Integration of genomic and ecological methods inform management of an undescribed, yet highly exploited, sardine species.

Assessing genetic diversity within species is key for conservation strategies in the context of human-induced biotic changes. This is important in marine systems, where many species remain undescribed while being overfished, and conflicts between resource-users and conservation agencies are common. Combining niche modelling with population genomics can contribute to resolving those conflicts by identifying management units and understanding how past climatic cycles resulted in current patterns of genetic diversity. We addressed these issues on an undescribed but already overexploited species of sardine of the genus Harengula. We find that the species distribution is determined by salinity and depth, with a continuous distribution along the Brazilian mainland and two disconnected oceanic archipelagos. Genomic data indicate that such biogeographic barriers are associated with two divergent intraspecific lineages. Changes in habitat availability during the last glacial cycle led to different demographic histories among stocks. One coastal population experienced a 3.6-fold expansion, whereas an island-associated population contracted 3-fold, relative to the size of the ancestral population. Our results indicate that the island population should be managed separately from the coastal population, and that a Marine Protected Area covering part of the island population distribution can support the viability of this lineage.

The Role of Hybridization in Species Formation and Persistence.

Hybridization, or interbreeding between different taxa, was traditionally considered to be rare and to have a largely detrimental impact on biodiversity, sometimes leading to the breakdown of reproductive isolation and even to the reversal of speciation. However, modern genomic and analytical methods have shown that hybridization is common in some of the most diverse clades across the tree of life, sometimes leading to rapid increase of phenotypic variability, to introgression of adaptive alleles, to the formation of hybrid species, and even to entire species radiations. In this review, we identify consensus among diverse research programs to show how the field has progressed. Hybridization is a multifaceted evolutionary process that can strongly influence species formation and facilitate adaptation and persistence of species in a rapidly changing world. Progress on testing this hypothesis will require cooperation among different subdisciplines.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp) kept in captivity.

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Out of Asia? Expansion of Eurasian Lyme borreliosis causing genospecies display unique evolutionary trajectories.

Vector-borne pathogens exist in obligate transmission cycles between vector and reservoir host species. Host and vector shifts can lead to geographic expansion of infectious agents and the emergence of new diseases in susceptible individuals. Three bacterial genospecies (Borrelia afzelii, Borrelia bavariensis, and Borrelia garinii) predominantly utilize two distinct tick species as vectors in Asia (Ixodes persulcatus) and Europe (Ixodes ricinus). Through these vectors, the bacteria can infect various vertebrate groups (e.g., rodents, birds) including humans where they cause Lyme borreliosis, the most common vector-borne disease in the Northern hemisphere. Yet, how and in which order the three Borrelia genospecies colonized each continent remains unclear including the evolutionary consequences of this geographic expansion. Here, by reconstructing the evolutionary history of 142 Eurasian isolates, we found evidence that the ancestors of each of the three genospecies probably have an Asian origin. Even so, each genospecies studied displayed a unique substructuring and evolutionary response to the colonization of Europe. The pattern of allele sharing between continents is consistent with the dispersal rate of the respective vertebrate hosts, supporting the concept that adaptation of Borrelia genospecies to the host is important for pathogen dispersal. Our results highlight that Eurasian Lyme borreliosis agents are all capable of geographic expansion with host association influencing their dispersal; further displaying the importance of host and vector association to the geographic expansion of vector-borne pathogens and potentially conditioning their capacity as emergent pathogens.

Assessment of Wild Rocket (Diplotaxis tenuifolia (L.) DC.) Germplasm Accessions by NGS Identified SSR and SNP Markers.

Rocket is the common designation for two baby-leaf salad crops of the Brassicaceae family: Eruca sativa (L.) Cav., usually referred to as annual garden rocket, and Diplotaxis tenuifolia (L.) DC. commonly named to as perennial wild rocket. E. sativa is used for human consumption since antiquity. However, the growing consumer preference for D. tenuifolia is being accompanied by the fast increase in its production area and commercialization of new cultivars. Nevertheless, the worldwide number of wild rocket accessions maintained in germplasm collections is very reduced, the solution for which situation the project "REMIRucula" intends to contribute, establishing a germplasm collection at the INIAV, Oeiras, Portugal. Herein, we report on the establishment via next generation sequencing (NGS) of the first genome assembly of D. tenuifolia and the identification of specific single sequence repeat (SSR) and single nucleotide polymorphisms (SNP) loci for the establishment of specific DNA-markers for this species. A representative set of 87 D. tenuifolia and 3 E. sativa accessions were assessed by 5 SSR and 9 SNP-CAPS markers, allowing a drastic discrimination between both species and the establishment of unequivocal molecular fingerprints for the analyzed accessions. The non-discrimination within six pairs and one trio of D. tenuifolia accessions is discussed.

Transcriptomic data reveals nuclear-mitochondrial discordance in Gomphocerinae grasshoppers (Insecta: Orthoptera: Acrididae).

The phylogeny of many groups of Orthoptera remains poorly understood. Previous phylogenetic studies largely restricted to few mitochondrial markers found many species in the grasshopper subfamily Gomphocerinae to be para- or polyphyletic, presumably because of incomplete lineage sorting and ongoing hybridization between putatively young lineages. Resolving the phylogeny of the Chorthippus biguttulus species complex is important because many morphologically cryptic species occupy overlapping ranges across Eurasia and serve important ecological functions. We investigated whether multispecies coalescent analysis of 540 genes generated by transcriptome sequencing could resolve the phylogeny of the C. biguttulus complex and related Gomphocerinae species. Our divergence time estimates confirm that Gomphocerinae is a very young radiation, with an age estimated at 1.38 (2.35-0.77) mya for the C. biguttulus complex. Our estimated topology based on complete mitogenomes recovered some species as para- or polyphyletic. In contrast, the multispecies coalescent based on nuclear genes retrieved all species as monophyletic clusters, corroborating most taxonomic hypotheses. Our results underline the importance of using nuclear multispecies coalescent methods for studying young radiations and highlight the need of further taxonomic revision in Gomphocerinae grasshoppers.

Extensive introgression at late stages of species formation: Insights from grasshopper hybrid zones.

The process of species formation is characterized by the accumulation of multiple reproductive barriers. The evolution of hybrid male sterility, or Haldane's rule, typically characterizes later stages of species formation, when reproductive isolation is strongest. Yet, understanding how quickly reproductive barriers evolve and their consequences for maintaining genetic boundaries between emerging species remains a challenging task because it requires studying taxa that hybridize in nature. Here, we address these questions using the meadow grasshopper Pseudochorthippus parallelus, where populations that show multiple reproductive barriers, including hybrid male sterility, hybridize in two natural hybrid zones. Using mitochondrial data, we infer that such populations diverged some 100,000 years ago, at the beginning of the last glacial cycle in Europe. Nuclear data show that contractions at multiple glacial refugia, and post-glacial expansions have facilitated genetic differentiation between lineages that today interact in hybrid zones. We find extensive introgression throughout the sampled species range, irrespective of the current strength of reproductive isolation. Populations exhibiting hybrid male sterility in two hybrid zones show repeatable patterns of genomic differentiation, consistent with shared genomic constraints affecting ancestral divergence or with the role of those regions in reproductive isolation. Together, our results suggest that reproductive barriers that characterize late stages of species formation can evolve relatively quickly, particularly when associated with strong demographic changes. Moreover, we show that such barriers persist in the face of extensive gene flow, allowing future studies to identify associated genomic regions.

A lizard with two tales: What diversification within Sceloporus occidentalis teaches us about species formation.

In 1859, Charles Darwin proposed that species are not fundamentally different from subspecies or the varieties from which they evolve. A century later, Dobzhansky (1958) suggested that many such lineages are ephemeral and are likely to reverse differentiation through introgression (Figure 1a); only a few evolve complete reproductive isolation and persist in sympatry. In this issue of Molecular Ecology, Bouzid et al. (2021) showed how new analytical methods, when applied to genomic data, allow us to more precisely determine whether or not species formation follows the paths outlined by Darwin and Dobzhansky (Figure 1b). The authors studied the diversification of the lizard Sceloporus occidentalis, finding a continuum of genetic interactions between the preservation of genetic identity to genetic merger, analogous to what is exemplified by ring species. In doing so, they teach us two tales about species formation: that lineages are fractal byproducts of evolutionary processes such as genetic drift and selection, and that lineages are often ephemeral and do not always progress into fully reproductively isolated taxa. Studying ephemeral lineages like those in S. occidentalis allows us to capture divergence at its earliest stages, and potentially to determine the factors that allow lineages to remain distinct despite pervasive gene flow. These lineages thus serve as a natural laboratory to address long standing hypotheses about species formation.

Ejaculate characteristics over seasons in five species of lancehead pitvipers (Bothrops spp ) kept in captivity

Due to their major medical importance in Latin America, lancehead pitvipers are frequently kept and bred in captivity for venom extraction to the production of antivenom serums. Nevertheless, despite the great contribution given to captive breeding, much of the knowledge of Bothrops' reproductive biology derived from sporadic and insufficient data provided by zoological collections. Thus, we aimed to investigate seasonal changes in gonadosomatic index (GSI) and seminal parameters (e.g., volume, concentration, motility, viability, and acrosome integrity) of five species of lancehead pitvipers from different biomes and phylogenetic groups, maintained in the indoors serpentarium at Butantan Institute (Brazil). Patterns of variation in GSI and semen parameters differed from one species to another, suggesting that captive populations should perhaps be managed distinctly to maximize reproductive success. Furthermore, in none of the studied species did changes in GSI occur concomitantly with seminal variations. GSI remained unaltered year-round for Jararaca (Bothrops jararaca) and Brazilian lancehead (Bothrops moojeni), whereas it peaked in the autumn for Common lancehead (Bothrops atrox), Jararacussu (Bothrops jararacussu), and Whitetail lancehead (Bothrops leucurus). But surprisingly, the scenario was inverted when we estimated the total number of motile spermatozoa per season, as Jararaca and Brazilian lancehead displayed seasonal differences and the other species did not vary throughout the year. Potential ecological and evolutionary factors underlying these differences were also discussed in the present article. Together, these findings can help to better define breeding management strategies for each species in captivity, in addition to optimizing the future use of artificial insemination and semen cryopreservation.

Assessing different liquid-storage temperatures for rooster spermatozoa.

Although liquid-storage is extensively used in poultry, there are still questions on how sperm physiology is affected and to what extent sperm functions are disrupted by storage temperature and time. There, therefore, was investigation of storage temperature and durations on multiple semen variables. The storage at 37 °C was the most damaging, affecting values for several variables within 4 h of storage, whereas most differences occurred between 5 and 25 °C after 8 h. Progressive motility and mitochondrial function started to decrease within 2 h at 25 and 37 °C, and within 4 h at 5 °C. Acrosomal damage only occurred in samples at 37 °C. Eosin-negrosin staining indicated there was damage to the plasma membrane at 37 °C, however, with use of propidium iodide there were differences between 5 and 25 °C following 24 h. Temperatures of 5 and 25 °C resulted in similar curves for chromatin dispersion although chromatin integrities differed with storage for periods longer than 4 h. At 37 °C, results using both chromatin evaluations indicated there was damage after 2 h of incubation. Oxidative stress at 5 and 25 °C was similar when there was 24 h of storage. Intriguingly, there were no interaction between temperature and storage duration for peroxidized sperm membrane and total peroxidation status. These findings indicated that with a prolonged storage at 5 °C there were not marked changes in chicken spermatozoa, whereas at 25 °C there did not appear to be sperm damage occurring as a result of short-term storage.

Genomic methods reveal independent demographic histories despite strong morphological conservatism in fish species.

Human overexploitation of natural resources has placed conservation and management as one of the most pressing challenges in modern societies, especially in regards to highly vulnerable marine ecosystems. In this context, cryptic species are particularly challenging to conserve because they are hard to distinguish based on morphology alone, and thus it is often unclear how many species coexist in sympatry, what are their phylogenetic relationships and their demographic history. We answer these questions using morphologically similar species of the genus Mugil that are sympatric in the largest coastal Marine Protected Area in the Tropical Southwestern Atlantic marine province. Using a sub-representation of the genome, we show that individuals are assigned to five highly differentiated genetic clusters that are coincident with five mitochondrial lineages, but discordant with morphological information, supporting the existence of five species with conserved morphology in this region. A lack of admixed individuals is consistent with strong genetic isolation between sympatric species, but the most likely species tree suggests that in one case speciation has occurred in the presence of interspecific gene flow. Patterns of genetic diversity within species suggest that effective population sizes differ up to two-fold, probably reflecting differences in the magnitude of population expansions since species formation. Together, our results show that strong morphologic conservatism in marine environments can lead to species that are difficult to distinguish morphologically but that are characterized by an independent evolutionary history, and thus that deserve species-specific management strategies.

Recovery from hybrid breakdown reveals a complex genetic architecture of mitonuclear incompatibilities.

Reproductive isolation is often achieved when genes that are neutral or beneficial in their genomic background become functionally incompatible in a foreign genomic background, causing inviability, sterility or other forms of low fitness in hybrids. Recent studies suggest that mitonuclear interactions are among the initial incompatibilities to evolve at early stages of population divergence across taxa. Yet, the genomic architecture of mitonuclear incompatibilities has rarely been elucidated. We employ an experimental evolution approach starting with low-fitness F2 interpopulation hybrids of the copepod Tigriopus californicus, in which frequencies of compatible and incompatible nuclear alleles change in response to an alternative mitochondrial background. After about nine generations, we observe a generalized increase in population size and in survivorship, suggesting efficiency of selection against maladaptive phenotypes. Whole genome sequencing of evolved populations showed some consistent allele frequency changes across three replicates of each reciprocal cross, but markedly different patterns between mitochondrial backgrounds. In only a few regions (~6.5% of the genome), the same parental allele was overrepresented irrespective of the mitochondrial background. About 33% of the genome showed allele frequency changes consistent with divergent selection, with the location of these genomic regions strongly differing between mitochondrial backgrounds. In 87% and 89% of these genomic regions, the dominant nuclear allele matched the associated mitochondrial background, consistent with mitonuclear co-adaptation. These results suggest that mitonuclear incompatibilities have a complex polygenic architecture that differs between populations, potentially generating genome-wide barriers to gene flow between closely related taxa.

Historical isolation facilitates species radiation by sexual selection: Insights from Chorthippus grasshoppers.

Theoretical and empirical studies have shown that species radiations are facilitated when a trait under divergent natural selection is also involved in sexual selection. It is yet unclear how quick and effective radiations are where assortative mating is unrelated to the ecological environment and primarily results from sexual selection. We address this question using sympatric grasshopper species of the genus Chorthippus, which have evolved strong behavioural isolation while lacking noticeable ecomorphological divergence. Mitochondrial genomes suggest that the radiation is relatively recent, dating to the mid-Pleistocene, which leads to extensive incomplete lineage sorting throughout the mitochondrial and nuclear genomes. Nuclear data shows that hybrids are absent in sympatric localities but that all species have experienced gene flow, confirming that reproductive isolation is strong but remains incomplete. Demographic modelling is most consistent with a long period of geographic isolation, followed by secondary contact and extensive introgression. Such initial periods of geographic isolation might facilitate the association between male signaling and female preference, permitting the coexistence of sympatric species that are genetically, morphologically, and ecologically similar, but otherwise behave mostly as good biological species.

Fecal sex steroids and reproductive behaviors in harpy eagles (Harpia harpyja).

Aiming to improve our reproductive knowledge of large birds of prey, behavioral data and fecal steroids were assessed in captive pairs of Harpy eagles, a keystone species that is monogamous and resides in the Neotropics year-round. Adult individuals exhibited different reproductive outcomes and a breeding season extending beyond summer solstice (5-9 months) suggests that harpy eagles may not be absolutely photorefractory. Comparisons among breeding stages in males revealed that mean androgen levels in courtship were higher than in copulation and incubation, but no differences were detected in fecal progestagens or estrogens. Females had higher mean estrogen concentrations in courtship and copulation, whereas mean progestagen levels peaked during egg laying. Mean androgen concentrations were not significantly different among breeding stages in females. Assessment of six egg-lay cycles from three females demonstrated that fecal estrogens peaked predominantly between 31 and 18 days before oviposition (-31 to -18 days), and then remained low until 45 days after laying the first egg (+45 days). In contrast, fecal progestagens raised mostly between -20 and +1 day, lowering to baseline concentrations by +3 days. To our knowledge, this is the first study to describe in detail endocrine and behavioral data regarding reproduction in tropical eagles, which may serve in the future as a reference to developing breeding programs.

Immunolocalisation and expression of oxytocin receptors and sex hormone-binding globulin in the testis and epididymis of dogs: correlation with sperm function.

The aim of this study was to confirm gene and protein expression of oxytocin receptor (OTR) and sex hormone-binding globulin (SHBG) in the testis and epididymis of dogs, correlating these data with sperm quality and production and testosterone concentrations. Positive correlations were found between OTR and SHBG expression in both the testis and epididymis. Testicular OTR expression was positively associated with plasma membrane and acrosome integrity in canine spermatozoa, whereas SHBG expression in the testis was positively correlated with various sperm characteristics, such as sperm concentration, total and progressive motility, plasma membrane integrity and acrosome integrity. Testicular expression of both OTR and SHBG was negatively correlated with low sperm mitochondrial activity. In the epididymis, SHBG expression was only positively correlated with plasma membrane integrity. Analysis of protein expression revealed that testicular OTR was positively correlated with testosterone concentrations and negatively correlated with the absence of sperm mitochondrial activity. In addition, SHBG expression in the testes was associated with epididymis SHBG expression and morphologically normal cells. Immunohistochemical (IHC) analysis revealed the presence of both OTR and SHBG in testicular smooth muscles and Leydig cells. However, in the epididymis, OTR was only located in smooth muscle cells, whereas neither IHC nor western blotting detected SHBG. Together, the results of this study suggest that OTR and SHBG play key roles in spermatogenesis and sperm maturation, being essential for male reproductive success.

Effect of simple and low-cost enrichment items on behavioral, clinical, and productive variables of caged laying hens.

Housing layers in battery cages is a practice still used by many countries but it has been criticized because of its influence on behavioral repertoire of birds. We investigated whether simple and affordable enrichment devices alone impact behavior, foot condition and performance of laying hens housed in conventional cages. Hens were divided into plain cages (CON), cages with perches (PER), and cages with tassels and scratch-pads (ENR), and parameters were evaluated before and after enrichment placement. After perch placement inactivity, drinking and competition for space reduced 35.6%, 40.8% and 70.3%, respectively, whereas social interaction increased 19.3%. Both modifications decreased locomotion (75.0% and 42.4% for PER and ENR respectively) and abnormal behaviors (62.5% and 43.9.4% for PER and ENR respectively). None of the performance variables were affected by ENR or PER. Thermography was more efficient than visual inspection in detecting subclinical bumblefoot, and it confirmed that PER reduced subclinical and clinical cases. Our findings indicate that perches increased welfare-related behaviors and foot health of hens, supporting the use of these inexpensive and highly adaptable alternatives for the enrichment of battery cages.

Semen collection by electro-stimulation in a variety of bird orders.

It is unquestinable that artificial insemination (AI) offers many benefits to avian conservation programs, but a serious impediment towards implementing AI for wild species is the development of effective techniques to consistently collect good quality ejaculates. Thus, we aimed to examine the success rate of electro-stimulation (ES) in collecting semen from 49 unconditioned males from orders Piciformes, Strigiformes, Accipitriformes, Cathartiformes, Galiformes, Anseriformes and Psittaciformes at different times of the year. Sixty out of 299 ES attempts provided ejaculates with sperm, but collection success rates varied widely (0-50%) depending on the species. Except for swans whose greater results were registered during spring-summer, males from most orders responded better to ES during winter-spring, suggesting seasonal variations on semen collection success rates. Overall, ES enabled successful semen collection from males of unproven and proven fertility under mixed pairing conditions. However, the highest success rate occurred in paired males with fertile clutches (40.6%) followed by unpaired males (22.1%), paired males without clutches (13.9%), and paired males with infertile clutches (6.8%). Behavioral responses of male birds to electrical impulses were also recorded to assess any discomfort during semen collection. Furthermore, macroscopic and microscopic analysis provided ejaculate parameters from several species, even from orders that hitherto have never been assessed for semen collection, which may serve as a starting point in the future. Altogether, these findings demonstrate the feasibility of ES in collecting semen from unpaired, unconditioned and non-imprinted males from a variety of bird orders. In the medium to long term, the use of this technique in both captive and free-ranging populations offers new perspectives to ensure genetic diversity in avian conservation programs.

Urofecal steroid profiles of captive Blue-fronted parrots (Amazona aestiva) with different reproductive outcomes.

Despite Psitaciformes (parrots) being the third largest nonpasserine order (398 species), it currently ranks second in number of threatened species (28%) according to the Internatinal Union for Conservation of Nature (IUCN) criteria. Since most of the literature concerning reproductive endocrinology in avian species derives from domestic and song birds, it is puzzling that advances in reproductive science for the Psitaciformes order lags far behind, in spite of the growing threats against them. In order to expand our knowledge of Neotropical parrots (Psittacidae), we examined annual changes in urofecal sex steroid metabolites of Blue-fronted amazon pairs (Amazona aestiva) exhibiting successful (nestlings) and unsuccessful breeding (infertile or no eggs). Urofecal samples were collected over a year from eight breeding pairs housed under the same environmental and management conditions. Fecal androgen and progestagen concentrations were determined in males and females, respectively, by enzyme immunoassays previously validated for this species. All eggs were registered between late winter and mid-spring, and egg-laying intervals varied between females (range: 1-8 days; average 3.60 ±â€¯0.51 days). Similar profiles of urofecal progestagens were observed in reproductively successful females and females producing infertile eggs, with progestagen peaks preceding egg laying events (1.77 ±â€¯0.50 days). In contrast, non-laying females had no rises in progestagens during the year. Successful and unsuccessful males did not displayed distinct annual patterns of androgen production, and apart from the peaks during the breeding season, more than half of the individuals intriguingly presented significant increases from late summer to early autumn, a period without reproductive activity. Finally, we noticed that samples with progestagen levels exceeding 40 ng/g had very high probability (>97.5%) to be from females in pre-laying or laying phases, suggesting a feasible application of this characteristic to noninvasively discriminate the reproductive status in amazon females with an accuracy and sensitivity of 94.55% and 58.13%, respectively. Our findings confirmed that urofecal progestagens and androgens are good indicators of the gonadal condition in Blue-fronted amazons, but there is still much to be done for their extensive use in artificial insemination or selection of the most suitable breeding birds for the season.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains.