histological analysis relative to cytological evaluation with the cytobrush technique.

O objetivo deste estudo foi determinar a prevalência de cervicite e endometrite citológicas e suas inter-relações em vacas leiteiras mestiças com mais de 60 dias pós-parto (DPP), bem como avaliar as técnicas de citologia (cytobrush) e histologia como métodos diagnósticos para estas doenças. Foram utilizados 149 tratos reprodutivos de vacas com mais de 60 DPP de abatedouro e avaliados macroscopicamente para determinar a involução uterina e a fase do ciclo estral. Posteriormente, realizou-se citologia cervical e uterina para o diagnóstico de inflamação utilizando como ponto de corte, contagem de polimorfonucleares (PMN) superior a 5% e 6%, respectivamente, para cervicite e endometrite citológica. Também foram coletadas amostras de cérvix e útero para histologia. Do total de vacas, identificaram-se 6% (9/149) com cervicite e 8,1% (12/149) com endometrite citológica, sendo três destes animais (2,1%) com ambas as inflamações. Histologicamente, dos animais com cervicite citológica, 66,6% (6/9) não apresentaram infiltrado inflamatório na mucosa cervical e 33,3% (3/9) apresentaram inflamação leve. A histologia uterina mostrou que de 12 vacas com endometrite citológica, 50% (6/12) apresentaram infiltrados inflamatórios leves a moderados, 8,3% (1/12) grave e 41,7% (5/12) não tinham Inflamação endometrial. Concluiu-se que a inflamação endometrial e cervical, por meio da técnica de citologia, persistiu em 12,1% das vacas com mais de 60 DPP. Quando se utiliza a histologia como método de diagnóstico, a detecção de inflamação cervical e uterina é inferior ao detectado pela técnica de citologia por meio do esfregaço endometrial (cytobrush).

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Productivity rates directly depend on the fertility of a herd, which in turn can be influenced by many factors. Semen deposited in the female reproductive tract is foreign to the body and, in response to this invasion, produces an inflammatory reaction, which is characterized by rapid infusion of polymorphonuclear (PMN) cells. Techniques to obtain an endometrial sample are usually invasive and can mask the true inflammatory response. Ultrasound is a noninvasive technique and can contribute to the diagnosis of postartificial insemination (AI) inflammatory response in cattle. The present study was divided into two experiments. The aim of experiment 1 was to compare two methods of endometrial cytology collection, uterine cytobrush (UC) and uterine lavage (UL), and their effects on uterine hemodynamics that provide information about blood flow. The two methods were evaluated by Doppler ultrasound using the spectral and color modes. For that purpose, 19 Nellore cows were synchronized for timed AI and subjected to UC (n = 9) or UL (n = 10). The techniques were performed 4 hours after AI. The results showed that both techniques allow collection of a good quality sample and with enough PMN cells to perform counting. More PMN cells were obtained by UL than UC. There was no difference in uterine blood flow between the UC and UL groups in any of the periods evaluated (34 hours before and 4, 24, and 48 hours after collection of uterine sample). On the basis of results of experiment 1, the effect of UL on fertility was studied in experiment 2. A total of 128 Nellore cows were synchronized for TAI; 35 cows were subjected to endometrial cytology by UL 4 hours after AI, and 93 were not submitted to any procedure (control). Pregnancy diagnosis was performed by transrectal ultrasound 30 days after AI. Pregnancy rates did not differ between UL (54.29%) and control (56.99%) groups. The results of this study showed that UL allows the collection of more representative cells of the surface of the uterus than UC technique and causes no damage to the reproductive tract. Moreover, UL did not affect pregnancy rate when performed 4 hours after AI.

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Com objetivo de estabelecer valores de referência para a citologia endometrial e avaliar histologicamente o endométrio de gatas clinicamente saudáveis e sem patologias uterinas, foram coletados 22 úteros de gatas que passaram por ovariohisterectomia eletiva. Após avaliação macroscópica dos úteros, coletou-se mostra para citologia endometrial pela técnica de escova endometrial (cytobrush) e fragmento para avaliação histológica. Quanto à apresentação das glândulas endometriais, 31,8% (7/22) das gatas apresentaram lâmina própria com poucas camadas de glândulas tubulares simples, revestidas por epitélio simples cúbico ou cilíndrico baixo, com diâmetro reduzido e sem secreção no seu lúmen. Proliferação glandular com algumas glândulas apresentando conteúdo no lúmen foi observada em 36,4% (8/22) dos animais. Presença de intensa proliferação glandular com glândulas revestidas por epitélio simples pavimentoso, a maioria com atividade secretora, foi observada em 31,8% (7/22) das gatas, sendo que em cinco destas verificou-se aparência cística com pronunciada dilatação glandular. A citologia endometrial, na leitura de 200 células, obteve os seguintes resultados: neutrófilos (10,77 ± 7,51), eosinófilos (1,93 ± 2,67), basófilos (0), linfócitos (6,58 ± 8,49), macrófagos (0,79 ± 2,05) e células de descamação (79,93 ± 11,44). Concluiu-se que em gatas clinicamente saudáveis há maior proporção de leucócitos na citologia endometrial quando comparado com outras espécies, mesmo quando não se evidencia inflamação na avaliação histológica. A colheita de material para citologia endometrial pela técnica de cytobrush apresenta celularidade satisfatória e sem presença de debris celulares em gatas.

This study evaluated the endometrial cytology and histology of clinically healthy cats without any uterine alteration in an attempt to establish reference parameters in dogs. Uteri were collected from 22 cats submitted to elective ovariohysterectomy. All uteri were macroscopically evaluated, after which an endometrial sample was obtained for cytological analysis using the endometrial brush (cytobrush) technique and a tissue fragment routinely processed for histological evaluation. Histological evaluation revealed that the endometrial glands of some 31.8% (7/22) cats consisted of a lamina propria with few layers of simple tubular glands, lined by simple cubical epithelium, with reduced diameter and without luminal secretion. Glandular proliferation with luminal content was observed in 36.4% (8/22) of the animals evaluated. The presence of intense proliferation with glandular glands lined by squamous simple epithelium, most with secretory activity was observed in 31.8% (7/22) of cats; in five of these, there was pronounced cystic glandular dilatation. The endometrial cytology of 200 cells revealed the following results: neutrophils (10.77 ± 7.51), eosinophils (1.93 ± 2.67), basophils (0), lymphocytes (6.58 ± 8.49), monocytes (0.79 ± 2.05), and cell shedding (79.93 ± 11.44). It was concluded that clinically healthy cats have a greater percentage of leukocytes in endometrial cytology when compared with other species, even when there is no evidence of inflammation by histological evaluation. The collection of material for endometrial cytology using the cytobrush technique provides adequate cellularity and without the presence of cellular debris in cats.

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A citologia conjuntival é um importante meio de diagnóstico de afecções da superfície ocular. Buscam-se técnicas que forneçam quantidade e qualidade celular, com uso de instrumentos que provoquem mínimo trauma e que diminuam as chances de danos iatrogênicos ao olho. Existem diversas técnicas de coleta de células, entre elas encontram-se: impressão, esfoliação e punção por agulha fina. Dentre os métodos utilizados para esfoliação, o uso da escova citológica fornece resultados superiores em vários parâmetros, incluindo a qualidade das células. Estudou-se a citologia conjuntival por esfoliação com escova citológica, utilizada para coleta de material da cérvix uterina, tendo como objetivos determinar se tal instrumento se adequaria à coleta de material da face palpebral da membrana nictitante e da conjuntiva palpebral de felinos sadios. Foram avaliados os seguintes parâmetros: facilidade de execução da técnica, possibilidade de ocorrência de danos iatrogênicos e quantidade e qualidade de células coletadas. Cinquenta gatos machos (58%) e fêmeas (42%), com ou sem raça definida, participaram do estudo. Apenas gatos isentos de alterações oculares no exame físico foram incluídos. A escova citológica se mostrou um instrumento de fácil utilização, que fornece células em quantidade satisfatória e com morfologia preservada. Comparada a conjuntiva palpebral, a face palpebral da membrana nictitante se mostrou um local mais adequado à realização da coleta de amostras citológicas, pela maior facilidade de execução da técnica e menor possibilidade de danos iatrogênicos. No que diz respeito à quantidade e qualidade celular, não houve diferença significativa entre os dois locais de coleta. Foi possível observar células provenientes das diferentes camadas do epitélio conjuntival com predomínio de células intermediárias e ausência de células caliciformes.(AU)

Conjunctival cytology is an important diagnostic method for ocular superficial diseases. There are several techniques to collect cells, including impression, exfoliation and fine needle aspiration. These techniques have shown individual variations depending on method and instrument used on the collection process. Techniques with instruments that cause low damage and decrease iatrogenic damage risks to the eyes, providing cellular quantity and quality, are wanted. Exfoliative cytology techniques are frequently used in human and veterinary medicine. Among the methods used for exfoliation, cytobrush provides better results on many parameters, including cell quality. The use of conjunctival exfoliation cytology using cytobrush (brush usually used for cervix cytology) was investigated. The aim was to determine if this technique would provide an adequate number and quality of conjunctival third eyelid cells of cats; to compare these samples with those collected by the same way from conjunctival area and to determine a cytologic pattern of this region. This comparison would provide information about technique security, possible damage risks and collected cells characteristics. Cats were evaluated through physical and routine ophthalmic exam. Fifty domestic cats, males (58%) and females (42%), of any breed were accepted. In the study were included only animals free of ocular and systemic changes. Cytobrush showed an easy and usefulness instrument, that provides satisfactory amount of cells with preserved morphology. Compared to palpebral conjunctiva, palpebral surface of nictitating membrane showed a most adequate site to cytological sample collect, greater facility of technical perform and smaller possibility of iatrogenic damage. In respect to quality and quantity of cells there was no significant difference between collect sites. It was possible to observe epithelial cells of conjunctiva from different stratum with predominance of intermediate cells and absence of goblet cells.(AU)

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RESUMO

A citologia conjuntival é um importante meio de diagnóstico de afecções da superfície ocular. Buscam-se técnicas que forneçam quantidade e qualidade celular, com uso de instrumentos que provoquem mínimo trauma e que diminuam as chances de danos iatrogênicos ao olho. Existem diversas técnicas de coleta de células, entre elas encontram-se: impressão, esfoliação e punção por agulha fina. Dentre os métodos utilizados para esfoliação, o uso da escova citológica fornece resultados superiores em vários parâmetros, incluindo a qualidade das células. Estudou-se a citologia conjuntival por esfoliação com escova citológica, utilizada para coleta de material da cérvix uterina, tendo como objetivos determinar se tal instrumento se adequaria à coleta de material da face palpebral da membrana nictitante e da conjuntiva palpebral de felinos sadios. Foram avaliados os seguintes parâmetros: facilidade de execução da técnica, possibilidade de ocorrência de danos iatrogênicos e quantidade e qualidade de células coletadas. Cinquenta gatos machos (58%) e fêmeas (42%), com ou sem raça definida, participaram do estudo. Apenas gatos isentos de alterações oculares no exame físico foram incluídos. A escova citológica se mostrou um instrumento de fácil utilização, que fornece células em quantidade satisfatória e com morfologia preservada. Comparada a conjuntiva palpebral, a face palpebral da membrana nictitante se mostrou um local mais adequado à realização da coleta de amostras citológicas, pela maior facilidade de execução da técnica e menor possibilidade de danos iatrogênicos. No que diz respeito à quantidade e qualidade celular, não houve diferença significativa entre os dois locais de coleta. Foi possível observar células provenientes das diferentes camadas do epitélio conjuntival com predomínio de células intermediárias e ausência de células caliciformes.

Conjunctival cytology is an important diagnostic method for ocular superficial diseases. There are several techniques to collect cells, including impression, exfoliation and fine needle aspiration. These techniques have shown individual variations depending on method and instrument used on the collection process. Techniques with instruments that cause low damage and decrease iatrogenic damage risks to the eyes, providing cellular quantity and quality, are wanted. Exfoliative cytology techniques are frequently used in human and veterinary medicine. Among the methods used for exfoliation, cytobrush provides better results on many parameters, including cell quality. The use of conjunctival exfoliation cytology using cytobrush (brush usually used for cervix cytology) was investigated. The aim was to determine if this technique would provide an adequate number and quality of conjunctival third eyelid cells of cats; to compare these samples with those collected by the same way from conjunctival area and to determine a cytologic pattern of this region. This comparison would provide information about technique security, possible damage risks and collected cells characteristics. Cats were evaluated through physical and routine ophthalmic exam. Fifty domestic cats, males (58%) and females (42%), of any breed were accepted. In the study were included only animals free of ocular and systemic changes. Cytobrush showed an easy and usefulness instrument, that provides satisfactory amount of cells with preserved morphology. Compared to palpebral conjunctiva, palpebral surface of nictitating membrane showed a most adequate site to cytological sample collect, greater facility of technical perform and smaller possibility of iatrogenic damage. In respect to quality and quantity of cells there was no significant difference between collect sites. It was possible to observe epithelial cells of conjunctiva from different stratum with predominance of intermediate cells and absence of goblet cells.

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This study compared quantitatively and qualitatively the DNA extracted from buccal cells collected from the upper or lower gutter areas. Buccal cells were collected from the upper (n=15) and lower gutter (n=15) region from 15 volunteers using a special cytobrush (Gentra), totaling 2 collections from each individual. DNA was extracted from the samples according to the manufacturer's instructions. The DNA obtained was qualitatively and quantitatively evaluated by 2 calibrated blind examiners using spectrophotometry and analysis of DNA bands (0.8 percent agarose gel electrophoresis). Data was statistically analyzed by one-way ANOVA (?=0.05). Means and standard derivation (SD) for total DNA yield from the upper and lower gutter area were 12.2 ?g (12.0) and 9.4 ?g (8.5), respectively (p=0.821). There was higher (p<0.05) DNA purity for the upper gutter (1.79; 0.05) when compared to lower gutter area (1.66; 0.10). Regarding to the DNA quality, no differences were observed between the 2 location sites, but all samples showed similar degree of degradation. In conclusion, it would be recommendable that buccal cells for DNA extraction be collected from the upper gutter area in the attempt to increase DNA purity.

O objetivo do presente estudo foi comparar quantitativamente e qualitativamente o DNA extraído de células epiteliais bucais coletadas do fundo de sulco superior e inferior. Foram coletadas células bucais do fundo de sulco superior (n=15) e inferior (n=15) de 15 voluntários utilizando escovas citológicas especiais (Gentra), totalizando 2 coletas por voluntário. Após a coleta o DNA foi extraído conforme o protocolo indicado pelo fabricante (Puregene DNA Buccal Cell Kit; Gentra Systems, Inc.). O DNA obtido foi avaliado quantitativamente e qualitativamente por dois examinadores calibrados cegos utilizando espectrofotometria e análise das bandas de DNA (gel de agarose 0,8 por cento, por eletroforese). Os dados foram submetidos a ANOVA a um critério, com p<0,05. As médias e desvio padrão (DP) para o rendimento total de DNA do fundo de sulco superior e inferior foram respectivamente 12,2 ?g (12,0) e 9,4 ?g (8,5) (p=0,821). Houve maior (p<0,05) pureza de DNA no fundo de sulco superior (1,79; 0,05) quando comparado com o fundo de sulco inferior (1,66; 0,10). Quanto à qualidade do DNA, não foi observado diferenças entre os dois locais testados, no entanto todas as amostras mostraram níveis de degradação semelhantes. Em conclusão seria recomendável coletar células bucais, para extração de DNA, do fundo de sulco superior na tentativa de aumentar a pureza do DNA.

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