A 2-step remote TUNEL approach for sperm DNA fragmentation assessment. Analysis in donors and patients.

OBJECTIVES: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters. STUDY DESIGN: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318). RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients. CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

OBJECTIVE: The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration. METHODS: Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers. RESULTS: The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%. CONCLUSIONS: Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

Separating the chaff from the wheat: antibody-based removal of DNA-fragmented sperm.

STUDY QUESTION: Is it possible to remove sperm with damaged DNA from a semen sample? SUMMARY ANSWER: By using immunomagnetic cell sorting that targets the sperm head-bound epididymal sperm-binding protein 1 (ELSPBP1), it was possible to produce an ELSPBP1(-) sperm fraction characterized by consistently lower levels of sperm DNA fragmentation (SDF). WHAT IS KNOWN ALREADY: In bovines, ELSPBP1 is bound to dead spermatozoa. Human ejaculates with high SDF have increased detected levels of sperm ELSPBP1 when compared to ejaculates with low native SDF. STUDY DESIGN, SIZE, DURATION: We recruited 267 patients who were referred to the clinic for conjugal infertility. After applying exclusion criteria, such as fever within 90 days of the study, history of systemic diseases, alterations or surgical interventions to the genital tract and use of cigarette or drugs, a total of 133 patients were included. A total of 52 samples were used for the evaluation of sperm ELSPBP1 levels (Sub-study 1), 41 samples for determination of ELSPBP1 location in human sperm (Sub-study 2), and 40 samples for immunomagnetic cell sorting targeting ELSPBP1, to produce ELSPBP1(-) (without ELSPBP1) and ELSPBP1(+) (with ELSPBP1) fractions (Sub-study 3). Samples were collected between July 2016 and September 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In Sub-study 1, sperm ELSPBP1 levels were assessed by western blotting. For Sub-study 2, ELSPBP1 was localized in sperm by immunocytochemistry. Finally, for Sub-study 3, sperm were selected based on incubation of semen samples with antibody-coated magnetic microspheres targeting ELSPBP1. Two fractions were produced (with or without ELSPBP1), and these sub-populations were submitted to an alkaline Comet assay for determination of SDF. MAIN RESULTS AND THE ROLE OF CHANCE: Men with high SDF presented higher sperm ELSPBP1 levels when compared to the control group (low SDF), while no difference between groups was observed in seminal plasma. ELSPBP1 was located in the head region of human sperm. The ELSPBP1(+) fractions presented high and variable levels of SDF, while their paired ELSPBP(-) fractions presented consistently low SDF. LIMITATIONS, REASONS FOR CAUTION: This work did not validate the levels of ELSPBP1 in other functional alterations of sperm, such as acrosome integrity or mitochondrial activity. Moreover, this is still a pre-clinical study, intended to demonstrate proof-of-concept that ELSPBP1 selects sperm with low DNA fragmentation; further investigation is warranted to demonstrate safety for use in ART. Sperm fractions were not assessed for sperm vitality. A clinical trial is still necessary for these findings to be extrapolated to outcomes in ART. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that ELSPBP1 is associated with sperm with higher levels of DNA fragmentation. The finding that the sperm membrane can reflect alterations in DNA integrity could give rise to a novel molecular method for sperm preparation prior to use of assisted reproductive procedures. Moreover, the detection of sperm-bound ELSPBP1 could serve as an indirect method for the determination of DNA fragmentation. STUDY FUNDING/COMPETING INTEREST(S): L.B.B. was a recipient of a Ph.D. scholarship from the Sao Paulo Research Foundation-FAPESP (process number 2016/05487-3). R.P.B. is a recipient of a Scientific Productivity scholarship from the Brazilian National Council for Scientific and Technological Development-CNPq (process number 306705/2017-6). The authors have no conflict of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Idiopathic subfertility in stallions. Clinical approaches to a complex problem

At least 30-40% of stallions in commercial breeding programs are moderately fertile and 8-12% are subfertile (0.5-3% with severe subfertility). From the total reported cases of the subfertility, in 2-20% of the stallions the cause is unknown or was not established. The objective of this work is to present the concept of subfertile stallion based on the current state of knowledge and advanced molecular diagnostic technologies. Low pregnancy rates have been reported in stallions with normal semen quality after conventional evaluation. Acrosome reaction (AR) is necessary for natural fertilization and impaired acrosome reaction (IAR) leads to subfertility or infertility in horses, however, AR test is not included in routine semen analysis. Genome-wide association study identified FKBP6 as a strong candidate gene responsible for this failure. The gene encodes for FK506 binding protein 6 (FKBP6) which is involved in sperm development and functions. We could conclude that the evaluation of the acrosomal status is essential in cases of stallions with good motility, concentration, morphology and viability but unexplained (idiopathic) subfertility or infertility. It is important to highlight the recent increase in reports of fertility problems in stallions related to disorders of genetic origin.(AU)

O que análises avançadas do sêmen podem contribuir de fato para a os resultados da IA em suínos? / What advanced semen analyzes can contribute to AI results in pigs?

O controle da qualidade seminal produzido por centrais e unidades de disseminação genética permite a maximização da produção de doses inseminantes por ejaculado, bem como organiza o acompanhamento da produtividade de cada macho. Este acompanhamento é crítico tanto quando o reprodutor jovem é introduzido na rotina de coleta de sêmen quanto na determinação do momento do descarte e reposição. Essencialmente, a avaliação do sêmen em suínos é a mesma há décadas, porém novas metodologias de análise individual da célula como a citometria de fluxo e de constituintes de tecidos e fluídos como a proteômica e metabolômica já estão trazendo avanços na andrologia animal. Na presente revisão, são abordados os aspectos técnicos, vantagens e limitações destas três análises avançadas aplicadas ao sêmen suíno, discutindo a sua implementação e impactos no manejo reprodutivo da espécie.(AU)

The quality control of the semen doses produced by artificial insemination centers and allows the maximization of the production of inseminating doses per ejaculate, as well as organizes the monitoring of the productivity of each male. This follow-up is critical when the young male is initiated in routine semen collection and in determining the moment of male replacement. Essentially, semen evaluation in pigs has been the same for decades, but new methodologies for individual cell analysis such as flow cytometry and tissue and fluid constituents such as proteomics and metabolomics are already bringing advances in animal andrology. In this review, the technical aspects, advantages, and limitations of these three advanced analyzes applied to swine semen are addressed, discussing their implementation and impacts on the reproductive management of the species.(AU)

Exame clínico andrológico em búfalos: particularidades e mercado / Clinical andrological examination in buffaloes: particularities and market

A bubalinocultura tem expandido mundialmente com grande importância socioeconômica na produção de leite, carne e na tração animal. No entanto, algumas características como: a sazonalidade, poucos reprodutores com genética superior para ser multiplicada e reduzido número de touros com sucesso de coleta constante de sêmen nas Centrais, dificultam a obtenção de maiores índices reprodutivos a nível de Brasil. Neste contexto, a eficiência reprodutiva do rebanho possui papel de destaque, consequentemente, o touro bubalino e a realização do exame andrológico são essenciais para o desenvolvimento da bubalinocultura. Apesar da realização de pesquisas ao longo dos anos relacionadas à andrologia em búfalos, na prática a campo ainda existe uma lacuna referente aos aspectos reprodutivos nessa espécie, fazendo com que haja uma demanda por conhecimento específico. Diante disso, objetivou-se abordar alguns dos principais aspectos da andrologia na espécie bubalina, para auxiliar o Médico Veterinário no atendimento clínico e andrológico visando alcançar uma boa eficiência reprodutiva.(AU)

Buffalo farming has expanded worldwide with great socioeconomic importance in the production of milk, meat and animal traction. However, some characteristics such as: seasonality, few sires with superior genetics to be multiplied and reduced number of bulls with constant semen collection success in the Centrals, make it difficult to obtain higher reproductive rates in Brazil. In this context, the reproductive efficiency of the herd plays a prominent role, consequently, the buffalo bull and the performance of the andrological examination are essential for the development of buffalo breeding. Despite conducting research over the years related to andrology in buffaloes, in field practice there is still a gap regarding reproductive aspects in this species, causing a demand for specific knowledge. In view of this, the objective was to address some of the main aspects of andrology in the buffalo species, to assist the veterinarian in clinical and andrological care in order to achieve good reproductive efficiency.(AU)

Reliability of the sperm chromatin dispersion assay to evaluate sperm deoxyribonucleic acid damage in men with infertility.

OBJECTIVE: To investigate the intraindividual agreement of the sperm chromatin dispersion (SCD) assay results to assess sperm DNA fragmentation (SDF) in men with infertility. DESIGN: Diagnostic test reliability study. SETTING: Andrology laboratories. PATIENT(S): A total of 219 men with infertility. INTERVENTION(S): Sperm DNA fragmentation assessment in two ejaculates of the same subjects within a 3-month interval, using the SCD assay performed and analyzed by the same observers under similar testing conditions. MAIN OUTCOME MEASURE(S): Cohen's κ statistics to assess the degree of agreement between the pairs of results after converting the nominal SCD values into categorical data, that is, normal (<20%), intermediate (21%-29%), and high (≥30%) SDF rates. We also assessed the pairs of results using reliability measures for numerical variables (intraclass correlation coefficient for consistency using the two-way mixed-effects model and Bland-Altman plots). RESULT(S): The degree of agreement in classifying patients according to normal and pathological SDF classes was overall substantial (κ = 0.632; 95% confidence interval [CI], 0.546-0.718). A total of 76.7% of individuals were classified under the same class using paired ejaculates. The agreement rate was highest (approximately 80%) in ejaculates initially classified as either normal or high and lowest (approximately 60%) among those with intermediate SDF levels. The frequency of intermediate SDF ejaculates downgraded to normal or upgrade to high SDF classes in the second test was similar (approximately 20%). The intraclass correlation coefficient was 0.856 (95% CI, 0.812-0.887), and the mean difference between the pairs of observations was 0.80% (95% CI, -0.72 to 2.23), indicating no systematic difference between paired observations. CONCLUSION(S): Our study indicates a substantial intraindividual agreement of paired SCD assay results to classify men with infertility into three SDF categories: normal, intermediate, and high. The reliability of the SCD assay was adequate and exceeded 0.80 using two ejaculates analyzed within a 3-month interval under similar conditions. Although this evidence overall supports a single SCD test for patient classification using predefined SDF thresholds, particularly when the first test shows normal or high SDF levels, one in four men will have discordant values in paired ejaculates. Clinicians should be judicious when using SDF test results in decision-making.

Quais são as dúvidas no momento do exame clínico andrológico bovino? / What are the doubts at the time of the bovine andrological clinical examination?

O exame clínico andrológico é a ferramenta de eleição utilizada para avaliar a fertilidade de um reprodutor. Normalmente utilizado em tourinhos jovens na pré-comercialização, como uma forma de garantia reprodutiva; no período pré-estação de monta; quando existem falhas ou desordens reprodutivas nos rebanhos; prévio ao ingresso nas centrais de inseminação; na determinação da puberdade, dentre outras. O exame clínico andrológico deve ser realizado por profissional com experiência suficiente para tomada de decisões sobre o real potencial reprodutivo dos animais, porém, em muitas ocasiões mesmo um profissional capacitado defronta-se com dúvidas, entre elas destacam-se as questões relacionadas ao valor cobrado e "tipo de exame"; a biometria testicular; exigências relacionadas a impotencia coeundi; e o que é necessário no certificado do exame. Buscou-se elencar, explanar e discutir essas questões em vista de diminuí-las e/ou encontrar possíveis soluções. O exame clínico andrológico é uma etapa fundamental e complexa para a seleção de reprodutores, e apesar de constantemente debatida, ainda permanecem lacunas a serem respondidas, visto que os profissionais necessitam de constante atualização.(AU)

The clinical andrological examination is the tool of choice used to assess the fertility of a sire. Usually used in young bulls in pre-marketing, as a form of reproductive guarantee; in the pre-mounting season period; when there are reproductive failures/disorders in herds; before entering insemination centers, in determining puberty, among others. The clinical andrological examination must be carried out by a professional with sufficient expertise to make decisions about the real reproductive potential of these animals, however, on many occasions even a trained professional is faced with doubts, among them the issues related to the amount charged and "type of exam"; testicular biometry; requirements related to impotencia coeundi; and what is required in the exam certificate. Based on the above context, we sought to list some possible doubts, explaining them and discussing them among veterinarians, so that such issues could be reduced and/or possible alternative solutions could be defined. The clinical andrological examination is a fundamental and complex step for the selection of sires, and despite being constantly debated, there are still gaps to be answered, since professionals need constant updating.(AU)

Avanços na andrologia de animais selvagens / Advances in wild animal andrology

As tecnologias de reprodução assistida (TRA) são de fundamental importância para a conexão de indivíduos em diferentes localidades, facilitando assim o intercâmbio genético e favorecendo a variabilidade genética de uma espécie. Por esta razão, as TRAS podem ser ferramentas importantes para a conservação de espécies ameaçadas de extinção. Apesar dos esforços nas últimas décadas, o avanço no desenvolvimento de tais tecnologias está aquém à urgência de reverter processos de baixa variabilidade genética em algumas espécies. A necessidade de refinamento das técnicas para as particularidades fisiológicas e comportamentais de cada espécie, somada à raridade de acesso aos animais são os principais fatores relacionados as dificuldades em se avançar com as TRAS. As técnicas mais recentes desenvolvidas para a recuperação de espermatozoides em animais selvagens são a colheita farmacológica, com uso de alfa-2-agonistas e a criopreservação / vitrificação testicular com posterior cultivo. Pouco de avançou, no entanto, em relação aos métodos de criopreservação, prevalecendo associação clássica de TRIS-gema-glicerol. Discutimos, então os métodos usados para acesso ao gameta masculino em espécies selvagens e suas aplicações na conservação animal.(AU)

Assisted reproduction technologies (ART) are of fundamental importance for connecting individuals in different locations, thus facilitating genetic exchange and favoring the genetic variability of a species. For this reason, TRAS can be important tools for the conservation of endangered species. Despite efforts in recent decades, the advance in the development of such technologies is short of the urgency of reversing processes of low genetic variability in some species. The need to refine the techniques for the physiological and behavioral particularities of each species, added to the rarity of access to animals, are the main factors related to the difficulties in advancing with TRAS. The most recent techniques developed for sperm collection in wild animals are pharmacological collection, with the use of alpha-2-agonists and testicular cryopreservation / vitrification with subsequent cultivation. Little progress has been made, however, in relation to cryopreservation methods, prevailing the classic association of TRIS-yolk-glycerol. We therefore discuss the methods used to access the male gamete in wild species and their applications in animal conservation.(AU)

Tourinhos genômicos: relação da qualidade e congelabilidade do sêmen / Genomic young bulls: relationship between semen quality and freezeability

A grande demanda por sêmen congelado para inseminação artificial no Brasil está ocasionando que criadores, programas genéticos, e centros de coleta e processamento de sêmen produzam touros cada vez mais jovens para doação de sêmen. A avaliação genômica é uma nova ferramenta para a seleção de touros jovens para os centros de coleta, porém, junto vieram os problemas na seleção de bons doadores de sêmen que muitas vezes não atingem a puberdade na idade desejada, principalmente na raça Nelore que é a mais importante para o mercado de inseminação no país. A maioria dos problemas com a produção de sêmen do touro jovem genômico podem ser resolvidos com o tempo, mas ainda ocasiona vários problemas não resolvidos. Os touros jovens genômicos da raça Nelore atingem a puberdade entre 13 a 16 meses e os touros jovens das raças Holandesa e Angus entre 10 a 12 meses de idade sendo que a maturidade sexual no geral é alcançada entre 18 a 24 meses quando passam a produzir sêmen rotineiramente e produzem grandes quantidades de unidades de sêmen criopreservado para uso na inseminação artificial. Este cenário ocasionará um problema para o mercado da inseminação artificial, com a demanda de mais touros com maturidade sexual.(AU)

The great demand for frozen semen in the artificial insemination in Brazil is causing breeders, genetic programs, and semen collection and processing centers to produce ever younger bulls for semen donation. The genomic evaluation is a new tool for the selection of young bulls for the collection centers, however, along came the problems in the selection of good semen donors that often do not reach puberty at the desired age, especially in the Nellore breed, the most important for the insemination market in the country. Most problems with genomic young bull semen production can be resolved over time, but there are still several unresolved problems. Young genomic Nellore bulls reach puberty between 13 to 16 months and young Holstein and Angus bulls between 10 to 12 months of age and sexual maturity is generally reached between 18 to 24 months when they start to produce semen routinely, so producing a large quantities of cryopreserved semen units for use in artificial insemination. This will cause a problem for the artificial insemination business where more sexually mature bulls will be needed.(AU)

Estratégias para melhorar a qualidade seminal em garanhões / Strategies to improve seminal quality in stallions

A grande quantidade de garanhões com alteração na qualidade seminal, seja pela idade, genética ou afecções do trato reprodutivo, resultou nos últimos anos, no rápido avanço do desenvolvimento de técnicas para recuperar e aumentar o potencial reprodutivo dos animais. Os detalhes antes e após a colheita seminal são fundamentais para alcançar o máximo da eficiência reprodutiva e obter índices de concepção adequados. Objetiva-se com esta revisão de literatura abordar as principais técnicas para melhorar a qualidade espermática em garanhões.(AU)

The large number of stallions with alterations in seminal quality, whether due to age, genetics or reproductive tract disorders, has resulted in the rapid advance of the development of techniques to recover and increase the reproductive potential of animals in recent years. Details before and after seminal collection are essential to achieve maximum reproductive efficiency and obtain adequate conception rates. The aim of this paper was to review the main techniques to improve sperm quality in stallions.(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)

Combination of nanoparticle green tea extract in tris-egg yolk extender and 39 °c thawing temperatures improve the sperm quality of post-thawed Kacang goat semen

Kacang goats are small ruminants produced by low-income households in smallholder and farm to reduce poverty and prevent undernutrition. Studies to find a cryopreservation protocol for Kacang goat semen are expected to multiplication of genetically superior animals selected by the paternal lineage. This study evaluated the effect of thawing temperature and supplementation of the green tea extract nanoparticle in skim milk-egg yolk (SM-EY) extender on post-thaw sperm quality of Kacang goat semen. Six ejaculates of Kacang goat were diluted in SM-EY supplemented or not (control group) with 0.001 mg/mL NPs GTE. The diluted semen was packaged with 0.25 mL straws (insemination dose: 60x106 sptz/mL) and cryopreserved. Then, six samples of the control group and NPs GTE groups were thawed at 37°C or 39°C sterile water for 30 s and submitted to sperm quality evaluations. The sperm viability, motility, and intact of the plasma membrane (IPM) were higher (p<0.05) in NPs GTE group than control group. In contrast, the NPs GTE group presented lower (p<0.05) malondialdehyde levels and sperm DNA fragmentation (SDF) compared with the control group. The catalase levels were not significantly different (p > 0.05) between the control and NPs GTE groups. Thawing at 39°C resulted in higher (p<0.05) sperm viability, motility, and IPM than thawing at 37°C. However, thawing at 39°C group presented lower (p<0.05) malondialdehyde levels compared with thawing at 37°C. SDF and catalase levels were similar (p>0.05) between thawing at 37°C and thawing at 37°C. In conclusion, supplementation of 0.001 mg/mL of NPs GTE in SM-EY extender and thawing temperature of 39°C resulted in a better quality of frozen-thawed Kacang goat semen.(AU)

Efficiency of CellROX deep red® and CellROX orange® fluorescent probes in identifying reactive oxygen species in sperm samples from high and low fertility bulls.

Detection of reactive oxygen species (ROS) is of great interest in semen analysis since their excess is detrimental to sperm function and male fertility. Fluorescence microscopy has achieved attention for providing broad possibilities of sperm evaluations and also for presenting substantial accessibility. In this context, this study investigated the efficiency of CellROX Deep Red® and Orange® probes in detecting ROS in bovine sperm cells and assessed their relationship with sperm fertility potential. First, 16 ejaculates were assigned in three treatments: T0 (no ROS production induced), T1x (ROS production induced once) and T2x (ROS production induced twice). Samples were incubated with Red and Orange probes and percentages of cells producing ROS were evaluated using fluorescence microscopy. Coefficient of determination was 0.61 for Red and 0.56 for Orange. Afterwards, frozen-thawed semen samples from high and low fertility bulls were evaluated regarding percentages of cells producing ROS detected by Red and Orange. Higher levels of ROS assessed by Red were detected in low fertility bovine samples. In conclusion, CellROX Red® and Orange® are both efficient in detecting ROS in bovine spermatozoa. Furthermore, higher sperm ROS detection by CellROX Red® might be associated with low fertility samples.

Investigating a method for pharmacologic semen collection in alpacas

While semen evaluation is standard practice prior to a sale or when infertility is suspected in other species, it is rarely done in camelids due to the difficulties involved in collecting a sample. The reproductive physiology of alpacas differs to that of other domestic animals and is still poorly understood. In the stallion, a technique was developed for semen collection that pharmacologically induces ejaculation without copulation (ex copula). This study investigates whether semen could be reliably collected by ex copula ejaculation in male alpacas. Eleven male Huacaya alpacas were used in this study, and six ex copula treatment protocols were evaluated: (1) saline (control); (2) xylazine only (0.1 mg/kg); (3) xylazine only (0.2 mg/kg); (4) imipramine only (1.0 mg/kg); (5) imipramine (1.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg); and (6) imipramine (2.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg). Each treatment protocol was repeated two to five times. Azoospermic samples obtained from ex copula ejaculation contained numerous epithelial cells but no sperm. A reliable treatment for pharmacologically inducing ejaculation in alpacas remains to be found.(AU)

Desafios e estratégias para identificação de machos subférteis em rebanhos suínos / Challenges and strategies for identification of subfertile boars

A subfertilidade de reprodutores suínos pode alcançar até 15% dos machos em um plantel, gerando perdas econômicas pela menor taxa de parto e menor número de leitões nascidos totais. O uso de doses ou inseminações heterospérmicas e o elevado número de espermatozoides para compensar características espermáticas do ejaculado, dificultam o diagnóstico desses machos na rotina produtiva. Além disso, há uma baixa associação dos exames espermáticos realizados nas centrais de produção de sêmen com os resultados in vivo. Ao longo dos anos, avaliações com a implementação dos sistemas computadorizados de análise de sêmen, proteômica do plasma seminal ou célula espermática, inseminação artificial em tempo fixo e a classificação dos machos pela capacidade de preservação das doses de sêmen durante o armazenamento foram metodologias utilizadas para classificar e identificar machos subférteis. O conhecimento de estratégias para classificação dos machos quanto a fertilidade pode auxiliar na tomada de decisão dos reprodutores a serem descartados e otimização dos melhores machos, proporcionando melhorias no desempenho reprodutivo.

Boar subfertility reach up to 15% of the males in a herd, which represent economic losses due to the lower farrowing rate, and fewer total piglets born. The use of heterospermic semen doses, heterospermic inseminations and the high number of sperm cells is used to compensate sperm characteristics of the ejaculates making difficult to identify these males in the productive routine. Furthermore, there is a low association of sperm analysis performed in the boar studs with in vivo results. Over the years, with the implementation of computerized systems for semen analysis, proteomics of seminal plasma or sperm cell, fixed-time artificial insemination, and classification of boars based on capacity for semen preservation during storage, were methodologies used to classify and identify subfertile boars. The knowledge of strategies for classification of boars regarding fertility aid the decision for culling or for optimizing the best boars allow an improvement on the reproductive performance. can help in the decision-making of breeders to be discarded and those where the user should be optimized, thus providing improvements in reproductive performance.

Técnicas alternativas para colheita de sêmen de cão / Alternative dog semen collection techniques

Métodos eficazes para a colheita de sêmen permitem avanços na inseminação artificial em cães domésticos e canídeos selvagens geneticamente valiosos, permitindo a manutenção de linhagens genéticas e possibilitando a propagação de material genético post-mortem, bem como, diminuindo os custos e os riscos de transporte de animais para reprodução. Dentre os métodos de colheita de sêmen utilizados em cães, podemos destacar os métodos ejaculatórios como a manipulação digital e a eletroejaculação; e os métodos não ejaculatórios, que compreendem aqueles que envolvem os espermatozóides coletados do epidídimo, em casos de óbito ou orquiectomia ou em animais submetidos a vasectomia. Mais recentemente a colheita farmacológica de sêmen foi descrita em cães como um método não ejaculatório eficiente. Essa nova metodologia de colheita de sêmen surge como mais uma alternativa para facilitar a colheita de sêmen em cães domésticos que não aceitam a manipulação digital e eliminar a necessidade da eletroejaculação em animais selvagens. No entanto, os protocolos anestésicos utilizados ainda apresentam alguns efeitos colaterais indesejáveis, indicando que novos protocolos, mais seguros para a colheita de sêmen via cateterismo uretral necessitam ser testados.

Effective methods for semen collection are determinant for artificial insemination advances in genetically valuable domestic dogs and wild canids, allowing the genetic maintenance and enabling the post-mortem genetic material use, as well as, reducing the animals transporting costs and risks for breeding. The semen collection methods used in canids are ejaculatory methods, such as digital manipulation in dogs and electroejaculation in wild canids; and non-ejaculatory methods, which include sperm collected from epididymis after death or vasectomy. More recently, pharmacological semen collection was described in dogs as an efficient non-ejaculatory method. This new semen collection methodology emerges as another alternative to facilitate semen collection in domestic dogs that do not accept digital manipulation and eliminate the electroejaculation in wild animals. However, side effects undesirable was observed indicating that new anesthetic protocols, safer for semen collection via urethral catheterization, need to be tested.

Diagnosis and management strategies for urospermia in stallions / Diagnóstico e estratégias de manejo na urospermia em garanhões

Urospermia, contaminação do sêmen com urina durante a ejaculação, é considerada a segunda causa mais frequente de disfunção ejaculatoria em garanhões. A presença de urina no sêmen pode afetar drasticamente a fertilidade através do pH alcalino e alta osmolaridade da urina, além de causar uma potencial inflamação uterina após a cobertura ou inseminação artificial. Esta condição pode estar associada a outras doenças como cistite, herpesvírus equino-1 e paralisia periódica hipercalêmica; no entanto, causas idiopáticas são as mais comuns. Por esse motivo, o tratamento da urospermia pode ser frustrante e o prognóstico para a resolução completa dessa condição costuma ser desfavorável. Garanhões que sofrem de urospermia são frequentemente diagnosticados após resultados frustrantes de fertilidade e baixa qualidade do sêmen. O diagnóstico de urospermia é baseado na aparência amarela e odor típico, presença de cristais de urina, pH alcalino, além do aumento da creatinina e níveis de nitrogênio. O tratamento da urospermia é desafiador, já que a maioria dos casos é idiopática, limitando as opções terapêuticas. No entanto, quando uma condição primária é diagnosticada, ela deve ser tratada para tentar resolver a causa da urospermia. Na maioria dos casos, as terapias direcionadas à redução da contaminação da urina incluem a redução da quantidade de urina na bexiga antes da cobertura ou coleta de sêmen, tratamento farmacológico para auxiliar o fechamento do colo da bexiga durante a ejaculação, ou a coleta apenas da porção rica em esperma através da coleta fracionada do ejaculado. O método mais simples e estabelecido para o manejo da urospermia é encorajar o garanhão a urinar antes da coleta de sêmen ou cobertura natural. No entanto, quando a coleta de sêmen livre de urina não é possível, o sêmen contaminado com urina pode ser processado para minimizar os efeitos adversos do pH e da osmolaridade ao esperma. O sêmen contaminado com urina deve ser diluído com diluente à base de leite no intuito de diminuir os efeitos deletérios da urina sobre os espermatozoides. A centrifugação com gradiente de densidade a fim de selecionar os espermatozoides com características superiores também é uma alternativa nesses casos. Além disso, a criopreservação de sêmen contendo baixos níveis de contaminação com urina pode ser realizada.

Urospermia is the contamination of semen with urine during ejaculation. Urospermia is the second most frequent ejaculatory dysfunction of stallions. It can drastically affect fertility mediated by alkaline pH, the high osmolarity of urine, and presumably excessive post-breeding inflammatory response. This condition can be associated with many other diseases (i.e., cystitis, equine herpesvirus-1, and hyperkalemic periodic paralysis); however, idiopathic causes appear to be predominant. For this reason, the treatment of urospermia can be frustrating, and the prognosis for complete resolution of this condition is often poor. Stallions suffering from urospermia are usually diagnosed after poor semen quality and fertility results. Diagnosis of urospermia is based on the yellow appearance, urine smell, urine crystal, alkaline pH, increased creatinine, and urea nitrogen levels. The treatment for urospermia is challenging. Most cases are idiopathic, thereby limiting therapeutic options. However, when a primary condition is diagnosed, it should be treated to solve the cause of urospermia. In most cases, therapies directed at reducing urine contamination include reducing the amount of urine in the bladder before breeding, pharmacological treatments to enhance bladder neck closure during ejaculation, or collecting only the sperm-rich portion of the ejaculate using an open-ended artificial vagina are used to manage stallions with urospermia. The simplest and most established method for managing urospermia is to encourage the stallion to urinate before semen collection or natural breeding. However, when the collection of semen free from urine is not possible, urine-contaminated semen can be processed to minimize the adverse effects of pH and osmolarity on the sperm. Semen contaminated with urine should be immediately extended with a milk-based extender to mitigate the deleterious effects of urine on the sperm. Single-layer gradient centrifugation can also be used to select sperm with superior traits for low urine contamination. In addition, semen cryopreservation can be performed in stallion semen with a low level of urine contamination.

Produção de Sêmen em Regime de Central – Muito além do espermograma / Semen production in Semen Collection and Processing Centres – Much more than an ordinary sperm evaluation

O mercado de inseminação artificial tem crescido de maneira exponencial nos últimos anos, resultando no inevitável aumento da demanda por sêmen das mais diferentes raças bovinas. Dessa forma, o número de touros em Centrais de Coleta e Congelação de Sêmen (CCCS) tem acompanhado o crescimento do mercado, sendo registrados recordes de produção de doses de sêmen bovino no país. No entanto, mesmo nesse cenário de crescente expansão, diferentes fatores como a própria idade dos reprodutores em coleta, problemas clínico-reprodutivos, estresse e dificuldades de adaptação ambiental e de manejo podem comprometer a plena produção de touros em regime de Central, justificando a abordagem desses temas no presente trabalho. Adicionalmente, algumas estratégias de manejo, diagnóstico ou tratamento de patologias reprodutivas de touros encontram-se abordadas nessa obra, denotando que a produção de sêmen de qualidade em CCCS vai muito além da condução do espermograma bovino.

The artificial insemination market in cattle has developed throughout the years, creating impact and greater demand for cattle semen production in several breeds. Furthermore, there was an increase in number of bulls in semen collection and processing centres (SCPC), followed by a record on production of frozen-thawed semen recently. Despite of this promising scenario, there are important factors that can affect semen production of bulls under SCPC regime, such as bull’s age, clinical and reproductive health, management of bulls and its association with stress and environmental adaptation. This study presents strategies for management, diagnosis, and treatment of reproductive pathologies, showing that production of quality semen in bovine SCPC can be affected by several factors which cannot be captured through the ordinary sperm evaluation.

Selenium supplementation prevents DNA damage in ram spermatozoa / Suplementação com selênio previne danos ao DNA espermático de ovinos

In the present study, we aimed to evaluate the effects of different concentrations of selenium (Se) ovine nutritional supplementation on spermatozoa DNA integrity. Thirty male ovines (age: 10 months) were used. They were fed with hay and ram food in an intensive system, which was divided into stalls (5 m long and 3 m wide) with feeding troughs, and had ad libitum access to food and water. Ovines in group 1 (G1, the negative control) received mineral salt supplementation without Se; ovines in G2 received the same mineral salt mixed with 5 mg Se (as sodium selenite)/kg mineral supplement;ovines in G3 received 10 mg Se/kg mineral supplement; ovines in G4 received 15 mg Se/kg mineral supplement; and ovines in G5 received 20 mg Se/kg mineral supplement. Ovines in all groups remained untreated for 14 days, followed by a treatment period of 56 days. Semen samples were obtained by electroejaculation. The DNA damage in semen samples was evaluated using the comet assay. The experimental design was implemented using a 5 × 5 Latin Square, i.e., five treatments and five experimental periods. The mean differences were compared using Tukeys test at a significance level of 5%. The control group (G1) showed a high percentage of DNA damage compared to the Se-treated groups (G2-G5). Therefore, Se supplementation could decrease the basal level of DNA damage in sperm cells, suggesting that Se might exert protective effects on sperm DNA.(AU)

O presente estudo teve por objetivo avaliar os efeitos da suplementação mineral com diferentes concentrações de selênio (Se) sobre a integridade de DNA espermático de ovinos. Utilizaram-se 30 machos, com 10 meses de idade. Eles foram mantidos em sistema intensivo, sendo alimentados com feno e ração própria para ovinos, divididos em baias (5 m x 3 m), com cochos e água ad libitum. Os ovinos do grupo 1 (G1=controle negativo) receberam suplementação de sal mineral sem a adição de Se, os animais do G2 receberam a mesma mistura mineral, porém com 5 mg de Se (selenito de sódio)/kg mistura mineral, os ovinos do G3 receberam 10 mg Se/kg mistura, os animais do G4 receberam 15 mg Se/kg mistura, os do G5 receberam 20 mg Se/kg mistura. Os ovinos de todos os grupos passaram por um período de adaptação de 14 dias, seguido por um período de tratamento de 56 dias. O sêmen foi colhido por meio de eletroejaculação. A integridade do DNA espermático foi avaliada por meio do teste de cometa. O modelo experimental utilizado foi Quadrado Latino 5 x 5, com cinco grupos e cinco períodos experimentais. A diferença entre as médias foi analisada pelo teste de Tukey, com 5% de nível de significância. O grupo controle (G1) apresentou elevada porcentagem de danos quando comparada aos demais grupos de tratamentos (G2 a G5). Portanto, a suplementação de Se diminui o nível de danos ao DNA espermáticos, sugerindo que o Se pode exercer efeitos protetores sobre o DNA dos espermatozoides de ovinos.(AU)