Biotecnologias na preservação de espermatozoides felinos / Biotechnologies in the preservation of feline sperm

O gato doméstico é a única espécie da família Felídea sem risco ou iminência de extinção, diferente da maior parte dos felinos selvagens. Desta forma, o desenvolvimento e aprimoramento de diferentes biotécnicas reprodutivas, são essenciais para a manutenção da qualidade reprodutiva, tendo em vista a preservação de espécies mais vulneráveis. Além disso, as biotécnicas do sêmen são para as tecnologias reprodutivas, como a inseminação artificial (IA) e a fertilização in vitro (FIV). Sendo assim, o objetivo deste compilado bibliográfico foi abordar as principais técnicas de colheita, análise e preservação de sêmen/espermatozoides felino, assim como o uso dessas células em IA e FIV. Para a colheita do sêmen felino, diferentes métodos têm sido aplicados: ejaculação farmacológica, eletroejaculação e vagina artificial. Em caso de óbito do reprodutor, os espermatozoides recuperados do epidídimo também apresentam viabilidade reprodutiva. Ademais, a cinética espermática avaliada pelo sistema CASA, a morfologia e a morfometria são as principais análises que demonstram a qualidade espermática e refletem na fertilidade do ejaculado. O sistema CASA também avalia a trajetória individual de cada espermatozoide, que ao se agrupar em clusters, demonstra a heterogeneidade do ejaculado nas subpopulações. Contudo, os diluentes para a conservação e refrigeração dos espermatozoides felinos e as curvas de congelação ainda não estão totalmente estabelecidos e influenciam diretamente a viabilidade dos espermatozoides criopreservados. Diante disso, os resultados da utilização do sêmen felino após criopreservação são inconsistentes, sendo necessários mais estudos para elucidar melhores curvas de congelação e meios de diluentes para viabilizar a preservação do material genético dos gatos.

The domestic cat is the only species of the Felidea family without risk or imminence of extinction, unlike most wild cats. Therefore, the development and improvement of different reproductive biotechnologies are essential for the maintenance of reproductive quality for the preservation of the most vulnerable species. Furthermore, semen biotechnologies are the basis for reproductive technologies such as artificial insemination (AI) and in vitro fertilization (IVF). Thus, the objective of this bibliographic compilation was to approach the main techniques of collection, analysis, and preservation of feline semen/sperm, as well as the use of these cells in AI and IVF. For feline semen collection, different methods have been applied: pharmacological ejaculation, electroejaculation, and artificial vagina. In case of death of the sire, sperm recovered from the epididymis also show reproductive viability. Moreover, the sperm kinetics evaluated by the CASA system, the morphology, and the morphometry are the main analyzes that demonstrate sperm quality and reflect on ejaculate fertility. The CASA system also evaluates the individual path of each sperm, which, when grouped into clusters, demonstrates the heterogeneity of the ejaculate in the subpopulations. However, diluents for the conservation and refrigeration of feline sperm and freezing curves are not yet fully established and directly influence the viability of cryopreserved sperm. Therefore, the results of using feline semen after cryopreservation are inconsistent, and further studies are needed to elucidate better freezing curves and diluents to enable the preservation of the genetic material of cats.

Sperm subpopulations influence the pregnancy rates in cattle.

This study aimed to study the characteristics and subpopulations of spermatozoa from bulls with low and high reproductive performance based on pregnancy rates. Based on historical records of pregnancy rate from four farms, 24 bulls were selected. Two groups were established, with low pregnancy rates (n = 12; LOW), including bulls that presented pregnancy rates <52.27% (33.33% to 51.81%); and a group with high pregnancy rates (n = 12; HIGH), with pregnancy rates >52.27% (52.27% to 69.64%), after fixed-time artificial insemination (FTAI). The thawed sperm straws were analysed to sperm kinetics, morphology, plasma membrane integrity and sperm subpopulations. The LOW group exhibited a higher proportion of static cells (p < .05). In contrast, the HIGH group showed greater percentages for membrane integrity and total and progressive motility, and cells with fast and medium velocity (p < .05). In the cluster procedures, four sperm subpopulations were established. The low-fertility bulls presented the highest percentage of subpopulation 2 (41.46%), characterized by slow and progressive spermatozoa. The high-fertility bulls exhibited the highest percentage of subpopulation 3 (37.17%), characterized by fast and nonlinear spermatozoa. Results from this study indicated that bulls with greater percentages of fast and nonlinear spermatozoa seem to have greater fertilization capacity and the subpopulations analysis can be considered a tool to identify ejaculates with high fertility.

Correlation of testicular artery Doppler velocimetry with kinetics and morphologic characteristics of epididymal sperm in dogs.

Sperm quality can be affected by a reduction in testicular blood flow, which can be measured by Doppler ultrasonography. The aim of this study was to correlate the Doppler velocimetry of the testicular artery with kinetics of the epididymal spermatozoa in dogs. Twenty-two dogs (44 testicles) were evaluated by Doppler ultrasonography in five regions of the testicular artery before orchiectomy. Spermatozoa were recovered by the epididymal tail compression technique and analysed for kinetics on a computer-assisted semen analysis (CASA system). Morphology (modified Karras) and sperm membrane integrity were analysed by eosin-nigrosine staining. Data were analysed by Pearson's correlation test (p < .01). The mean total motility was 69.0% ± 17.7, progressive motility was 43.7% ± 14.7, average path velocity (VAP) was 127.0 µm/s ± 20.7, curvilinear velocity (VCL) was 221.0 µm/s ± 31.1, and sperm velocity index (SVI) was 389.9 ± 56.1. There were positive correlations between the peak systolic velocity (PSV) in the proximal supratesticular region with the SVI (r = .529), VCL (r = .555) and VAP (r = .473), and a negative correlation with the percentage of slow spermatozoa (r = -.463). The results suggest that the testicular artery blood flow velocity can positively affect the speed of spermatozoa movement. For the first time, we have correlated sperm kinetics with the Doppler evaluation of the testicular artery in dogs.

Influência da cinética espermática sobre a taxa de prenhez na inseminação artificial em tempo fixo em bovinos / Influence of sperm kinetics on pregnancy rate in fixed-time artificial inseminations in cattle

The aim of the study was to evaluate the influence of hyperactivated sperm kinetics on pregnancy rate in IATF. Two experimental groups were established, based on the results of the semen analysis in the CASA system: groups of bulls with hyperactivated sperm (N = 10; HIPER) and bulls with non-hyperactivated sperm (N = 14; N HIPER). Differences between groups were estimated by the t test, and a significance level <5% was considered. Highervalues for the variables were identified in the HIPER group: VAP; VSL; VCL; ALH; RAPID cells and SLOW cells. On the other hand: BCF; STR; LIN; WOB and MEDIUM cells, which had higher values in the N HIPER group. No difference was found for the pregnancy rate variable between groups (p=0.454). Therefore, although the CASA system is an objective method of analysis, we can consider that it alone is not sufficient to predict seminal fertility, but when associated with other techniques such as: specific software and multivariate statistics that identify subpopulations, if makes it an important methodology for assessing semen quality.

Características do comportamento hiperativado de espermatozoides de felinos domésticos / Characteristics of the cat sperm hyperactivated behavior

Sperm hyperactivity is a physiological behavior, and in the feline species it is characterized by an increase in the curvilinear velocity (VCL) and a greater head beat (ALH) evaluated by the CASA system. The study aimed to compare and correlate kinetic parameters of Hyperactive and Non-Hyperactive feline ejaculates when considering the means of VCL and ALH. Ejaculates were collected by electro ejaculation from 21 cats. The seminal samples had the kinetic parameters evaluated by the CASA system. From the average values of VCL and ALH, the ejaculates were classified in group HP (Hyperactivated, when VCL>190.17µm/s and ALH>6.44µm) and NHP (Non-Hyperactivated, when VCL<190.17 and ALH<6.44 µm). A T test and Pearson's correlation were performed with a significance of p<0.05. Among the groups, were detected higher values of total (HP: 68,2% vs NHP: 35,9%) and progressive (HP:40,1% vs NPH: 17,18%) motility, VAP (HP: 165,85µm/s vs NHP: 97,72 µm/s), VSL (HP:137,63µm/s vs NHP 82,6µm/s), VCL (HP: 237,31µm/s vs NHP: 147,94µm/s) and ALH (HP: 7,28µm vs NHP:5,42µm) for the HP group. There was a correlation in the HP ejaculates between total and progressive motility. In the NHP group, a correlation was observed between motility and progressive motility, and between progressive motility and STR and LIN. It was concluded that HP spermatozoa have a higher curvilinear speed, while NHP spermatozoa stand out due to their straight path.

Influência da cinética espermática sobre a taxa de prenhez na inseminação artificial em tempo fixo em bovinos / Influence of sperm kinetics on pregnancy rate in fixed-time artificial inseminations in cattle

The aim of the study was to evaluate the influence of hyperactivated sperm kinetics on pregnancy rate in IATF. Two experimental groups were established, based on the results of the semen analysis in the CASA system: groups of bulls with hyperactivated sperm (N = 10; HIPER) and bulls with non-hyperactivated sperm (N = 14; N HIPER). Differences between groups were estimated by the t test, and a significance level <5% was considered. Highervalues for the variables were identified in the HIPER group: VAP; VSL; VCL; ALH; RAPID cells and SLOW cells. On the other hand: BCF; STR; LIN; WOB and MEDIUM cells, which had higher values in the N HIPER group. No difference was found for the pregnancy rate variable between groups (p=0.454). Therefore, although the CASA system is an objective method of analysis, we can consider that it alone is not sufficient to predict seminal fertility, but when associated with other techniques such as: specific software and multivariate statistics that identify subpopulations, if makes it an important methodology for assessing semen quality.(AU)

Características do comportamento hiperativado de espermatozoides de felinos domésticos / Characteristics of the cat sperm hyperactivated behavior

Sperm hyperactivity is a physiological behavior, and in the feline species it is characterized by an increase in the curvilinear velocity (VCL) and a greater head beat (ALH) evaluated by the CASA system. The study aimed to compare and correlate kinetic parameters of Hyperactive and Non-Hyperactive feline ejaculates when considering the means of VCL and ALH. Ejaculates were collected by electro ejaculation from 21 cats. The seminal samples had the kinetic parameters evaluated by the CASA system. From the average values of VCL and ALH, the ejaculates were classified in group HP (Hyperactivated, when VCL>190.17µm/s and ALH>6.44µm) and NHP (Non-Hyperactivated, when VCL<190.17 and ALH<6.44 µm). A T test and Pearson's correlation were performed with a significance of p<0.05. Among the groups, were detected higher values of total (HP: 68,2% vs NHP: 35,9%) and progressive (HP:40,1% vs NPH: 17,18%) motility, VAP (HP: 165,85µm/s vs NHP: 97,72 µm/s), VSL (HP:137,63µm/s vs NHP 82,6µm/s), VCL (HP: 237,31µm/s vs NHP: 147,94µm/s) and ALH (HP: 7,28µm vs NHP:5,42µm) for the HP group. There was a correlation in the HP ejaculates between total and progressive motility. In the NHP group, a correlation was observed between motility and progressive motility, and between progressive motility and STR and LIN. It was concluded that HP spermatozoa have a higher curvilinear speed, while NHP spermatozoa stand out due to their straight path.(AU)

Central de IA em suínos: Uma análise prática do processo de produção de sêmen de alta qualidade / Boar AI Center: A practical analysis of the process of producing high quality semen doses

A suinocultura é uma atividade em constante busca por altos índices de produtividade, e consequentemente, de animais com elevado potencial genético. Entretanto, essa atualização genética dos plantéis comerciais precisa ser realizada de forma rápida, econômica, prática e sanitariamente segura. Nesse contexto, a aquisição de doses de sêmen via centrais de inseminação artificial (CIA) especializadas é uma das formas mais eficientes para alcançar esse objetivo. No entanto, além da necessidade de que as doses sejam produzidas utilizando reprodutores de máximo potencial genético, exige-se um rigoroso controle na rotina de produção, para que o sêmen apresente ótima qualidade espermática e potencial fecundante. Nesse sentido, o objetivo do presente artigo é revisar alguns dos fatores mais relevantes na rotina de produção do sêmen em uma CIA em suínos, bem como citar as estratégias que visem minimizar os fatores que possam interferir na eficiência do processo.

The pig production is a sector in constant search for high rates of productivity, and consequently, of animals with high genetic potential. However, this genetic update of commercial farms needs to be done quickly, economically, practically, and safe from a sanitary point of view. In this context, the acquisition of semen doses via specialized artificial insemination (AI) centers is one of the most efficient ways to achieve this goal. However, in addition to the need for doses to be produced using boars of maximum genetic merit, strict control of the production routine is required so that the semen presents optimum sperm quality and fertilization potential. In this sense, the objective of the present article is to review some of the most relevant factors in customary semen production in an AI center in pigs, as well as to cite the strategies that aim to minimize the factors that may interfere in the process efficiency.

Central de IA em suínos: Uma análise prática do processo de produção de sêmen de alta qualidade / Boar AI Center: A practical analysis of the process of producing high quality semen doses

A suinocultura é uma atividade em constante busca por altos índices de produtividade, e consequentemente, de animais com elevado potencial genético. Entretanto, essa atualização genética dos plantéis comerciais precisa ser realizada de forma rápida, econômica, prática e sanitariamente segura. Nesse contexto, a aquisição de doses de sêmen via centrais de inseminação artificial (CIA) especializadas é uma das formas mais eficientes para alcançar esse objetivo. No entanto, além da necessidade de que as doses sejam produzidas utilizando reprodutores de máximo potencial genético, exige-se um rigoroso controle na rotina de produção, para que o sêmen apresente ótima qualidade espermática e potencial fecundante. Nesse sentido, o objetivo do presente artigo é revisar alguns dos fatores mais relevantes na rotina de produção do sêmen em uma CIA em suínos, bem como citar as estratégias que visem minimizar os fatores que possam interferir na eficiência do processo.(AU)

The pig production is a sector in constant search for high rates of productivity, and consequently, of animals with high genetic potential. However, this genetic update of commercial farms needs to be done quickly, economically, practically, and safe from a sanitary point of view. In this context, the acquisition of semen doses via specialized artificial insemination (AI) centers is one of the most efficient ways to achieve this goal. However, in addition to the need for doses to be produced using boars of maximum genetic merit, strict control of the production routine is required so that the semen presents optimum sperm quality and fertilization potential. In this sense, the objective of the present article is to review some of the most relevant factors in customary semen production in an AI center in pigs, as well as to cite the strategies that aim to minimize the factors that may interfere in the process efficiency.(AU)

as / Sperm subpopulations

A biotecnologia da reprodução é importante para melhorar a eficiência reprodutiva desenvolvendométodos que identifiquem os melhores reprodutores. Embora a motilidade espermática seja a variável maisavaliada, é necessária a avaliação das características da cinética espermática, as quais se alteram conformeespécie, indivíduos, ejaculados e desafios a que são submetidos. Atualmente sabe-se que o ejaculado não écomposto de um grupo homogêneo de espermatozoides, e sim por subpopulações espermáticas heterogêneas, asquais apresentam respostas variadas quanto aos padrões de motilidade, e a identificação dessas subpopulaçõespode auxiliar na escolha do reprodutor mais eficiente.(AU)

Reproductive biotechnology is important for improving reproductive efficiency by developing methodsthat identify the best breeding. Although sperm motility is the most evaluated variable, it is necessary to evaluatethe characteristics of sperm kinetics, which change according to species, individuals, ejaculates and thechallenges that are submitted. It is known that ejaculate is not composed of a homogeneous group ofspermatozoids, but by heterogeneous sperm subpopulations, such as varied kiosks for motility patterns, and asubpopulation identifier may aid in the selection of most efficient breeders.(AU)

as / Sperm subpopulations

A biotecnologia da reprodução é importante para melhorar a eficiência reprodutiva desenvolvendométodos que identifiquem os melhores reprodutores. Embora a motilidade espermática seja a variável maisavaliada, é necessária a avaliação das características da cinética espermática, as quais se alteram conformeespécie, indivíduos, ejaculados e desafios a que são submetidos. Atualmente sabe-se que o ejaculado não écomposto de um grupo homogêneo de espermatozoides, e sim por subpopulações espermáticas heterogêneas, asquais apresentam respostas variadas quanto aos padrões de motilidade, e a identificação dessas subpopulaçõespode auxiliar na escolha do reprodutor mais eficiente.

Reproductive biotechnology is important for improving reproductive efficiency by developing methodsthat identify the best breeding. Although sperm motility is the most evaluated variable, it is necessary to evaluatethe characteristics of sperm kinetics, which change according to species, individuals, ejaculates and thechallenges that are submitted. It is known that ejaculate is not composed of a homogeneous group ofspermatozoids, but by heterogeneous sperm subpopulations, such as varied kiosks for motility patterns, and asubpopulation identifier may aid in the selection of most efficient breeders.

Estudio del semen humano: implementación de un método objetivo / Study of human semen: implementation of an objective method / Estudo do sÛmem humano: implementaþÒo de um método objetivo

El objetivo del presente trabajo fue estandarizar y validar un sistema computarizado de análisis de semen CASA, SCA (Sperm Class Analyzer) para los parámetros de concentración y movilidad espermática de acuerdo a normativas internacionales. Se estandarizó el tipo de velocidad para la clasificación en grados y la profundidad adecuada de la cámara. Para la validación se determinó precisión, límite de detección, intervalo de medición y estudio de comparación de métodos. Se halló alta correlación lineal en la clasificación del movimiento empleando Velocidad Curvilínea o Velocidad Promedio (r=0,99; p< 0,001), estableciendo el uso de la primera para obtener resultados comparables con otros sistemas objetivos. También, se normatizó el empleo de cámaras de 10 Am de profundidad debido a que se obtienen mejores imágenes para el análisis sin afectar la movilidad. La imprecisión media fue 13,29%, 14,26% y 18,75% para recuento, móviles progresivos y móviles grado (a) respectivamente. Se halló alta correlación con el método manual, tanto en concentración (r=0,97, p>< 0,0001), móviles progresivos (r=0,84; (p>< 0,001) y móviles grado (a) (r=0,82; p>< 0,0001). Se comprobó linealidad (r=0,99; p>< 0,001), entre 0,98 x 106 y 125 x 106 esp/mL. Se concluye que el método propuesto cumple con los requisitos necesarios para su empleo en la clínica, siendo indispensable la edición de las imágenes por parte de un operador calificado.(AU)

The aim of this study was to standardize and validate a CASA system, SCA (Sperm Class Analyzer) for the parameters of sperm concentration and motility according to international standards. The type of speed for the classification of degrees and the proper depth of the camera were standardized. For the validation, accuracy, detection limit, measuring range and method comparison study were determined. High linear correlation was found in the classification of motion using curvilinear velocity or average speed (r=0.99, p< 0.001), establishing the use of the first to obtain results comparable with other objectives. Likewise, the use of cameras 10 microns in depth was also standardized since better images are obtained for the analysis without affecting mobility. Average imprecision was 13.29%, 14.26% and 18.75% for counting, progressive and mobile grades respectively. High correlation was found with the manual method, both in concentration (r=0.97, p>< 0.0001), progressive mobiles (r=0.84, (p>< 0.001) and mobile grade a (r=0.82, p>< 0.0001). Linearity (r=0.99, p>< 0.001) was proved between 0.98 and 125 x 106 x 106 sperm/mL. It is concluded that the proposed method meets the requirements for use in the clinic, being image editing by a qualified operator indispensable.(AU)

O objetivo do presente trabalho foi padronizar e validar um sistema computadorizado de análise de sÛmen CASA, SCA (Sperm Class Analyzer) para os parÔmetros de concentraþÒo e motilidade espermática de acordo com normativas internacionais. Foi padronizado o tipo de velocidade para a classificaþÒo em graus e a profundidade adequada da cÔmara. Para a validaþÒo se determinou precisÒo, limite de detecþÒo, intervalo de mediþÒo e estudo de comparaþÒo de métodos. Foi encontrada alta correlaþÒo linear na classificaþÒo do movimento utilizando Velocidade Curvilínea ou Velocidade Média (r=0,99; p< 0,001), estabelecendo o uso da primeira para obter resultados comparáveis com outros sistemas objetivos. Também, foi normatizado o uso de cÔmaras de 10 Am de profundidade visto que se obtÛm melhores imagens para a análise sem afetar a mobilidade. A imprecisÒo média foi de 13,29%, 14,26% e 18,75% para recontagem, móveis progressivos e móveis grau (A) respectivamente. Achou-se alta correlaþÒo com o método manual, tanto em concentraþÒo (r=0,97; p>< 0,0001), móveis progressivos (r=0,84; (p>< 0,001) e móveis grau (A) (r=0,82; p>< 0,0001). Foi comprovada linearidade (r=0,99; p>< 0,001), entre 0,98 x 106 e 125 x 106 esp/mL. Conclui-se que o método proposto cumpre com os requisitos necessários para sua utilizaþÒo na clínica, sendo indispensável a ediþÒo das imagens por parte de um operador qualificado.(AU)