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.

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.

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.

Impact of induced levels of specific free radicals and malondialdehyde on chicken semen quality and fertility.

Over the past decades, scientists endeavored to comprehend oxidative stress in poultry spermatozoa and its relationship with fertilizing ability, lipid peroxidation (LPO), free-radical scavenging systems, and antioxidant therapy. Although considerable progress has been made, further improvement is needed in understanding how specific reactive oxygen species (ROS) and malondialdehyde (MDA, a toxic byproduct of LPO) disrupt organelles in avian spermatozoon. Hence, this study examined functional changes in chicken spermatozoa after incubation with different ROS, and their implications for the fertility. First, semen samples from 14 roosters were individually diluted and aliquoted into five equal parts: control, superoxide anion, hydrogen peroxide (H2O2), hydroxyl radicals, and MDA. After incubation with these molecules, aliquots were analyzed for motility, plasma membrane and acrosome integrity, mitochondrial activity, and LPO and DNA damage. Hydrogen peroxide was more detrimental for sperm motility than hydroxyl radicals, whereas the superoxide anion and MDA exhibited no differences compared with controls. In turn, plasma membrane and acrosome integrity, mitochondrial activity, LPO and DNA integrity rates were only affected by hydroxyl radicals. Thereafter, semen aliquots were incubated under the same conditions and used for artificial insemination. In accordance to our in vitro observations, H2O2 and hydroxyl radicals sharply reduced egg fertility, whereas superoxide anion and MDA only induced slight declines. Thus, chicken sperm function was severely impaired by H2O2 and hydroxyl radicals, but their mechanisms of action seemingly comprise different pathways. Further analysis regarding susceptibility of spermatozoon organelles to specific radicals in other poultry will help us to understand the development of interspecific differences in scavenging systems and to outline more oriented antioxidant approaches.

Conservation of avian germplasm by xenogeneic transplantation of spermatogonia from sexually mature donors.

Approximately 12.5% of all 9,920 extant bird species in the world are threatened with extinction, and yet conservation efforts through natural breeding of captive species continue to encounter difficulties. However, sperm cryopreservation and artificial insemination offer potential benefits over natural breeding, but their applicability is still limited in nondomestic species. In this study, we aimed to exploit the potential of germ cell xenotransplantation as an alternative tool for preserving germplasm of endangered birds. The study was designed to investigate whether transfer of either spermatogonia-enriched cell fraction (SEF) or crude testicular cell fraction (CTF) from adult Japanese quails (as a model for wild species) would result in recolonization of gamma-irradiated gonads of adult recipient chickens. One month after transplantation, 75% of recipients injected with SEF and 25% of recipients injected with CTF resumed spermatogenesis. However, it took more than 3 months for 33% of the negative controls to resume marginal production of sperm. Some SEF recipients produced more spermatozoa bearing head morphology compared with donor controls. DNA analysis using quail-specific primers did not detect donor's DNA in these recipients' semen. However, 6 months after xenotransplantation, presence of quail germ cells was demonstrated by polymerase chain reaction and by immunohistochemistry in 1 rooster injected with SEF. These findings indicate that spermatogonia from adult quails were capable of colonizing immunocompetent testis of adult chickens but failed to produce sufficient sperm. Despite this limitation, the present approach represents a potential conservation tool that may be used to rescue germ cells of endangered adult male birds.

RAD51 plays a crucial role in halting cell death program induced by ionizing radiation in bovine oocytes.

Reproductive health of humans and animals exposed to daily irradiants from solar/cosmic particles remains largely understudied. We evaluated the sensitivities of bovine and mouse oocytes to bombardment by krypton-78 (1 Gy) or ultraviolet B (UV-B; 100 microjoules). Mouse oocytes responded to irradiation by undergoing massive activation of caspases, rapid loss of energy without cytochrome-c release, and subsequent necrotic death. In contrast, bovine oocytes became positive for annexin-V, exhibited cytochrome-c release, and displayed mild activation of caspases and downstream DNAses but with the absence of a complete cell death program; therefore, cytoplasmic fragmentation was never observed. However, massive cytoplasmic fragmentation and increased DNA damage were induced experimentally by both inhibiting RAD51 and increasing caspase 3 activity before irradiation. Microinjection of recombinant human RAD51 prior to irradiation markedly decreased both cytoplasmic fragmentation and DNA damage in both bovine and mouse oocytes. RAD51 response to damaged DNA occurred faster in bovine oocytes than in mouse oocytes. Therefore, we conclude that upon exposure to irradiation, bovine oocytes create a physiologically indeterminate state of partial cell death, attributed to rapid induction of DNA repair and low activation of caspases. The persistence of these damaged cells may represent an adaptive mechanism with potential implications for livestock productivity and long-term health risks associated with human activity in space.

Chemotherapy-induced late transgenerational effects in mice.

To our knowledge, there is no report on long-term reproductive and developmental side effects in the offspring of mothers treated with a widely used chemotherapeutic drug such as doxorubicin (DXR), and neither is there information on transmission of any detrimental effects to several filial generations. Therefore, the purpose of the present paper was to examine the long-term effects of a single intraperitoneal injection of DXR on the reproductive and behavioral performance of adult female mice and their progeny. C57BL/6 female mice (generation zero; G0) were treated with either a single intraperitoneal injection of DXR (G0-DXR) or saline (G0-CON). Data were collected on multiple reproductive parameters and behavioral analysis for anxiety, despair and depression. In addition, the reproductive capacity and health of the subsequent six generations were evaluated. G0-DXR females developed despair-like behaviors; delivery complications; decreased primordial follicle pool; and early lost of reproductive capacity. Surprisingly, the DXR-induced effects in oocytes were transmitted transgenerationally; the most striking effects being observed in G4 and G6, constituting: increased rates of neonatal death; physical malformations; chromosomal abnormalities (particularly deletions on chromosome 10); and death of mothers due to delivery complications. None of these effects were seen in control females of the same generations. Long-term effects of DXR in female mice and their offspring can be attributed to genetic alterations or cell-killing events in oocytes or, presumably, to toxicosis in non-ovarian tissues. Results from the rodent model emphasize the need for retrospective and long-term prospective studies of survivors of cancer treatment and their offspring.

Enhancing survival of mouse oocytes following chemotherapy or aging by targeting Bax and Rad51.

BACKGROUND: Therapeutic approaches to preserve fertility in females undergoing cancer treatments are currently ineffective. This is partly due to limited knowledge of the molecular mechanisms that injured germ cells elicit to repair damage and survive or to abort repair and activate biochemical pathways leading to death. So far, we know that following spontaneously occurring or drug-induced DNA damage, the efficiency of DNA repair is a critical determinant of the cell's fate. The protein encoded by the Rad51 gene is one of several components recruited for homologous recombination-dependent DNA double-strand break repair in both somatic cells and germ cells. Recently, we showed that microinjection of recombinant Rad51 into AKR/J mouse oocytes decreased the extent of spontaneous DNA double-strand breaks, suppressed apoptosis, and restored the developmental competence in AKR/J embryos. Herein we characterized the nature of chemotherapy-induced lesions in oocytes, and the associated individual components of the DNA damage sensor and repair apparatus. For comparison, we also assessed parallel spontaneous changes in aging oocytes. METHODS: Data collected were derived from: analysis of apoptosis; immunodepletion; oocyte microinjections; immunocytochemistry; immunofluorescence; and CHIP-like assays. RESULTS: Our data show that: (i) DNA damage in oocytes can be induced by both chemotherapy and spontaneously by the aging process; (ii) oocytes possess the machinery and capability for repairing such DNA damage; (iii) Rad51 is a critical player in the repair of both chemotherapy-induced and spontaneously-sustained DNA damage; and (iv) in response to damage, oocytes exhibit an inverse functional relationship between presence of Bax and activity of Rad51. CONCLUSION/SIGNIFICANCE: Our results establish Rad51 and/or Bax as potential candidates that can be targeted for development of individualized chemotherapeutic interventions that are effective, but minimal in toxicity. The use of Rad51 and Bax modulating compounds could offer women the opportunity to maintain fully functional germ cells despite cancer treatments or aging.