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Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

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Estudos sobre a fisiologia espermática demonstram que padrões de fertilidade e funcionalidade espermática pós-criopreservação possuem relação importante com a eficiência do metabolismo energético destas células, bem como com a sua capacidade de manter a homeostase oxidativa. Os conhecimentos sobre a relação entre perfil fisiológico de espermatozoides e fertilidade foram e, ainda estão sendo, aprimorados, com o uso de análises de perfis moleculares, com destaque para a metabolômica. As análises moleculares permitiram a identificação de classes de metabólitos importantes na fisiologia espermática, bem como de potenciais biomarcadores de fertilidade, inclusive em bovinos. No entanto, ainda não há disponível uma avaliação isolada capaz de estimar o padrão de fertilidade de amostras seminais. Há vários desafios a serem superados para a validação de biomarcadores de fertilidade, principalmente considerando-se as diferenças entre perfis metabólicos de raças distintas de touros e a heterogeneidade dos ejaculados. A superação destes desafios pode ser iniciada com um maior aproveitamento dos resultados já obtidos e futuros, com a aplicação de análises mais avançadas e com eficácia em elevado número de dados. Para tal, podem ser utilizados modelos estatísticos de inteligência artificial, cuja aplicação pode aumentar a acurácia das observações obtidas, bem como aproximá-las da aplicação pelo setor de produção animal.(AU)

Studies on sperm physiology demonstrate that fertility outcomes and sperm post-cryopreservation have an important relation with sperm energy metabolism efficiency and ability to maintain oxidative homeostasis. Knowledge on sperm physiology has been enhanced, continually, with the application of molecular profiling analysis, focusing on metabolomics. Molecular analysis allowed the identification of important classes of metabolites in sperm physiology, as well as potential fertility biomarkers, including in bovine. Despite all developments, there is still no isolated assessment available capable of estimating fertility on sperm samples. There are several challenges to be overcome for the validation of fertility biomarkers, especially considering the metabolic profiles differences between bull breeds and the ejaculate heterogeneity. Overcoming these challenges could start with the application of more advanced and effective data analysis from research database already obtained, as well as future ones. To this end, statistical models of artificial intelligence can be used, whose application can increase the accuracy of the observations obtained, as well as bringing them closer to the application by the animal production sector.(AU)

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Benign Prostatic Hyperplasia (BPH) is a pathological condition that directly interferes with the reproductive potential of senile dogs, by leading to prostate enlargement and sperm injury, which in turn may compromise sperm freezeability. Moreover, albeit finasteride treatment reduces prostatic volume and blood supply and maintains seminal quality and testicular integrity, the effects of sperm samples submitted to cryopreservation after the finasteride treatment are still unknown. Thus, the aim of this study was to evaluate frozen-thawed semen of BPH dogs, as well as dogs subjected to BPH pharmacological treatment with finasteride. For such purpose, 20 dogs were previously selected and assigned to three experimental groups, according to BPH diagnosis and treatment with finasteride: Control (n = 9), BPH Group (n = 5) and BPH-Finasteride Group (n = 6). Semen was subjected to one-step cryopreservation protocol with tris-fructose-citric acid extender with 5% glycerol and thawed at 37°C for 30 sec. Fresh and post-thaw sperm samples were evaluated for macroscopic parameters, sperm concentration, sperm motility kinetics, sperm mitochondrial activity and potential, oxidative stress, plasmatic and acrosome membrane integrity, sperm DNA fragmentation and sperm binding test on perivitelic membrane of chicken egg yolk. Regarding fresh semen, BPH-Finasteride group had the lowest ejaculate visual aspect (opacity), higher frequency of sperm flagellar beating (BCF) and percentage of sperm with medium velocity. Control group had the highest percentage of sperm DNA integrity compared to BPH group. For the frozen-thawed semen, Control group presented the highest percentage of spermatozoa with high mitochondrial activity. However, the BPH-Finasteride group showed higher number of sperm bound to the perivitelline membrane of chicken egg yolk compared to the BPH Group. Conversely, BPH group had higher percentage of DNA damage. In conclusion, the ejaculate of BPH dogs has higher susceptibility to cryoinjury, whereas finasteride-treated dogs have increased spermatozoa functional performance, suggesting a promising use of BPH dogs as semen donors in sperm cryopreservation programs.

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Semen quality is one of the criteria used for the selection of bulls with relatively greater fertility. In addition, bull fertility depends on the integrity and function of all sperm structures. The aim of this study, therefore, was to determine associations when there was conducting of conventional and functional techniques for the evaluation of sperm samples from bulls with known fertility history as determined when semen from these bulls was used for fixed-time artificial insemination programs. The study was designed in a 2 × 2 factorial arrangement, with one factor being breed (Angus x Nellore) and the other fertility (greater x lesser). Greater fertility groups were composed of ten Angus and 11 Nellore bulls, while lesser fertility groups were composed of ten Angus and seven Nellore bulls. Sperm were analyzed, in four cryopreserved distinct batches for each animal, for morphology, kinetics, plasma and acrosomal membrane integrity, mitochondrial activity and mitochondrial membrane potential, DNA integrity and oxidative status. There was no difference in characteristics commonly used in sperm quality conventional analysis. The results from functional analysis indicated an important association between mitochondrial dysfunctions, oxidative stress, and damage to sperm structures in lesser fertility bulls. Greater fertility bulls had greater sperm quality and indicators of functional cell structures. The associations, when there were evaluations using different techniques, indicate the importance of evaluation and correlation between different sperm functions to understand effects of distinct parameters on sperm fertilization capacity.

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Although bovine embryo in vitro production (IVP) is a common assisted reproductive technology, critical points warrant further study, including sperm traits and oxidative status of sperm for in vitro fertilization (IVF). Our aim was to evaluate whether the lipid peroxidation index of commercial bull semen is influenced by sperm traits and oxidative status of sperm populations selected using Percoll® gradient. Semen straws from 48 batches from 14 Nelore bulls were thawed individually, analyzed for motility and subjected to Percoll selection. After Percoll, the lipid peroxidation index of the extender was evaluated, whereas selected sperm were analyzed for motility, acrosome and membrane integrity, mitochondrial membrane potential, chromatin resistance and oxidative potential under IVF conditions. Batches were divided retrospectively in four groups according to lipid peroxidation index. Sperm from Group 4 with the lowest index of lipid peroxidation had, after Percoll selection, greater plasma membrane integrity (81.3%; P = 0.004), higher mitochondrial potential (81.1%; P = 0.009) and lower oxidative potential (135.3 ng thiobarbituric acid reactive substances (TBARS)/ml; P = 0.026) compared with Group 1 with highest lipid peroxidation index (74.3%, 73% and 213.1 ng TBARS/ml, respectively). Furthermore, we observed negative correlations for the lipid peroxidation index with motility, membrane integrity and mitochondrial potential, and positive correlations with oxidative potential. In conclusion, oxidative stress in semen straws, as determined using lipid peroxidation in the extender, is associated with sperm traits and their oxidative potential under IVF conditions. These results provided further insights regarding the importance of preventing oxidative stress during semen handling and cryopreservation, as this could affect sperm selected for IVF. Finally, Percoll selection did not completely remove sperm with oxidative markers.

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The giant anteater (Myrmecophaga tridactyla) is being threatened by natural habitat destruction and fragmentation, illegal hunting and road kills. In this context, the generation of basic information on the reproductive parameters of this species is vital, aiming to improve reproductive management via, amongst others, assisted reproductive technologies. This study aimed to describe the morphological and functional features of semen collected from captive giant anteaters. Electroejaculation was performed in 13 animals housed in zoos located in São Paulo state, Brazil. Semen samples were collected from 13 animals in 16 procedures. Samples were evaluated for volume, motility, vigor, pH, concentration, sperm morphology, and functional tests. The following mean values were obtained: volume 1.28 ± 0.27 mL; motility 28.3 ± 6.2%; vigor 2.4 ± 0.25; concentration 129.4 ± 36.1 × 106 sperm/mL; pH 7.4 ± 0.2. Total acrosome, head, midpiece, and tail sperm abnormalities were 3.2 ± 0.8%, 25.4 ± 3.6%, 20.7 ± 3.2%, and 14.7 ± 2.6%, respectively. Intact acrosome was found in 83.7 ± 3.1% and intact membrane in 81.1 ± 4.0% of all samples collected. Mitochondrial activity was 66.4 ± 6.0% (Class I), 18.7 ± 2.9% (Class II), 8.0 ± 2.0% (Class III), 3.9 ± 1.0% (Class IV), and 3.0 ± 0.9% (Class V). Sperm DNA fragmentation rate was 13.2 ± 3.7%. These results indicated that electroejaculation is a feasible method for semen collection in giant anteaters, allowing a more detailed description of the semen in this species.

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The female reproductive tract, in particular the composition of the uterine and oviduct fluids, is responsible, at least in part, for triggering sperm cell modifications, essential for the acquisition of fertilization ability. Hyaluronic acid (HA) is a glycosaminoglycan present in these fluids, and its role in the fertilization process and sperm functionality is still barely understood. This work was designed to (a) determine the rheological characteristics of the fertilization medium by the addition of HA and (b) determine the HA influence on sperm motility and functional status. To that end, the in vitro fertilization medium was supplemented with 4 doses of HA (6, 60, 600 and 6,000 µg/ml) and analysed for viscosity and adhesion strength characteristics. Then, thawed semen from 6 bulls were incubated in these media and assessed at 4 different moments for morphological and functional parameters (plasma and acrosomal membrane integrities, mitochondrial membrane potential, capacitation, acrosomal reaction, and motility). The rheological evaluation showed that the addition of HA was able to increase both the viscosity and the adhesion strength of the fertilization medium, especially in the 6,000 µg/ml group in which the effect was more pronounced. No influence of HA could be observed on mitochondrial potential, and acrosomal and plasma membrane integrities. However, HA supplementation, at lower doses, led to an increase in the number of reacted sperm, as well as changes in motility parameters, with increase in the number of motile, rapid and progressive spermatozoa. In conclusion, the addition of HA alters the rheological properties of the fertilization medium and leads to the improvement of the properties related to sperm motility and capacitation, without compromising other functional aspects of the cell.

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The golden-headed lion tamarin (Leontopithecus chrysomelas) is an endangered primate that, according to timing of births, there seems to have seasonal reproductive patterns in captive populations inhabiting the Southern Hemisphere. The extent to which male tamarins have circannual cyclic fluctuations in testicular functions, however, remains unknown. Changes in testis size and seminal variables, therefore, were evaluated during the rainy and dry seasons of the year in seven males. There was estimation of mating and birth seasons from the breeding colony using a 27 year database (229 birth records). Births were distributed throughout the year with peaks occurring in August-October and January-March (39.7 % and 30.5 % of all births, respectively). Semen collection using penile vibrostimulation was successfully induced in more than 96 % of the attempts regardless of the season (total of 75 ejaculates). Body mass did not vary significantly between seasons, but relative testes size was larger during the dry season. Values for none of the seminal variables (total sperm count, total and progressive motility, plasma membrane and acrosome integrity, and total sperm defects), however, were different during the rainy and dry seasons. These results indicate that testicular function in golden-headed lion tamarins may not be affected by daylength changes, and that seasonal patterns of female reproduction is perhaps more relevant for the reproductive timing of the species. Furthermore, the possibility of year-round production of ejaculates containing viable sperm broadens our perspective of preserving genetic diversity within the species because there is a greater opportunity for semen collection and freezing.

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Testicular heat stress affects sperm quality and fertility. However, the chronology of these effects is not yet fully understood. This study aimed to establish the early sequential effects of heat stress in bull sperm quality. Semen and blood samples of Nellore breed bulls were collected and distributed into control and testicular heat stress (scrotal bags/96 h) groups. Semen samples were evaluated for sperm motility, abnormalities, plasma membrane integrity, acrosomal membrane integrity, mitochondrial membrane potential, sperm lipid peroxidation, seminal plasma lipid peroxidation, and DNA fragmentation. Blood plasma was also evaluated for lipid peroxidation. An increase in sperm abnormalities was observed 7 days following heat stress. After 14 days, sperm lipid peroxidation increased and mitochondrial membrane function, sperm motility, and plasma membrane integrity decreased. Heat stress effects were still observed after 21 days following heat stress. An increase in sperm DNA fragmentation was observed as a late effect after 28 days. Thus, the initial effects of heat stress (i.e., increasing sperm abnormalities and lipid peroxidation) suggest the presence of oxidative stress in the semen that alters mitochondrial function, sperm motility, plasma membrane integrity, and belatedly, DNA fragmentation. Although sperm abnormalities persisted and increased over time, sperm lipid peroxidation, in turn, increased only until 21 days after heat stress. In this regard, these findings provide a greater understanding of the chronological effects of experimentally induced heat stress on bovine sperm, providing valuable insights about spermatogenesis during the first 28 days following heat stress.

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Wild animal genetic resource banking (GRB) represents a valuable tool in conservation breeding programs, particularly in cases involving endangered species such as the golden-headed lion tamarin (Leontopithecus chrysomelas). Thus, we aimed to assess a sperm freezing protocol for golden-headed lion tamarins using two different exenders: BotuBOV® (BB) and Test Yolk Buffer® (TYB). Ejaculates were collected by penile vibrostimulation from animals housed at São Paulo Zoological Park Foundation, São Paulo, Brazil, and after immediate analysis, two aliquots were diluted in BB and TYB. Postthawing samples were evaluated for total and progressive motility, plasma membrane and acrosome integrities, mitochondrial activity, susceptibility to oxidative stress, and sperm-egg-binding. No differences between BB and TYB were found for most seminal parameters, except for acrosome integrity and susceptibility to oxidative stress (in both cases BB showed higher values). However, in spite of these differences and regardless of the extender used, postthaw sperm motility and viability with the described protocol were encouraging (on average >50% and >80%, respectively), indicating that sperm cryopreservation may be a short-term measure for the conservation of golden-headed lion tamarins.

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