The embryo non-invasive pre-implantation diagnosis era: how far are we?

Advancements in assisted reproduction (AR) methodologies have allowed significant improvements in live birth rates of women who otherwise would not be able to conceive. One of the tools that allowed this improvement is the possibility of embryo selection based on genetic status, performed via preimplantation genetic testing (PGT). Even though the widespread use of PGT from TE biopsy helped to decrease the interval from the beginning of the AR intervention to pregnancy, especially in older patients, in AR, there are still many concerns about the application of this invasive methodology in all cycles. Therefore, recently, researchers started to study the use of cell free DNA (cfDNA) released by the blastocyst in its culture medium to perform PGT, in a method called non-invasive PGT (niPGT). The development of a niPGT would bring the diagnostics power of conventional PGT, but with the advantage of being potentially less harmful to the embryo. Its implementation in clinical practice, however, is under heavy discussion since there are many unknowns about the technique, such as the origin of the cfDNA or if this genetic material is a true representative of the actual ploidy status of the embryo. Available data indicates that there is high correspondence between results observed in TE biopsies and the ones observed from cfDNA, but these results are still contradictory and highly debatable. In the present review, the advantages and disadvantages of niPGT are presented and discussed in relation to tradition TE biopsy-based PGT. Furthermore, there are also presented some other possible non-invasive tools that could be applied in the selection of the best embryo, such as quantification of other molecules as quality biomarkers, or the use artificial intelligence (AI) to identify the best embryos based on morphological and/or morphokitetic parameters.(AU)

Cooling of porcine semen in an extender supplemented with isoespintanol / Resfriamento de sêmen suíno em um diluente suplementado com isoespintanol

ABSTRACT: Spermatozoa experience oxidative, osmotic, chemical, and thermal stresses when cooled, which degrade the quality and fertilizing capacity of the cells. Adding antioxidants to the sperm extender mitigates these alterations. This study evaluated the effect of isoespintanol (ISO) on boar semen subjected to cooling. Fifteen ejaculates from five boars (Susscrofadomestica) were extended in Beltsville thawing solution (BTS) supplemented with 0 µM (control), 5 µM (ISO5), 10 µM (ISO10), 15 µM (ISO15), 20 µM (ISO20), 25 µM (ISO25), and 30 µM (ISO30) of ISO, which were then cooled for five days at 16 °C. Sperm kinetics, total motility (TM), and progressive motility (PM) were evaluated every 24 h using an IVOS computer-assisted sperm analysis (CASA) system. On day 1 and day 5 of cooling, a hypoosmotic test, spectrofluorometry, and flow cytometry were performed to evaluate the following: membrane functionality, measured as a function of hypoosmotic swelling (HOS); total antioxidant capacity (TAC); reactive oxygen species (ROS); and mitochondrial membrane potential (Δ¥M). Regression analysis and comparison of means using the Duncan test were performed. The ISO added had a slight impact on sperm motility, as evidenced by a reduction in TM at 24 h of cooling (but not prior) with the addition of 20 µM of ISO. Similarly, no effect of the ISO on the kinetics and functional integrity of the sperm membrane was observed at 96 h of cooling; however, the regression coefficients indicated that the ISO lowered the rate of decrease in sperm motility and the proportion of rapid spermatozoa relative to the concentration of ISO used. The ISO did not affect the TAC of the cooled semen; however, different concentrations of ISO lowered ROS production in the semen after 96 h of cooling. ISO also impacted the Δ¥M of the spermatozoa at 0 h of cooling, increasing the proportion of low Δ¥M cells and decreasing the proportion of high Δ¥M cells. In conclusion, ISO can reduce the loss of quality and oxidative stress occurring in boar semen during cooling and can modulate the mitochondrial activity of sperm.

RESUMO: Durante a refrigeração, os espermatozoides sofrem estresse oxidativo, osmótico, químico e térmico, que diminuem sua qualidade e afetam sua capacidade de fertilização. A adição de antioxidantes ao diluente espermático é uma alternativa para mitigar essas alterações. O objetivo desta pesquisa foi avaliar o efeito do isospintanol (ISO) na refrigeração do sêmen suíno. Quinze ejaculados de cinco varrascos (Sus scrofa domestica) foram diluídos em BTS suplementado com ISO a 0 (controle), 5 (ISO5), 10 (ISO10), 15 (ISO15), 20 (ISO20), 25 (ISO25) e 30 (ISO30) µM e foram refrigerados por cinco dias a 16 °C. A motilidade total (MT), motilidade progressiva (MP) e cinética dos espermatozóides foram avaliadas a cada 24 h com um sistema CASA IVOS. Nos dias um e cinco de refrigeração, foram avaliadas a funcionalidade da membrana, a capacidade antioxidante total (CAT), as espécies reativas de oxigênio (ROS) e o potencial de membrana mitocondrial (Δ¥M), através do teste hiposmótico (HOS), espectrofluorimetría e citometria de fluxo. Foram realizadas análises de regressão e comparação de médias, pelo teste de Duncan. A adição de ISO teve pouca influência na motilidade espermática, apresentando apenas redução na MT em 24 h de refrigeração, devido à adição de 20 µM. Da mesma forma, não foi observada influência de ISO na cinética e integridade funcional da membrana em 96 horas de refrigeração; porém, os coeficientes de regressão mostraram que ISO produziu menor taxa de diminuição da motilidade e proporção de espermatozoides rápidos dependendo da concentração utilizada. ISO não influenciou significativamente na CAT do sêmen refrigerado; entretanto, diferentes concentrações de ISO reduziram a produção de EROs a partir do sêmen após de 96 h de refrigeração. ISO também influenciou o Δ¥M dos espermatozóides em 0 h de refrigeração, com aumento das células de baixo Δ¥M e diminuição das células de alto Δ¥M. Em conclusão, o isospintanol pode reduzir a perda da qualidade e o estresse oxidativo do sêmen suíno durante a refrigeração e pode modular a atividade mitocondrial do esperma.

Morphometric descriptive report of scleral ossicle rings, by ultrasound and computed tomography, in three Testudines specimens / Relato descritivo morfobiométrico dos anéis de ossículos esclerais, por ultrassonografia e tomografia computadorizada, em três espécimes de Testudines

The scleral ossicle rings function has been related to mechanical protection, muscle fixation, support for eyeball shape and visual accommodation. There are few morphobiometric reports on these rings in different Testudines species, and we performed ultrasound (US) and computed tomography (CT) of the scleral ossicle rings in one green turtle (Chelonia mydas), one black-bellied slider (Trachemys dorbigni) and one red-footed tortoise (Chelonoidis carbonarius). The US and CT of the ossicle rings were performed for anatomical identification. The thickness, density, width, and diameters of each ring were measured. The US and CT of the scleral ossicle rings of three animals showed single and continuous circular structures, located in the anterior pole. These structures were easily observed in C. mydas, whose rings were the biggest, thickest and widest. The T. dorbigni CT presented decreased dimensions and the ossicles were the most difficult to identify. Bone density in the superior region was greater than in the inferior of each ring in all animals. Non-invasive imaging exams are good tools to study the anatomy of the ocular skeleton. The scleral ossicle rings of the three specimens presented general morphological similarities and CT enabled visualizing a greater number of details of the ring bone morphology.

Os anéis de ossículos esclerais têm sua função relacionada à proteção mecânica, fixação muscular, suporte para o formato do bulbo ocular e acomodação visual, contudo existem poucos relatos morfobiométricos sobre esses anéis em diferentes espécies de Testudines. Desta forma, foi realizada a avaliação morfobiométrica, por ultrassom (US) e tomografia computadorizada (TC), dos anéis de ossículos esclerais em uma tartaruga verde (Chelonia mydas), um tigre-d'água (Trachemys dorbigni) e um jabuti-piranga (Chelonoidis carbonarius). Foram realizadas US e TC dos anéis esclerais dos três animais para identificação anatômica, espessura, densidade, largura e diâmetros. A US e a TC dos três animais mostraram estruturas circulares únicas e contínuas, localizadas no polo anterior. Estas estruturas foram facilmente observadas na C. mydas, cujos anéis eram os maiores, mais espessos e mais largos. A TC da T. dorbigni apresentou dimensões reduzidas e os ossículos foram dificilmente identificados. A densidade óssea na região superior foi maior comparativamente a parte inferior de cada anel, em todos os animais. Exames de imagem não invasivos mostraram-se bons instrumentos para estudo do esqueleto escleral. Os anéis de ossículos esclerais dos três espécimes apresentaram semelhanças morfológicas gerais e a TC permitiu visualizar um maior número de detalhes da morfologia óssea do anel.

A new species of barred Sternopygus (Gymnotiformes: Sternopygidae) from the Orinoco River

A new species of Sternopygus is described from the Orinoco River of Venezuela using traditional methods of morphometrics and meristics, and micro-computed tomography (micro-CT) imaging for osteological analysis. The new species is readily separated from all congeners in having broad, vertical pigment bars that extend from the mid-dorsum to the ventral margin of the pterygiophores. A similar color pattern, characterized by subtle differences in the densities and sizes of chromatophores, is also present in juveniles of S. obtusirostris from the Amazon River, juveniles of S. sabaji from rivers of the Guiana Shield, and S. astrabes from clearwater and blackwater terra firme streams of lowlands around the Guiana Shield. The new species further differs from other congeners in the Orinoco basin by having a reduced humeral pigment blotch with poorly defined margins, a proportionally smaller head, a longer body cavity, a more slender body shape in lateral profile, and in having vertical pigment bars that extend ventrally to the pterygiophores (vs. pigment saddles not reaching the pterygiophores). The description of this species raises to three the number of Sternopygus species in the Orinoco basin, and to 11 the total number of Sternopygus species.(AU)

Se describe una nueva especie de Sternopygus del río Orinoco de Venezuela utilizando métodos tradicionales de morfometría y merística, y microtomografía computarizada (micro-CT) para análisis osteológico. La nueva especie se distingue fácilmente de todos los congéneres por tener barras de pigmento verticales anchas que se extienden desde la parte media del dorso hasta el margen ventral de los pterigióforos. Un patrón de color similar, caracterizado por diferencias sutiles en las densidades y tamaños de los cromatóforos, también está presente en juveniles de S. obtusirostris del río Amazonas, juveniles de S. sabaji de ríos del Escudo Guayanés y S. astrabes de aguas claras y arroyos de tierra firme de aguas negras de las tierras bajas alrededor del Escudo Guayanés. La nueva especie se diferencia aún más de otros congéneres en la cuenca del Orinoco por tener una mancha de pigmento humeral reducida con márgenes mal definidos, una cabeza proporcionalmente más pequeña, una cavidad corporal más larga, una forma corporal más delgada en el perfil lateral y por tener barras de pigmento verticales que extenderse ventralmente a los pterigióforos (frente a las monturas de pigmentos que no llegan a los pterigióforos). La descripción de esta especie eleva a tres el número de especies de Sternopygus en la cuenca del Orinoco y a 11 el número total de especies de Sternopygus.(AU)

Relation of fibrocartilaginous embolism and acute and non-compressive nucleus pulposus extrusion with imaging tests: case report / Relação de embolia fibrocartilaginosa e extrusão aguda e não-compressiva do núcleo pulposus com testes de imagem: relato de caso

Fibrocartilaginous embolism (FCEM) and acute, non-compressive nucleus pulposus extrusion (ANNPE) are non-compressive myelopathies that are difficult to differentiate. The definitive diagnosis is obtained only with histology, but the presumptive diagnosis is made through clinical signs and imaging tests. The aim of this study is to report the imaging tests performed for the diagnosis of a neurological clinical case and discuss the best diagnostic method. After attending the patient, complementary tests were requested. Radiography results showed no change. The computed tomography diagnostic impression indicated distal protrusion between C6-C7, T11-T12, T13-L1 followed by mild spinal cord compression defined by the presence of a ventral hyperattenuating region. Magnetic resonance (RMI), showed a slight T2W hypersignal, well delimited in the gray matter, lateralized to the right, over the cranial third of C7. Concluding that the magnetic resonance is the method that brought more information for the diagnosis, in which the others were not described medullary alterations pertinent to FCEM and ANNPE. With their fair prognosis, the absence of histological diagnosis of these diseases may be a limiting factor in this study and, in relation to the RMI alterations being very similar between FCEM and ANNPE it is not possible to diagnose fully accurately.

A embolia fibrocartilaginosa (EFC) e a extrusão aguda não compressiva do núcleo pulposo (EANCNP) são mielopatias não compressivas de difícil diferenciação. O diagnóstico definitivo é obtido apenas com a histologia, mas o diagnóstico presuntivo é feito por meio de sinais clínicos e exames de imagem. O objetivo deste trabalho é relatar os exames de imagem realizados para o diagnóstico de um caso clínico neurológico e discutir o melhor método diagnóstico. Após o atendimento do paciente, foram solicitados exames complementares. Os resultados da radiografia não mostraram nenhuma alteração. A impressão diagnóstica da tomografia computadorizada indicou protrusão distal entre C6-C7, T11-T12, T13-L1, seguida de leve compressão medular definida pela presença de região hiperatenuante ventral. À ressonância magnética (RM), apresentava discreto hipersinal em T2W, bem delimitado na substância cinzenta, lateralizado à direita, sobre o terço cranial de C7. Concluiu-se que a ressonância magnética é o método que mais trouxe informações para o diagnóstico, os demais métodos não foram descritos alterações medulares pertinentes à EFC e à EANCNP. Com seu prognóstico favorável, a ausência de diagnóstico histológico dessas doenças pode ser um fator limitante neste estudo. Em relação às alterações do RM serem muito semelhantes entre EFC e EANCNP, não é possível diagnosticar com total precisão.

Sperm quality and morphometry characterization of cryopreserved canine sperm in ACP-106c or TRIS

Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.(AU)

Separation of coriander seeds by red, green and blue image processing / Separação de sementes de coentro por processamento de imagens red, green e blue

Coriander seeds have high socio-economic value in several regions of Brazil, especially in the North and Northeast. Seed maturation determined by color influences the seed quality. With this, digital image processing has become an important tool for separating seeds by color since this classification is usually performed by humans and is highly susceptible to error. The study established parameters for separating coriander seeds by red green and blue (RGB) image analysis, seeking a better selection of coriander seeds according to their color, and evaluating the physiological quality by the germination test. Separation was carried out from two coriander seed lots to obtain samples of 20 g each in three shades: yellowish, gray, and mixed. Images were acquired by the HP C4480 Scanner and processed in the MATLAB software; then, a histogram was constructed for each color analyzed in each sample by the RGB system. ANOVA tested the averages of the scales to ratify the difference in the components' distributions. The germination test was performed to confirm the results of seed separation using image analysis. The best selection of coriander seeds was achieved by the blue scale, and the germination test indicated that yellow seeds have a higher physiological quality than brownish/greyish seeds.

As sementes de coentro possuem alto valor socioeconômico em diversas regiões do Brasil, especialmente no Norte e Nordeste. A maturação de sementes determinada pela coloração influencia a qualidade da semente. Com isso, o processamento digital de imagens se tornou uma importante ferramenta para separação de sementes por meio da sua coloração, tendo em vista que essa separação normalmente é realizada por humanos e está altamente suscetível ao erro. O estudo tem como objetivo estabelecer parâmetros para separação de sementes de coentro por meio de processamento de análises de imagem red green and blue (RGB), buscando uma melhor seleção de sementes de coentro em função da sua coloração, assim como, avaliar a qualidade fisiológica por meio do teste de germinação. A partir de dois lotes de sementes de coentro, realizou-se a separação para obter amostras de 20g cada, em três tonalidades: amareladas, cinzas e mistas. As imagens foram adquiridas através da Scanner HP C4480 e processadas através do programa MATLAB, em seguida gerou-se um histograma para cada coloração analisada em cada amostra, por meio do sistema RGB. Na sequência, os resultados das médias das escalas foram analisados por ANOVA com a finalidade de ratificar a diferença das distribuições das componentes. Realizou-se o teste de germinação para ratificar os resultados da separação de sementes de coentro por meio de análises de imagens. A melhor seleção das sementes de coentro ocorreu através da escala em azul e o teste de germinação indicou que as sementes amarelas possuem qualidade fisiológica superior as sementes amarronzadas/acinzentadas.

The effect of adjusting settings within a Computer-Assisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results

Abstract Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.

Adrenal glands of Chinchilla lanigera - study of anatomical features with computed tomography and magnetic resonance imaging

Background: Since chinchilla (Chinchilla lanigera) is frequently used as a laboratory animal, satisfactory data about the imaging anatomical appearance of its adrenal glands, such as their anatomical location and closeness with other abdominal soft tissue and vessels, are important. The aim of this study to determine anatomical features of the chinchilla adrenal gland's using computed tomography and magnetic resonance imaging. Materials, Methods & Results: We used 12 chinchillas (6 males and 6 females), aged 18 months. The animals were in supine recumbency when contrast-enhanced computed tomography (CT) was performed. Transverse, sagittal and dorsal images of the adrenal glands were obtained with iodinated contrast medium, and 3D reconstruction of the obtained images was applied. The craniocaudal (CrCc - length), dorsoventral (DV - height) and lateromedial (LM - width) diameters were measured using an electronic calliper. Magnetic resonance imaging was performed, and coronal T1-weighted images were obtained. The transverse CT anatomical image at the level of the 3rd lumbar vertebra demonstrated the location of the both adrenal glands in accordance with the grey-white scale's variation. The right adrenal gland was hypo-attenuated and elliptic compared to the right kidney and in close contact to it and to the caudal vena cava. The left adrenal gland was oval and at a distance to the abdominal aorta. The dorsal MRI anatomical study of the chinchilla's abdominal organs at a distance of 10 mm from the spine and in a T1-weighted sequence showed that both adrenal glands were retroperitoneal organs. Discussion: Post-contrasted CT defined the topography of both glands. The right adrenal gland has an oval shape and is cranially situated to the left gland, whose shape is cylindrical and elongated. The LM diameter of the right gland is higher than that measured in the left gland. Both DV and CrCc diameters of the right gland are lower compared to those of the left gland. The right adrenal gland is in close contact to the caudal vena cava, the right kidney and the liver, and the left adrenal gland is in a distance to the abdominal aorta. The right adrenal gland was close to the caudal vena cava and the right kidney and medially to the left kidney. The successful comparative analysis of the images in 3D reconstruction and post-contrast CT in 2D allowed us to conclude that 3D reconstruction is suitable to obtain detailed information in a summary form regarding the closeness of the glands and their shape, mainly because the results are in a real time and highly comprehensive. Our data are in in agreement with previous findings about the advantages of 3D reconstruction. The research algorithm applied was based on the dorsal visualization of the glands in T1-weighted sequence, achieving a comprehensive and high-quality MRI imaging of the examined organs in chinchillas. Both adrenal glands were retroperitoneal organs and with low signal. The dorsal MRI anatomical study of the chinchilla's abdominal organs at a distance of 10 mm from the spine and in a T1-weighted sequence showed the whole profile of the right and left glands and the cranial position of the right gland to the left one, the close contact between the right gland and the kidney and the distance between the left gland and the left kidney. The MRI results are detailed and comprehensive for interpretation. In conclusion, the results of the present study are comprehensive, detailed and with high resolution. We present data for the anatomical relationships of the studied organs, their shape and macrometric parameters, concluding that the above mentioned modalities are very important tools for studying the chinchilla's adrenal glands to create a morphological base, which is necessary to investigate specific diseases.

The effect of adjusting settings within a ComputerAssisted Sperm Analysis (CASA) system on bovine sperm motility and morphology results

Semen motility is the most widely recognized semen quality parameter used by Artificial Insemination (AI) centers. With the increasing worldwide export of semen between AI centers there is an increasing need for standardized motility assessment methods. Computer-Assisted Sperm Analysis (CASA) technology is thought to provide an objective motility evaluation; however, results can still vary between laboratories. The aim of present study was to verify the impact of different setting values of the CASA IVOS II on motility, concentration, and morphology of bovine semen samples frozen in an extender with or without egg yolk and then decide on optimal settings for a further validation step across AI centers. Semen straws from 30 different bulls were analyzed using IVOS II with twelve modified settings. No significant changes were observed in semen concentration, percentage of motile sperm or kinetic results for either extender type. However, increasing settings for both STR and VAP progressive (%) from Low, Medium, and High cut-off values significantly (p<0.05) reduced the percentage of detected progressive spermatozoa, in egg yolk extender from 49.5±15.2, 37.2±11.9 to 11.9±5.3%, and in clear extender from 51.9±9.1, 35.8±7.3 to 10.0±2.4%, respectively. In clear extender only, the modification of droplet proximal head length significantly affected the detection of normal sperm percentages (88.0± 4.7 to 95.0±0.6 and 96.0±0.6%) and of the percentage of detected proximal droplets (12.2±4.7, 2.5±2.7 to 0.6±0.2%) for Low, Medium and High values respectively (p<0.05). The identification of sensitivity within the CASA system to changes in set parameters then led to the determination of an optimal IVOS II setting. The existing variability among centers for these phenotypes was reduced when the standardized settings were applied across different CASA units. The results clearly show the importance of applied settings for the final CASA results and emphasize the need for standardized settings to obtain comparable data.(AU)

Action of swim-up and caffeine on equine frozen sperm

Cryopreservation of equine semen is crucial to semen commercialization. However, it reduces sperm motility and longevity. Thus, sperm selection methods and addition of motility-activating substances to sperm, such as caffeine, may improve sperm quality of equine frozen semen. The objective of the current work was to evaluate the effects of caffeine on recovery and quality parameters of frozen-thawed sperm subjected to swim-up selection to be used in intracytoplasmic sperm injection (ICSI) in assisted reproductive techniques. Stallion semen were frozen and after thawing different caffeine concentrations were added to the samples performing four treatments control (no caffeine), 3, 5, and 7.5 mM caffeine. Sperm kinematic and motility were assessed by computer-assisted sperm analysis (CASA). Then, the four treated samples were submitted to the swim-up sperm selection, and the number of recovered sperm and morphology were evaluated at four times 20, 40, 60, and 80 min. The swim-up increased the recovery proportion of normal morphology sperm without (80.1±1%) or with caffeine addition (3mM: 81.2±1%, 5mM: 79.9±1% and 7.5 mM 78.9±1%) compared to the thawed semen (70±2%). However, the addition of 5 mM caffeine induced an increase in sperm motility (38.9±2.8 vs. 32.6±3.4%, P<0.05), and sperm recovery after swim-up (7.9x106 vs. 3.4x106 sperm/ml, P<0.05) compared to the control. The addition of 5 mM caffeine to frozen-thawed equine semen before swim-up selection improved sperm motility and increased the sperm recovery rate while not decreasing the percentage of morphologically normal sperm. Thus, caffeine addition to frozen-thawed equine semen before swim-up selection has potential clinical application in improving sperm quality for use in ICSI.(AU)

Effect of quercetin or butylated hydroxytoluene on cooled or frozen-thawed ram sperm quality / Efeito da inclusão de quercetina ou butil-hidroxitolueno na qualidade do sêmen de carneiro resfriado ou congelado

Cooling and freezing processes cause physical and chemical damage to sperm by cold shock and oxidative stress. This study aimed to evaluate the effect of two antioxidants on sperm parameters of cooled and frozen-thawed ram semen diluted in an egg yolk-based extender. Semen was collected from 30 rams and processed in two consecutive experiments to test the inclusion of different concentrations of quercetin and butylated hydroxytoluene (BHT) in an egg yolk-based semen extender. Dimethyl sulfoxide (DMSO) was added as a solvent to the semen extender in a ratio of 1 mL DMSO for 90 mg of quercetin and 1 mL DMSO for 880 mg of BHT. After collection, semen was diluted at 200 × 106 motile sperm/mL (control) and split into different groups in each experiment. In experiment 1, semen was diluted with the extender containing quercetin (Q5, 5 µg/mL; Q10, 10 µg/mL; Q15, 15 µg/mL) or DMSO alone (DMSO1, 0.055 µL DMSO per mL; DMSO2, 0.165 µL DMSO per mL). In experiment 2, semen was diluted with the extender with BHT (BHT1, 0.5 µg/mL; BHT2, 1 µg/mL; BHT3, 1.5 µg/mL) or DMSO alone (DMSO3, 0.375 µL DMSO per mL; DMSO4, 1.125 µL DMSO per mL). After dilution, the semen was divided into two aliquots. Treated ram sperm samples were also subjected to different storage methods. The first set of samples was cooled at 5 °C for 24 h, whereas the second set of samples was frozen-thawed. Sperm motility parameters and plasma membrane integrity (PMI) were evaluated immediately after dilution (0h) and 24 h after cooling and in the frozen-thawed samples via computer-assisted sperm analysis and epifluorescence microscopy, respectively. The inclusion of quercetin or BHT did not affect sperm motility parameters or PMI of fresh, cooled, or frozen-thawed sperm in this study (P < 0.05). However, further studies are needed to test the effects of these antioxidants on the fertility of cryopreserved ram semen.(AU)

O resfriamento e o congelamento causam danos físicos e químicos aos espermatozoides por choque térmico e estresse oxidativo. Portanto, este estudo teve como objetivo avaliar o efeito da inclusão de dois antioxidantes em um diluente à base de gema de ovo sobre os parâmetros espermáticos do sêmen ovino resfriado e congelado. Trinta carneiros tiveram o sêmen coletado e processado em dois experimentos consecutivos para testar a inclusão de diferentes concentrações de quercetina e hidroxitolueno butilado (BHT) em diluente de sêmen à base de gema de ovo. O DMSO foi adicionado como solvente ao diluente de sêmen em uma proporção de 1 mL de DMSO parra 90 mg de quercetina e 1 Ml de DMSO para 880 mg de BHT. Após a coleta, o sêmen foi diluído a 200 × 106 espermatozoides móveis/mL (Controle) e dividido em diferentes grupos em cada experimento. Experimento 1, Quercetina (Q5, 5 µg / mL; Q10, 10 µg / mL; Q15, 15 µg / mL) ou DMSO (DMSO1, 0,055 µL de DMSO por ml; DMSO2, 0,165 µL de DMSO / mL) foram adicionados ao extensor. Experimento 2, BHT (BHT1, 0,5 µg / mL; BHT2, 1 µg / mL; BHT3, 1,5 µg / mL) ou DMSO (DMSO3, 0,375 µL de DMSO por ml; DMSO4, 1,125 µL de DMSO / mL) foram adicionados à o extensor. Após a diluição, o sêmen foi dividido em duas alíquotas. O primeiro foi resfriado a 5 ° C por 24h, enquanto o segundo foi congelado. Os parâmetros de motilidade espermática e integridade da membrana plasmática (PMI) foram avaliados, imediatamente após a diluição (0h) e 24h após o resfriamento e nas amostras congeladas, pelo CASA e microscopia de epifluorescência, respectivamente. A inclusão de quercetina ou BHT não afetou os parâmetros de motilidade espermática e PMI de espermatozoides frescos, resfriados ou congelados (P < 0,05). Portanto, a inclusão de quercetina e BHT não beneficiou os parâmetros espermáticos do sêmen ovino submetido a armazenamento líquido a 5 ° C por 24h ou protocolo de congelamento no presente estudo. No entanto, mais estudos são necessários para testar o efeito desses antioxidantes na fertilidade do sêmen ovino criopreservado.(AU)

Growth performance, reproductive parameters and fertility measures in young Nellore bulls with divergent feed efficiency

The growth, sexual maturity and fertility-related parameters related of young Nellore bulls with divergent residual feed intake (RFI) raised on pasture were evaluated. After classification of 48 young males as low and high RFI (more and less efficient, respectively), the animals were evaluated for growth and reproductive parameters at 28-day intervals from 14.3 to 24.6 months of age. The semen was cryopreserved in the last sampling and fresh and post-thaw semen samples were evaluated. Low RFI bulls exhibited higher initial and final body weight (P < 0.05), but feed intake, body condition score and growth measures evaluated by carcass ultrasound were unaffected by RFI (P > 0.05). The scrotal circumference, sperm concentration, defects, and quality of fresh semen, and ultrasonographic testicular characteristics were unaffected by RFI (P > 0.05). However, velocity parameters such as average path and curvilinear velocities determined by computer-assisted sperm analysis of thawed semen submitted to the rapid thermoresistance test were improved (P < 0.05) in low RFI bulls, but this improvement in quality did not enhance in vitro sperm fertilizing ability. Our results demonstrated significant differences in metabolism and growth performance between bulls of divergent RFI. In addition, there was slight improvement in the semen quality of bulls with low RFI bulls, but this did not enhance in vitro fertilizing ability. Selection of beef bulls for RFI can be performed, which will result in economic benefits by improving the growth performance of the animals without affecting reproductive parameters.(AU)

Análises computadorizadas da motilidade espermática (CASA): conceitos e possibilidades de padrões / Computerized analysis of sperm motility (CASA): concepts and possibilities of standards

O melhoramento genético possibilitou a evolução da eficiência e taxas de desfrute da pecuária nacional. A biotécnica da IATF (Inseminação Artificial por Tempo Fixo) consolidou-se como a principal ferramenta de disseminação de genética melhorada em rebanhos de leite e de corte. A avaliação espermática laboratorial tem o objetivo de analisar o potencial de reprodutores/partidas de sêmen e buscar a desafiadora tarefa de determinar e estabelecer características confiáveis que possam ser utilizadas como indicadores de fertilidade. Nesse contexto emerge o sistema CASA (Computer-Assisted Sperm Analysis) como ferramenta promissora para obtenção de parâmetros indicativos de fertilidade seminal.(AU)

Genetic improvement enabled the evolution and efficiencies of national livestock activity. The IATF (Fixed Time Artificial Insemination) biotechnology, has established itself as the main tool for the dissemination of improved genetics in dairy and beef herds. Laboratory sperm evaluation aims to analyze the potential of a sires/semen batches and seek the challenging task of determining and establishing reliable characteristics that can be used as fertility indicators. In this context, the computerized system of sperm motility (CASA) emerges as a promising tool for obtaining parameters indicative of seminal fertility.(AU)

Paniculite nodular com esteatonecrose subcutânea em Golden Retrivier: relato de caso / Nodular panniculitis with subcutaneous steatonecrosis in a Golden Retrivier: case report

Um paciente canino, da raça Golden Retriever, foi atendido em clínica veterinária particular, com uma extensa formação nodular cutânea em região ventro-lateral direita, com evolução de um ano. A lesão apresentava consistência mole e presença de nodulações sólidas e retilíneas no seu interior, entremeadas ao meio líquido. Foi realizada colheita do material, por meio de punção por aspiração com agulha fina e o resultado citopatológico foi inconclusivo para neoplasia. Não foram observadas alterações dignas de nota na ultrassonografia abdominal. Após a realização de tomografia computadorizada da formação cutânea, as imagens eram compatíveis com abscesso ou hematoma entre áreas de mineralização. O animal foi submetido ao procedimento cirúrgico para biópsia excisional, tendo como diagnóstico morfológico paniculite nodular estéril com esteatonecrose, processo de morte celular dos adipócitos. O procedimento cirúrgico foi considerado curativo, uma vez que após um ano não houve recidiva no local ou formação de outras nodulações cutâneas. Essa patologia é incomum em cães, principalmente da raça Golden Retrevier e em região abdominal do tecido subcutâneo. Embora sejam correlacionadas algumas hipóteses, a causa dessa patologia permanece idiopática.(AU)

Canine intraventricular pneumocephalus: a case report / Pneumoencéfalo intraventricular em cão: relato de caso

Pneumocephalus is a well described disease; it is commonly diagnosed in humans, but the condition is rarely encountered in veterinary medicine. Computed tomography (CT) is the gold-standard diagnostic method for identifying this disease, and other methods (such as necropsy) are rarely described. In this report, we describe necropsy findings of a 10-month-old, mixed-breed dog with intraventricular pneumocephalus. The dog was referred to Laboratory of Animal Pathology of the Federal University of Uberlândia, Brazil, for necropsy after being diagnosed with pneumocephalus upon CT. In the examination, the brain had dilation of both lateral ventricles with empty spaces. Histopathology showed congestion and mineralization only near the lateral ventricles, leading to the diagnosis of pneumocephalus based on the macroscopic findings. The animal also showed sinusitis characterized by nasal discharge and neutrophilic infiltration of nasal sinuses. However, bacterial culture was not conclusive because of contamination of the sample. This is therefore an important report that shows necropsy findings of intraventricular pneumocephalus, which is a rare condition in dogs. By documenting the necropsy findings, we hope to help veterinary pathologists, including those with limited access to diagnostic imaging.

Pneumoencéfalo é uma condição bem descrita e comumente diagnosticada em humanos, mas raramente encontrada na medicina veterinária. A tomografia computadorizada (TC) é o método diagnóstico padrão-ouro para identificar tal alteração, e outros métodos (como a necropsia) raramente são descritos. Neste relato, são apresentados os achados de necropsia de um cão sem raça definida de 10 meses de idade com pneumoencéfalo intraventricular. O cão foi encaminhado ao Laboratório de Patologia Animal da Universidade Federal de Uberlândia, Brasil, para necropsia após ser diagnosticado com pneumoencéfalo na TC. No exame, o cérebro apresentava dilatação de ambos os ventrículos laterais com espaços vazios; à histopatologia, foram observadas congestão e mineralização próximas aos ventrículos laterais. Com isso foi estabelecido o diagnóstico de pneumoencéfalo, com base nos achados macroscópicos. O animal também apresentou sinusite caracterizada por secreção nasal e infiltração neutrofílica dos seios nasais. No entanto, a cultura bacteriana não foi conclusiva devido à contaminação da amostra. Este é, portanto, um importante relato que mostra achados de necropsia de pneumoencéfalo intraventricular, que é uma condição rara em cães. Ao documentar os achados da necropsia, esperamos ajudar os patologistas veterinários, incluindo aqueles com acesso limitado a técnicas de diagnóstico por imagem.

Pneumocephalus and suppurative meningoencephalitis in a dog after craniofacial trauma

Background: Pneumocephalus is characterized by the presence of gas in the intracranial compartment, and it can be developed by trauma, craniofacial surgery or spontaneously. Clinical signs start within days or months after the injury and vary according to the site of involvement. Computed tomography is the ideal diagnostic tool, however skull radiography can also be used. Treatment varies according to the severity of the case, and it can be conservative or associated with surgical intervention in the most severe cases. The purpose of this report is to describe the case of a dog that developed pneumocephalus and suppurative meningoencephalitis after head trauma caused by a bite from another dog. Case: A 2-month-old bitch, mixed breed, with 3.2 kg, was referred to the Veterinary Hospital because it had been bitten on the head by another dog. Shortly after the incident, the animal showed no clinical signs. However, 2 days later, the bitch became depressed and in persistent lateral decubitus. A lesion with a crust of approximately 0.5 cm was found close to the occipital region, with bone irregularity on palpation. The animal was in lateral decubitus with muscular hypotonia, bilateral mydriasis unresponsive to light and stupor. Radiographic images showed parietal fracture and pneumocephalus. Based on the findings of physical and laboratorial exams, diagnosis of suppurative meningoencephalitis and pneumocephalus secondary to craniofacial trauma was established. Empirical broad-spectrum antimicrobial therapy was started in addition to mannitol, corticoids, and analgesics. The animal was referred for surgical debridement by trepanation, when samples were collected to bacterial culture, which was negative. Despites the care, the animal died 14 h after the surgical procedure. Histopathological examination of the frontal cortex was performed, being the histological changes compatible with suppurative meningoencephalitis. Discussion: Dog bites on the head and neck are particularly severe, and can create intracranial bleeding, disfigurement of the face, damage to peripheral structures or cranial fractures. In this report, through radiographic images, it was found that the patient had an intracerebral aerocele, since there was presence of gas in the intracranial compartment. This alteration should always be considered in animals with neurological alterations and a history of craniofacial trauma. The main neurological changes observed in the reported case were unresponsive to mydriasis and altered mental status 2 days after the trauma, and this delay in the onset of clinical signs is frequently reported in cases of pneumocephalus. Neutrophilia and leukocytosis observed can be justified by the suppurative meningoencephalitis, confirmed by the histopathological exam. Antimicrobial therapy should be started as soon as possible, and the choice must be based on their capacity to cross the blood-brain barrier and the broad spectrum. The administration of antibiotics before collecting the material for bacterial culture may explain the negative result of this test, so that it is not possible to determine whether the intracranial gas observed on the radiograph may have developed from the trauma or because of gas-producing bacteria. Head trauma can induce suppurative meningoencephalitis and pneumocephalus even in the absence of perforating wounds at the time of the consultation. The neurological signs can start days after the trauma. Besides the clinical and surgical treatments, the prognosis of any bacterial infection of the central nervous system is poor.

Extraction of antioxidant and antimicrobial phytochemicals from corn stigma: a promising alternative to valorization of agricultural residues / Extração de fitoquímicos antioxidantes e antimicrobianos do estigma do milho: uma alternativa promissora para valorização de resíduos agrícolas

The discovery of new natural additives from agro-industrial waste is considered an important research topic. This study investigated the feasibility of ultrasound-assisted extraction (UAE) of antioxidant compounds from corn stigma (CS) and the effect of independent variables (time and solid-solvent ratio) and their interaction in the extraction of CS. Results indicated that the UAE method increases the antioxidant activity and reduces the extraction time by 67%. Optimized conditions for the simultaneous extraction of antioxidants and polyphenols from CS were obtained using 5 min and a solid-solvent ratio of 0.05 g mL-¹. The CS extract obtained by UAE was characterized by ESI-ToF-MS and 27 phytochemicals were reported. The extract showed promising antifungal and antibacterial activities against 23 of the studied microorganisms. Therefore, the CS extract obtained by the UAE can be used as a source of bioactive and antimicrobial compounds for use as a functional ingredient in the food and pharmaceutical industry.

A descoberta de novos aditivos naturais a partir de resíduos agroindustriais é considerada um importante tópico de pesquisa. Este estudo teve como objetivo investigar a viabilidade da extração assistida por ultrassom (EAU) de compostos antioxidantes do estigma do milho (EM) e o efeito de variáveis independentes (tempo e relação sólido-solvente) e sua interação na extração de EM. Os resultados indicaram que a EUA aumenta a atividade antioxidante e reduz o tempo de extração em 67%. Condições otimizadas para a extração simultânea de antioxidantes e polifenóis do EM foram obtidas com 5 min e uma relação sólido-solvente de 0,05 g mL-¹. O extrato de EM obtido pela EUA foi caracterizado por ESI-ToF-MS e 27 fitoquímicos foram encontrados. O extrato apresentou atividades antifúngicas e antibacterianas promissoras contra 23 dos micro-organismos estudados. Portanto, o extrato de EM obtido pela extração assistida por ultrassom pode ser utilizado como fonte de compostos bioativos e antimicrobianos para uso como ingrediente funcional na indústria alimentícia e farmacêutica.

Retrospective study on the incidence of cats and dogs' spinal injuries by computed tomographic scan. Part I: Cervical and Cervicothoracic / Estudo retrospectivo sobre incidência de injúrias em coluna vertebral de cães e gatos por meio de exame tomográfico. Parte I: Cervical e Cervicotorácica

Injuries involving the spine are frequent in dogs and knowledge of them is important to define the patient's treatment and prognosis. The objective of this retrospective study was to describe the epidemiological profile of animals and lesions in the cervical (C1-5) and cervicothoracic (C6-T2) spine diagnosed through tomographic examination. Compilation of computed tomography (CT) reports for the referred regions was carried out in a diagnostic center between 01/04/2017 and 30/04/2020, with or without contrast, from the clinical routine, in order to relate the most common lesions and their locations, as well as the species, breeds and ages most affected. A total of 1164 CT scans were performed in the period, 57.56% (n=670/1164) for the spine, with 89.7% (n=601/670) reports accessed, where both regions referred to here totaled 26.95% of the studies (n=162/601). Male mixed-breed dogs (MBD) showed the most lesions. For the cervical spine, the most identified lesion was disk extrusion and the site was C3-C4, while the mean age for lesions was 8.09±3.55 years. As for cervicothoracic, disk mineralization was more frequent and the mean age for lesions was 6.96±2.93 years. It was concluded that the spine is the main target of CT scans, that lesions related to the intervertebral disk were the main ones identified, and older MBD animals are the main ones affected.

As lesões envolvendo coluna vertebral são frequentes em animais de companhia, podendo ocorrer à nível vertebral, medula espinal, disco intervertebral, meninges ou raízes nervosas, e o conhecimento das mesmas é importante para definir o tratamento e prognóstico do animal. O objetivo desse estudo retrospectivo foi descrever o perfil epidemiológico dos animais e das lesões em coluna vertebral cervical (C1-5) e cervicotorácica (C6-T2) diagnosticadas por meio de exame tomográfico. Realizou-se a compilação de laudos de tomografias computadorizadas para as referidas regiões realizadas em centro diagnóstico comercial, entre 01/04/2017 a 30/04/2020, contrastadas e não-contrastadas, provenientes da rotina clínica, a fim de relacionar as lesões, raças e locais mais comuns. Foram realizados 1164 exames tomográficos no período avaliado, sendo 57,56% (n=670/1164) para a coluna, com 89,7% dos laudos acessados (n=601/670), sendo que as regiões cervical e cervicotorácica somaram 26,95% dos estudos (n=162/601). Em ambas regiões, os cães sem raça definida (SRD) machos foram os que mais demonstraram lesões. Para a coluna cervical, a lesão mais identificada foi extrusão de disco e o local mais afetado foi C3-C4, enquanto a média de idade para lesões foi 8,09±3,55 anos. Já para cervicotorácica, a mineralização de disco foi mais frequente e média de idade para ocorrência de lesões foi de 6,96±2,93 anos. Concluiu-se que a coluna vertebral foi o principal alvo de tomografias, as lesões relacionadas ao disco intervertebral foram as mais identificadas, sendo animais SRD com idade avançada os mais acometidos.

Retrospective study on the incidence of cats and dogs' spinal injuries by computed tomographic scan. Part II: Thoracolumbar and lumbosacral / Estudo retrospectivo sobre incidência de injúrias em coluna vertebral de cães e gatos por meio de exame tomográfico. Parte II: Toracolombar e Lombossacral

For vertebromedullary injuries, epidemiological knowledge is essential to guide the choice of the most effective diagnostic method. The objective of this retrospective study was to describe the epidemiological profile of animals and thoracolumbar and lumbosacral spine lesions diagnosed by computed tomography scan. A compilation of CT scan reports for the aforementioned regions performed in a comercial diagnostic center from 04/01/2017 to 04/30/2020, contrasted and non-contrasted, from routine, was performed in order to list the most common lesions and their locations, as well as the species, breeds and ages most affected. There were 1164 CT scans performed in the period evaluated, all of them in dogs, 57,56% (n=670) for the spine, with 89,7% reports accessed (n=601), where the regions mentioned here added up to 73,05% studies (n=439). In both regions, male French Bulldog dogs had the most injuries. For the thoracolumbar region, disk mineralization, followed by extrusion, were the most frequent injuries, while the site was T12-13 and the mean age 6,5±3,63 years old. In the lumbosacral, disk protrusion was most frequent, the most affected site was L7-S1 and age 6,65±3,79 years. It was concluded that the spine is the main target of CT scans, and that lesions related to the intervertebral disk were the main ones identified, with chondrodystrophic animals being more predisposed to injury.

Para lesões vertebromedulares é indispensável o conhecimento epidemiológico para direcionar a escolha do método diagnóstico mais eficaz. O objetivo desse estudo retrospectivo foi descrever o perfil epidemiológico dos animais e das lesões em coluna vertebral toracolombar e lombossacral diagnosticadas através de exame tomográfico. Realizou-se a compilação de laudos de tomografias computadorizadas para as referidas regiões realizadas em centro diagnóstico comercial, entre 01/04/2017 a 30/04/2020, contrastadas e não-contrastadas, provenientes da rotina, a fim de relacionar as lesões e seus locais de ocorrência, bem como a espécie, raças e idades mais comuns. Foram realizados 1164 exames tomográficos no período avaliado, sendo 57,56% (n=670) para a coluna, com 89,7% laudos acessados (n=601), nos quais as regiões toracolombar e lombossacral somaram 73,05% dos estudos (n=439). Em ambas as regiões, os cães Bulldog Francês machos foram os que mais apresentaram lesões. Para a região toracolombar, a mineralização de disco, seguida pela extrusão, foram as lesões mais frequentes, enquanto o local foi T12-13 e a média de idade 6,5±3,63 anos. Na lombossacral, a protusão de disco foi mais frequente, o local mais afetado foi L7-S1 e idade de 6,65±3,79 anos. Concluiu-se que a coluna vertebral é o principal alvo de tomografias, e que as lesões relacionadas ao disco intervertebral foram as principais identificadas, sendo os animais condrodistróficos mais predispostos a lesões.