Evaluation of Chilled Dog Semen Extended With Sperm Activator.

Within modern biotechnology, different tools and methodologies have been developed to maximize canine semen conservation protocol to optimize reproductive results. In the last decades, the survival of chilled semen has been prolonged from 2 to 3 days with the first basic diluents, to 10-14 days with the modern extenders. However, their main limitation is that sperm quality decreases during cold storage. Sperm activators (SA) have been produced to provide the molecules necessary to maximize the sperm survival and quality with the aim to enhance fertility and prolificacy. In this study, the effect of commercial extender SA (Theriosolution® Canine AI extender -Chile-) was recorded by daily evaluation of chilled semen for 14 days. In this experiment, sperm-rich ejaculate fraction was collected from six adult healthy Neapolitan Mastiff dogs. The semen evaluation started immediately after collection (d0), and after that a next generation extender was added (d0) for every 24 h from d1 (with and without SA) to d14, to determine spermatozoa progressive motility, velocity of forward progression (VFP), morphology, and integrity of the spermatic membrane. The initial sperm concentration of extended semen was 417.3 ± 170.4 x 106/mL (mean ± SEM) with 85.89 ± 4.76% of MNS (morphologically normal sperm), 84.47 ± 5.22 % live sperm, and pH of 6.2 ± 2.8. The initial VFP was 3.83 ± 0.48, but after 1 min with SA, it rises to 4.45 ± 0.45 (P < 0.001). The sperm progressive motility parameter increases significantly (P < 0.05) in experimental trial, respect to control, starting to d2 at finish (except for d7). The VFP analysis significantly increases in experimental trial (P < 0.05) during most days of the study with the exclusion of d3 and d14. To evaluate the seminal characteristics over time, the experiment was divided into T1 (d0-d5), T2 (d6-d10), and T3 (d11-d14) (P < 0.001) in evaluation of morphology and membrane functionality. The MNS reached 70% at d10 and finally 65% at d14, being considered normal and possibly fertile. With Host-s, 65% of MNS were also achieved at d14. The presence of glucose and fructose in the diluents used for refrigeration can exert very important effects given the fact that metabolic routes have been found in both sugars, providing both different and complementing effects. It can be concluded that the use of SA prior to artificial insemination improves the quality of chilled semen significantly, although it does not reverse the effects of deterioration due to cellular metabolism over time.

Sperm selection in IVF: the long and winding road from bench to bedside.

Spermatozoa wage battle to conquer fertilization but the traits needed to succeed remain elusive. The natural advantageous qualities that enable only a few selected sperm cells to reach the site of fertilization remain unknown. Although in vitro fertilization (IVF) facilitates the job of spermatozoa, a universally acceptable means of sperm selection is yet to be developed. No objective or reliable sperm quality indicators have been established and sperm selection is, to a great extent, based on subjective qualitative evaluation. The best method for sperm selection in IVF presents several challenges: intrinsic sperm qualities cannot be evaluated and the ideal endpoint for these studies is debatable. An ideal method for sperm selection in ART should be noninvasive and cost-effective, and allow the identification of high-quality spermatozoa and yield better outcomes in terms of pregnancy and live birth rates. This narrative review included 85 papers and focused on the new available methods and technologies that might shed some light on sperm selection in IVF. It discusses the available data on microfluidic devices, omics profiling, micronuclei studies, sperm plasma membrane markers, and other techniques, such as Magnetic Activated Cell Sorting (MACS), Raman micro-spectroscopy, and artificial intelligence systems. The new techniques herein reviewed offer fresh approaches to an old problem, for which a definite solution has yet to cross the bridge from bench to IVF clinics around the world, since clinical usefulness and application remain unproven.

Semen evaluation techniques and their relationship with fertility

This review summarizes those methods - established and emerging of semen assessment whose outcom e intents revealing its potential fertility and, as a carry over concept, that of the sire whose semen we examined. The review does not, however, focus on the wide display of current techn iques designed to explore specific or multiple sets of sperm attributes essential for fertilization but on two basic con cerns present: the heterogeneity of the sperm suspension and the multitude of attributes required for each spermatozoon to be fertile; concepts that shadow our diagnostic capabilities. The review points out advancements in the exploration of the genome, the transcriptome, and the proteome of both spermatozoa and the seminal plasma which unveil how spermatozoa modulate their own survival an d signal to the environment when displaying degenerative changes. Specific seminal plasma components, both among individuals and portions of the ejaculate, not only relate to survival but also signal differential immune tolerance by the female with a previ ously unattended linkage to fertility. Lastly it foresees how Cytomics, combining novel designed motility analyzers, flow cytometers and enhanced digital imaging shall dominate the landscape of andrological laboratories and enable quick determinations on huge sperm numbers for markers highly relevant to sperm function and hence, for fertility.(AU)

Semen evaluation techniques and their relationship with fertility

This review summarizes those methods - established and emerging of semen assessment whose outcom e intents revealing its potential fertility and, as a carry over concept, that of the sire whose semen we examined. The review does not, however, focus on the wide display of current techn iques designed to explore specific or multiple sets of sperm attributes essential for fertilization but on two basic con cerns present: the heterogeneity of the sperm suspension and the multitude of attributes required for each spermatozoon to be fertile; concepts that shadow our diagnostic capabilities. The review points out advancements in the exploration of the genome, the transcriptome, and the proteome of both spermatozoa and the seminal plasma which unveil how spermatozoa modulate their own survival an d signal to the environment when displaying degenerative changes. Specific seminal plasma components, both among individuals and portions of the ejaculate, not only relate to survival but also signal differential immune tolerance by the female with a previ ously unattended linkage to fertility. Lastly it foresees how Cytomics, combining novel designed motility analyzers, flow cytometers and enhanced digital imaging shall dominate the landscape of andrological laboratories and enable quick determinations on huge sperm numbers for markers highly relevant to sperm function and hence, for fertility.

Fluorescent probes: an advance on the evaluation of structural and functional sperm integrity / Sondas fluorescentes: um avanço na avaliação da integridade estrutural e funcional de espermatozoides

The semen quality is a determining factor for obtaining good results with reproductive biotechnologies, such as artificial insemination (AI). Thus, the evaluation of sperm parameters is essential to determine the fertility of gametes. Assessment techniques that best predict in vitro fertility of sperm have been relentlessly pursued with the purpose of ensuring better results after AI. However, until now, no evaluation methodology alone was efficient for this purpose. Then it is recommended to associate the use of these techniques to better predict the fertilizing ability of spermatozoa. In recent years a great number of new strategies were developed for semen evaluation, with the highlighted use of fluorescent probes. These techniques have limited use due to unfamiliarity with their employment. The aim of this paper is to review the advances on semen evaluation and prediction of their fertilising ability, with emphasis on those based on fluorescent probes.

A qualidade do sêmen é um fator determinante para se obter bons resultados com a utilização de biotécnicas reprodutivas como a inseminação artificial (IA). Desta forma, torna-se essencial a avaliação dos parâmetros espermáticos que determinem a fertilidade destes gametas. Técnicas de avaliação que melhor predigam a fertilidade in vitro dos espermatozoides têm sido buscadas incessantemente, a fim de garantir melhores resultados após IA. Entretanto, até o momento, nenhuma metodologia de avaliação mostrouse efi ciente para este fim, quando utilizada isoladamente, sendo recomendado o emprego conjunto destas técnicas para a melhor predição da capacidade fertilizante dos espermatozoides. Nos últimos anos uma série de novas estratégias foram desenvolvidas para avaliação de sêmen, dentre as quais se destaca o emprego das sondas fluorescentes. Estas técnicas são utilizadas com limitações em função do desconhecimento de seu emprego. Esta revisão tem como objetivo abordar os avanços na avaliação do sêmen e a predição de sua capacidade fertilizante, com ênfase nas que se baseiam no uso de sondas fluorescentes.

Fluorescent probes: an advance on the evaluation of structural and functional sperm integrity / Sondas fluorescentes: um avanço na avaliação da integridade estrutural e funcional de espermatozoides

The semen quality is a determining factor for obtaining good results with reproductive biotechnologies, such as artificial insemination (AI). Thus, the evaluation of sperm parameters is essential to determine the fertility of gametes. Assessment techniques that best predict in vitro fertility of sperm have been relentlessly pursued with the purpose of ensuring better results after AI. However, until now, no evaluation methodology alone was efficient for this purpose. Then it is recommended to associate the use of these techniques to better predict the fertilizing ability of spermatozoa. In recent years a great number of new strategies were developed for semen evaluation, with the highlighted use of fluorescent probes. These techniques have limited use due to unfamiliarity with their employment. The aim of this paper is to review the advances on semen evaluation and prediction of their fertilising ability, with emphasis on those based on fluorescent probes.

A qualidade do sêmen é um fator determinante para se obter bons resultados com a utilização de biotécnicas reprodutivas como a inseminação artificial (IA). Desta forma, torna-se essencial a avaliação dos parâmetros espermáticos que determinem a fertilidade destes gametas. Técnicas de avaliação que melhor predigam a fertilidade in vitro dos espermatozoides têm sido buscadas incessantemente, a fim de garantir melhores resultados após IA. Entretanto, até o momento, nenhuma metodologia de avaliação mostrouse efi ciente para este fim, quando utilizada isoladamente, sendo recomendado o emprego conjunto destas técnicas para a melhor predição da capacidade fertilizante dos espermatozoides. Nos últimos anos uma série de novas estratégias foram desenvolvidas para avaliação de sêmen, dentre as quais se destaca o emprego das sondas fluorescentes. Estas técnicas são utilizadas com limitações em função do desconhecimento de seu emprego. Esta revisão tem como objetivo abordar os avanços na avaliação do sêmen e a predição de sua capacidade fertilizante, com ênfase nas que se baseiam no uso de sondas fluorescentes.

Efeito de três diferentes diluidores sobre o sêmen canino submetido a dois protocolos de descongelamento / Effect of three different diluent on canine semen submitted to two thawing protocols

Com objetivo de avaliar o efeito de dois protocolos de descongelação do sêmen canino congelado em três diluidores foram coletados ejaculados de nove cães. Cada ejaculado foi dividido em 3 amostras, centrifugado e os pellets tratados sob diferentes protocolos: 1°- meio à base de glicina e gema de ovo; 2° - meio à base de TRIS e 3° - meio M9. Foram envasadas 28 palhetas de O,5ml segundo cada protocolo. Amostras tratadas sob cada protocolo foram avaliadas (M1) quanto a motilidade, vigor e integridade das membranas. As palhetas foram refrigeradas a 5°C por 60 min. e congeladas em N2. Uma amostra de cada protocolo foi avaliada quanto: motilidade, vigor e integridade das membranas após o equilíbrio (M2). Eram sempre descongeladas três pares de palhetas, sendo que cada par havia sido congelado sob um mesmo protocolo. Uma das palhetas de cada par era descongelada a 37°C por 30 segundos e outra a 72°C por 8 segundos. Cada grupo foi avaliado após o descongelamento (M3) quanto a motilidade e vigor espermático, avaliação computadorizada do movimento, integridade das membranas e avaliação acrossomal por meio de FitcPNA e PI. As amostras foram mantidas em banho Maria a 37°C por 1h para o teste de longevidade espermática e reavaliadas (M4) quanto à motilidade e vigor espermáticos, integridade das membranas. Após análise estatística concluímos que as células espermáticas caninas descongeladas a 72°C por 8 segundos apresentaram um maior somatório de bons resultados quanto aos testes de viabilidade espermática in vitro empregados.(AU)

To verify the effect of three different extenders and two thawingtemperatures on frozen-thawed canine sperm characteristics, oneejaculate from nine dogs were separately collected and processed (n=9).Each ejaculate was divided into 3 equal samples and centrifuged. Thepellets were re-suspended using: 1st pellet Glicyne-egg yolk extender(GEY), 2nd pellet - TRIS extender and 3rd pellet - M9 extender and allsamples were filled in 0.5ml straws. A total of 84 straws (28 for eachprotocol) were done. The sperm motility, vigour and plasmamembrane integrity from each protocol were immediately evaluated(M1) and the straws were brought to a refrigerator at 5ºC for 60minutes. After that, a sample from each protocol was warmed up ina water bath 37ºC for 5 min. and sperm motility, vigour and plasmamembrane integrity were evaluated (M2). The straws were frozen inliquid nitrogen. One straw from each protocol was thawed at 37ºCfor 30 sec and another at 72ºC for 8 sec and the sperm motility;vigour, CASA and plasmatic membrane integrity and acrossomalstatus using FITC-PNA stain were evaluated (M3). Plasma membrane,sperm motility and vigour were evaluated after a 1-hour incubation at37ºC (M4). Statistical analysis showed that the higher temperaturehad positive effect on froze-thawed canine sperm characteristics. Thebest results were taken when canine semen was frozen in GEYextender and thawed 72ºC for 8 sec. (AU)