Evaluation of ImageJ software in ultrasonic image analysis: Follicular and luteal morphological characteristics of cattle.

This study was conducted to compare the effectiveness of two methodologies in evaluating B- and Doppler-mode ultrasonic images: analysis using ultrasonic software and utilizing a computer with ImageJ software. To determine if ImageJ software utilization is an efficacious alternative to ultrasonic software device- analysis, there were comparisons of values when using the two methods for morphological and vascular characteristics of follicular dynamics and luteal function in 18 crossbred cattle. From day 8 of an ovarian dynamics synchronization treatment regimen period until the time of ovulation (Day 10), B-mode and power-flow ultrasonic cineloops were obtained every 12 h to assess follicular diameter, wall area, and wall blood perfusion area. On Day 14 after ovulation, US cineloops of ovaries were obtained in B mode and power flow to evaluate various morphological and vascular characteristics of the corpus luteum (CL), including luteal diameter, luteal area, and CL blood perfusion area. Cineloops were evaluated and analyzed using ultrasonic software, and in a computer with ImageJ software. To evaluate consistency in results between the two methods, there was evaluation utilizing paired t-test, Pearson correlation coefficient, Bland-Altman plot, and Linear Regression Test procedures to calculate proportion of bias between values for measurements of variables evaluated. Results indicated none of the values for variables before and after ovulation differed (P > 0.05). It, therefore, was concluded that utilization of ImageJ software is an efficacious biomedical technique to analyze ultrasonic images of morphological and vascular characteristics before and after ovulation in cattle.

Sperm kinematic subpopulations of the American crocodile (Crocodylus acutus).

There has been very limited use of computer assisted semen analysis (CASA) to evaluate reptile sperm. The aim of this study was to examine sperm kinematic variables in American crocodile (Crocodylus acutus) semen samples and to assess whether sperm subpopulations could be characterized. Eight ejaculates (two ejaculates/male) from four sexually mature captive crocodiles were obtained. An ISAS®v1 CASA-Mot system, with an image acquisition rate of 50 Hz, and ISAS®D4C20 counting chambers were used for sperm analyses. The percentages of motile and progressively motile spermatozoa did not differ among animals (P > 0.05) but there was a significant animal effect with regards to kinematic variables (P < 0.05). Principal component (PC) analysis revealed that kinematic variables grouped into three components: PC1, related to velocity; PC2 to progressiveness and PC3 to oscillation. Subpopulation structure analysis identified four groups (P < 0.05), which represented, on average, 9.8%, 32.1%, 26.8%, and 31.3% of the total sperm population. Males differed in the proportion of sperm in each of the kinematic subpopulations. This new approach for the analysis of reptile sperm kinematic subpopulations, reflecting quantifiable parameters generated by CASA system technology, opens up possibilities for future assessments of crocodile sperm and will be useful in the future development of assisted reproduction for these species.

Assisted breeding technology in the saltwater crocodile Crocodylus porosus: a review and look to the future.

This review reports the current status of artificial breeding technology in the Crocodylia and the future requirements for the establishment of AI in the saltwater crocodile. Although there are challenges regarding safe restraint and immobilisation, semen collection of the saltwater crocodile by manual stimulation has proven effective in yielding sufficient volume and sperm concentrations for empirical and molecular analyses of sperm preservation and physiology. Nevertheless, there is still much to learn with respect to fundamental anatomy, physiology and behaviour in both sexes, but particularly in the female. Although lessons can be learned from successful AI in the alligator, the details of this research are not readily accessible. Future research needs to focus on the proximate factors of seasonality and the underlying control of the female's annual reproductive cycle; this will require novel and innovative ways to collect blood samples without causing stress or injury, and ideally a dedicated crocodile research breeding colony. Because the saltwater crocodile is a farmed species, there is likely to be sufficient impetus for the application of assisted breeding technology to drive future productivity in the industry. These developments will also have benefits for the genetic and reproductive management of endangered captive populations.

Quality of domestic cat semen collected by urethral catheterization after the use of different alpha 2-adrenergic agonists.

OBJECTIVES: We compared the effects of two alpha (α)2-adrenergic agonists on semen traits. METHODS: In this study, 13 adult domestic cats were divided into two experimental groups, according to the chemical ejaculation protocol used: the first group received medetomidine hydrochloride (100 µg/kg) and ketamine (5000 µg/kg); the second group received dexmedetomidine hydrochloride (25 µg/kg) and ketamine (5000 µg/kg), both by the intramuscular route. RESULTS: The animals responded positively (P >0.05) to chemical collection. Seminal parameters evaluated included volume, sperm vigor, total motility, progressive motility, sperm concentration, and the structural and functional integrity of the plasma membrane; sperm morphology values did not differ between groups (P >0.05). CONCLUSIONS AND RELEVANCE: The results indicated that dexmedetomidine is a more viable and economical alternative to medetomidine in domestic cats submitted to semen collection by urethral catheterization. Semen collection by urethral catheterization after using α2-adrenergic agonists is a recently developed technique in feline species that is considered to be quick and highly applicable to assisted reproduction programs in felids.

Effects of equine chorionic gonadotropin administered via the Baihui acupoint on follicular ovarian dynamics and the luteal function of cattle during an ovulation synchronization treatment regimen for fixed-time artificial insemination.

Objectives were to evaluate effects of a smaller than typically used dose of equine chorionic gonadotropin (eCG) during a fixed-time artificial insemination (FTAI) treatment regimen. Transrectal ultrasonic (US) examinations were conducted on dairy cows on Day 0 (D0) and the treatment regimen was initiated with administrations of an intravaginal progesterone (P4) implant, estradiol benzoate (im), and prostaglandin F2α (PGF2α; im). On D8, the P4 implant was removed and PGF2α and estradiol cypionate were administered to all animals. Subsequently, cows were randomly assigned to three groups and eCG was administered to Groups 1, 2, and 3 in doses of 300 (im); 100 (im); and 100 (Baihui acupoint) IUs, respectively. The B-mode and power-flow US cineloops were performed to assess follicular dynamics and evaluate various morphological and vascular characteristics of the corpus luteum. Blood samples were collected to quantify serum P4 concentrations. There were no differences between the ovulation synchronization treatment regimens for all follicular dynamic variables tested; however, cows in Group 3 differed from Group 2 having a larger follicle diameter (FD) on D10 (P = 0.06) and larger preovulatory FD (P = 0.09), as well as a blood perfusion area of the dominant follicle wall on D8 (P = 0.07). There were no differences in responses to the ovulation synchronization treatment regimens for the luteal variables evaluated subsequent to ovulation. In conclusion, the Baihui acupoint was effective as an alternative route for eCG dose reduction when FTAI treatment regimens were imposed without detrimentally affecting values for reproductive variables.

Can amides be alternative cryoprotectors for the preservation of feline semen?

Sperm cryopreservation is a tool for the conservation of the genetic material of animals of genetic importance or for species preservation. In the case of domestic cats, this can be used to generate information about seminal harvest, evaluation and preservation, which is especially important due to its applicability to wild felids. This study evaluated seminal samples harvested by urethral catheterisation from 13 adult domestic cats. Samples were cryopreserved with experimental groups of extenders were defined by the penetrating cryoprotectant: 6% glycerol (GLY6%), 3% dimethylacetamide (DMA3%) and 3% dimethylformamide (DMF3%). The samples were thawed and evaluated by conventional microscopy and by computer-assisted sperm analysis (CASA). The structural and functional membrane integrity was assessed by supravital tests (EOS), hypoosmotic swelling tests (HOST) and flow cytometry (FC). There was a correlation (P < 0.05) between total motility and EOS (r = 0.54), HOST and FC (r = -0.62) and total motility and flow cytometry (r = 0.63), indicating that these are complementary parameters that increase the accuracy of the feline sperm quality evaluation post-thaw. The results regarding the structural and functional integrity of the sperm plasma membrane did not differ (P > 0.05) among groups. However, the DMA3% group had a lower (P < 0.05) percentage of morphological changes in the sperm tail compared to samples cryopreserved with GLY6% and DMF3%. Additionally, DMA3% provided lower values of immobile sperm post-thaw when compared to DMF3%. DMA is an interesting alternative to GLY and superior to DMF for the cryopreservation of feline semen at the studied concentrations.

Analysis of testicular variables, semen motility and kinematics-derived indexes in boar using a CASA-Mot system.

The present study aimed to evaluate differences in area, volume and testicular weight among groups related to breed and age of boar and establish associations between testicular parameters and seminal characteristics. Seminal quality has been studied using computer-assisted semen analysis (CASA) systems that reduce the subjectivity of kinematic parameters. Based on the data obtained through this system, sperm quality indexes and their derived parameters have been assessed to identify a better estimator for determining movement and velocity parameters. We also established indexes of velocity and sperm movement for boar semen. To this end, we evaluated 191 ejaculate samples from 63 boars. Differences related to the effects of season and age on quality boar semen were found (p ≤ .05) in the animal groups. Indexes were established for velocity and sperm movement based on CASA-Mot, and we proposed a sperm movement index (SMI) and sperm velocity index (SVI) with the CASA-Mot variables. Correlations were found (p < .05) among sperm indexes and CASA-Mot variables. Boar characteristics were found to be associated with some CASA-Mot variables, and the estimated SMI and SVI indexes predicted a greater variation in kinematic characteristics compared to the parameters evaluated separately using the CASA-Mot system.

Análisis de subpoblaciones cinéticas y morfométricas de espermatozoides con sistemas CASA: revisión

La valoración subjetiva de la calidad seminal ha dado paso al uso de las técnicas objetivas de valoración por medio de la tecnología CASA (computer-assisted semen analysis). Se pueden aplicar una serie de pruebas cinéticas, morfométricas, morfológicas o de integridad del ADN para caracterizar y entender la biología reproductiva del espermatozoide. En los últimos años, se ha dado un cambio significativo de paradigma conceptual respecto de qué es un eyaculado. Cabe decir que, hasta tiempos recientes, se consideró que una población representativa (miles de millones) de espermatozoides estaba formada por células "equivalentes" con un objetivo común: ser el que finalmente pudiese fecundar el ovocito. La comprobación de que el conjunto de espermatozoides se agrupa en subpoblaciones bien definidas de acuerdo con sus características cinéticas y/o morfométricas, ha abierto el camino hacia una visión más cooperativa. Además, se ha visto que la distribución subpoblacional es diferente entre individuos, lo que parece indicar diferentes estrategias que se pueden entender dentro de otro paradigma: el de la competencia espermática entre diferentes eyaculados. A pesar de que aún no se conoce el papel de las diferentes subpoblaciones, se deben continuar los trabajos en esa dirección. En esta revisión, se caracterizó la evaluación de la fertilidad con la tecnología CASA y se examinaron los principales métodos multivariados en la evaluación de subpoblaciones de espermatozoides analizados mediante un sistema CASA.

In recent years, there has been a substantive change in the conceptual paradigm regarding what an ejaculate is. Until recently, it was considered that a representative population (billions) of sperm was made up of "equivalent" cells with a common goal: to be the one that could finally fertilize the oocyte. New data suggesting that a set of spermatozoa is grouped in distinct subpopulations, according to their kinematic and morphometric characteristics, has opened the way towards a more cooperative vision. In addition, recently, it has been established that the subpopulation distribution is different among males, which seems to indicate that there are different strategies that can be understood within another paradigm: sperm competition between different ejaculates. These heterogeneous subpopulations of spermatozoa in the ejaculate, that show kinematics and morphometric patterns, has been widely known for a while, but the biological meaning of these different sperm subpopulations is still not clear. For this reason, the subjective evaluation of seminal quality is being displaced by objective assessment techniques using CASA technology (computer-assisted semen analysis). Also, the application of principal components (PC) and clustering methods to reveal subpopulations of spermatozoa are complementary tools used to characterize raw semen and processed cell suspensions. Despite the advances in such powerful tools, researchers are not well versed on such techniques and its advantages. For instance, PC analysis is a multivariate statistical method that reduces the number of variables used in subsequent calculations for describing the data. By integrating the original data into new complex mathematical variables, homogenous subpopulations of spermatozoa can define clearly. Furthermore, kinematic, morphometric, morphological or DNA integrity tests can be also applied to characterize and understand the reproductive biology of the spermatozoon. Although the role of the different subpopulations of spermatozoa remains unknown, the work should continue by implementing novel technologies and coordinated tools. In this review, we characterize the fertility evaluation with CASA technology and examine the main multivariate methods in the assessment of sperm subpopulations analyzed by CASA systems.

Biometría testicular y características seminales en felinos neotropicales (Carnivora: Felidae) sometidos a cateterismo uretral

Introducción: La administración de un fármaco agonista de un alfa- 2-adrenérgicos asociado con la técnica del cateterismo uretral, es un método de desarrollo reciente en la recolección seminal en felinos, ofreciendo las ventajas sobre la recolecta por electroeyaculación y la recolecta epididimaria. La técnica se ha probado en Panthera onca y felinos de pequeño porte. Objetivos: Realizar la recolección seminal en felinos neotropicales utilizando la técnica de cateterización uretral. Determinar los parámetros testiculares y seminales de muestras recolectadas por la técnica de cateterización uretral. Metodología: En este estudio se utilizó un individuo por especie de Panthera onca, Puma yagouaroundi yLeopardus wedii localizados en el parque Zoobotánico Getúlio Vargas, Salvador, Bahia, Brasil. El semen fue recolectado por medio del cateterismo uretral y se utilizaron los farmácos: clorhidrato de medetomidina (0.1 mg /kg) y ketamina (5.0 mg /kg). Durante el desarrollo del procedimiento, se realizó la recolecta de los datos biométricos corporales y testiculares, así como la ultrasonografía de los testículos. Mediante microscopia óptica, en las muestras seminales se determinó: el vigor espermático, motilidad total, motilidad progresiva, la integridad estructural y funcional de la membrana plasmática y la morfología espermática. Las muestras se sometieron al proceso de congelación - descongelación utilizando tres diferentes crioprotectores. Las muestras descongeladas fueron evaluadas por microscopía convencional, análisis seminal computarizado y citometría de flujo. Resultados: En este estudio fue realizado con éxito la técnica de recolección seminal en las tres especies, siendo el primer estudio que informa de los parámetros seminales deP. yagouaroundiy el primero que reporta la recolección seminal uretral en L. wiedii. Los datos presentados describen de forma pionera la morfocinética espermática de las especies incluidas. No hubo diferencias (P > 0.05) en la calidad seminal evaluando por grupo de crioprotector, ni diferencias en la integridad de las membranas pré e post-congelamento. Fue posible solamente la comparación con otras técnicas de recolección seminal o con resultados de calidad seminal obtenidos de muestras de gato doméstico. Conclusión: La cateterización uretral asociada al fármaco alfa- 2-adrenérgicos es una técnica eficiente para la recolección seminal en felinos neotropicales. El establecimiento de una potencial asociación de las características físicas del animal y la calidad seminal, es información de suma importancia en la aplicabilidad para la elección de reproductores en los programas de reproducción asistida.

Introduction: The urethral catheterization associated with the application of alfa- 2 adrenergic agonist drug is a recent development and application technique for the collection of feline semen, with advantages over electro-ejaculation harvesting and epididymal harvesting. Objetives: Seminal collection in neotropical felines using the urethral catheterization technique. Determine the testicular and seminal parameters of samples collected by the urethral cataterization technique. Methods: The technique has been tested on Panthera onca and others small cats. In this study, individuals of Panthera onca, Puma yagouaroundi and Leopardus wiedii were used in captivity at Getulio Vargas Zoobotanical Park, Salvador, Bahia, Brazil. Medetomidine hydrochloride (0.1 mg/kg) and ketamine (5.0 mg/kg) were used as sedatives, the semen was collected by urethral catheterization. In the process were checked the testicular biometry was performed with percutaner and ultrasonography technique. The fresh samples were evaluated for conventional microscopy in the parameters of volume, sperm vigor, total motility, progressive motility, structural and functional integrity of the plasmatic membrane and sperm morphology. The samples were subjected to a freeze - thaw process using three different extenders. The thawed samples were tested using conventional microscopy, automated system analysis and flow cytometry. Results: In this study, the urethral harvest technique was successfully used in the three species. In the literature used don't have report about seminal parameters in P. yagouaroundi and urethral seminal collection in L. wiedii. The data presented describe the spermatic morphocinetics of the included species in a pioneer study. There were no differences (P > 0.05) in the seminal quality evaluating the extender, nor differences in the integrity of pre and post-freeze membranes of the samples. Conclusion: The urethral catheterization associated with the application of alfa- 2 adrenergic agonist drug it's an efficient technique for semen collection in neotropical cats. The establishment of a potential association of physical characteristics with seminal quality is extremely important information on the applicability of reproductive traits in assisted reproduction programs.

Kinematic and head morphometric characterisation of spermatozoa from the Brown Caiman (Caiman crocodilus fuscus).

The development of analytical methods for the evaluation of crocodilian semen is an important component for the assessment of male breeding soundness and the development of assisted breeding technology in this taxon. Computer-Assisted Semen Analysis (CASA) technology is becoming an increasingly common technique in seminal evaluations for animals but there has been no application of this technique for reptilian spermatozoa. The aim of this study was to analyse sperm kinematic and morphometric variables in Caiman crocodilus fuscus semen samples and to determine whether there were sperm subpopulations. Four ejaculates from four sexually mature captive caimans were used for this study. A CASA-Mot and CASA-Morph system was used with an image acquisition rate of 50 Hz for 2 s of capture. The ISAS®D4C20 counting chambers were used and spermatozoa incubated at 25 °C. Total and progressive motilities did not differ among animals (P > 0.05). There was a significant animal effect in the model with respect to sperm morphometry, and kinematic indices including linearity (LIN) and straightness (STR) (P < 0.05). Results for principal component (PC) analysis indicated variables were grouped into four components: PC1 related to velocity, PC2 to progressivity, PC3 to oscillation and PC4 to sperm path cross-linking. Subpopulation (SP) structure analysis indicated there were four groups, namely, rapid non-progressive (SP1), slow non-progressive (SP2), rapid progressive (SP3) and medium progressive (SP4), representing 14.5%, 45.4%, 18.7%, and 21.4% respectively. Findings in the present study indicate the importance of continuing development of reliable protocols regarding the standardisation of computer-based semen analyses in reptilian species.