Cryopreservation-Induced Morphological Changes in Freshwater Fish Sperm: A Systematic Review.

A systematic review was performed to summarize the scientific evidence and critically evaluate the effects of cryopreservation on sperm morphology in freshwater fish, and to assess the methodologies for sperm morphology classification. The search strategy was applied to four electronic databases (CAB Direct, Pub Med, Scopus, and ISI Web of Science). The main inclusion criteria involved studies on semen from freshwater fish subjected to the cryopreservation process and evaluation of sperm quality through morphology. The risk of bias was assessed with respect to randomization, allocation concealment, blinding, incomplete outcome data, and selective reporting. A total of 6 publications reporting sperm cryopreservation from 4 species with a total 74 fish individuals were included in this review. A high methodological variability among the results of the studies was observed due to the species-specific protocols and diversity of freshwater fish species studied. All included studies reported negative effects of cryopreservation on sperm quality, especially morphology, highlighting the increase in incidence of sperm abnormalities. However, only five studies statistically compared abnormalities between groups (fresh and cryopreserved sperm). Our results suggest the need to elaborate on a new morphological classification of fish spermatozoa, by considering the structure and physiology of fish sperm. This classification should be developed based on the sperm characterization and observing damage caused by different cryopreservation protocols.

Preservation of fertility in female and male prepubertal patients diagnosed with cancer.

Over the past two decades, the importance of fertility preservation has grown not only in the realm of medical and clinical patient care, but also in the field of basic and applied research in human reproduction. With advancements in cancer treatments resulting in higher rates of patient survival, it is crucial to consider the quality of life post-cure. Therefore, fertility preservation must be taken into account prior to antitumor treatments, as it can significantly impact a patient's future fertility. For postpubertal patients, gamete cryopreservation is the most commonly employed preservation strategy. However, for prepubertal patients, the situation is more intricate. Presently, ovarian tissue cryopreservation is the standard practice for prepubertal girls, but further scientific evidence is required in several aspects. Testicular tissue cryopreservation, on the other hand, is still experimental for prepubertal boys. The primary aim of this review is to address the strategies available for possible fertility preservation in prepubertal girls and boys, such as ovarian cryopreservation/transplantation, in vitro follicle culture and meiotic maturation, artificial ovary, transplantation of cryopreserved spermatogonia, and cryopreservation/grafting of immature testicular tissue and testicular organoids.

From in vivo to in vitro: exploring the key molecular and cellular aspects of human female gametogenesis.

Human oogenesis is a highly complex and not yet fully understood process due to ethical and technological barriers that limit studies in the field. In this context, replicating female gametogenesis in vitro would not only provide a solution for some infertility problems, but also be an excellent study model to better understand the biological mechanisms that determine the formation of the female germline. In this review, we explore the main cellular and molecular aspects involved in human oogenesis and folliculogenesis in vivo, from the specification of primordial germ cells (PGCs) to the formation of the mature oocyte. We also sought to describe the important bidirectional relationship between the germ cell and the follicular somatic cells. Finally, we address the main advances and different methodologies used in the search for obtaining cells of the female germline in vitro.

Evolutionary dynamics of autopolyploids in natural populations: the case of Turnera sidoides complex / Dinámica evolutiva de autopoliploides en poblaciones naturales: el caso del complejo Turnera sidoides

ABSTRACT Turnera sidoides (x=7) is one of the few well-studied South American autopolyploid complexes. Since polyploidy has played a prominent role within this complex, ongoing studies in T. sidoides focus on understanding the mechanisms involved in the origin and the establishment of polyploids using integrative approaches. This paper synthesises the results of more than 20 years of research on this topic. Cytogenetics analysis provided evidences for the production of unreduced male and female gametes, supporting the hypothesis of bilateral sexual polyploidization as the mechanism of origin of polyploids in T. sidoides. The finding of viable triploids suggested that unilateral sexual polyploidization could also be an important mechanism for the origin of tetraploids in T. sidoides. The occurrence of plants continuously forming many unreduced gametes would play a key role in the establishment of neopolyploids in natural populations. Also, the higher number of propagules that tetraploids contribute to subsequent generations, the ability to multiply asexually by rhizomes, and the occurrence of occasional cases of self-compatibility and successful illegitimate crosses in polyploids increase the likelihood that a low frequency of neopolyploids can be maintained in natural populations of T. sidoides. In addition, integration of cytogeographic and genetic divergence data together with past niche modelling provided further insights supporting the hypothesis that historical climatic and geomorphological events provided favourable conditions for the establishment of autopolyploids, with the wider distribution of tetraploids of T. sidoides being the result of their range expansion.

RESUMEN Turnera sidoides (x=7) es uno de los pocos complejos autopoliploides sudamericanos bien estudiados. Como la poliploidía ha tenido un papel destacado en el complejo, los estudios en curso en T. sidoides se centraron en la comprensión de los mecanismos implicados en el origen y el establecimiento de los poliploides mediante diferentes enfoques. En este trabajo se sintetizan los resultados de más de 20 años de investigación sobre este tema. El análisis citogenético proporcionó evidencias de la producción de gametos masculinos y femeninos no reducidos, sustentando la hipótesis de la poliploidización sexual bilateral como mecanismo de origen de los poliploides en T. sidoides. Sin embargo, el hallazgo de triploides fértiles sugirió que la poliploidización sexual unilateral también sería un mecanismo importante de origen de tetraploides en T. sidoides. La ocurrencia de plantas que forman continuamente gametos no reducidos desempeñaría un papel clave en el establecimiento de neopoliploides. Además, el mayor número de propágulos que los tetraploides aportan a las siguientes generaciones, la capacidad de multiplicación asexual por rizomas y los casos ocasionales de autocompatibilidad y cruzamientos ilegítimos exitosos aumentarían la probabilidad de que se mantenga una baja frecuencia de neopoliploides en las poblaciones naturales de T. sidoides. Asimismo, la integración de datos citogeográficos y de divergencia genética junto con el modelado de nicho en el pasado aportó información que sustenta la hipótesis de que los eventos climáticos y geomorfológicos históricos proporcionaron las condiciones favorables para el establecimiento y expansión de los tetraploides de T. sidoides.

Morphological description of gametes in cave and surface populations of Astyanax mexicanus (De Filippi, 1853).

The Mexican tetra Astyanax mexicanus presents two contrasting morphs, a widely distributed surface morph and a cave-adapted morph. These cave-adapted morphs have evolved independently from two different lineages (i.e. 'old' and 'new' lineages); therefore, this model system gives a unique opportunity to explore parallel adaptive evolution in biological traits. The present study corresponds to the first morphological description of the Astyanax mexicanus maturation process of the spermatozoa and oocytes, using thermal and hormonal stimuli to promote spermatogenesis and oogenesis, considering surface and cave morphs from both lineages. We corroborate the relevance of thermal and hormonal stimuli to promote gamete maturation. The hormone Ovaprim (GnRHa + Domperidone) is an effective promoter of ovarian development, maturation end in oocytes and spawning in Astyanax mexicanus. The sperm morphology of Astyanax mexicanus includes the sperm head, the midpiece, and tail or flagellum. We found differences in the spermatozoan total length between environments (F = 9.929, P = 0.05) and linages (F = 49.86, P = 0.005). The oocytes showed a spherical conformation with a mean diameter of 822.4 ± 194.1 µm for the surface populations, and 604.6 ± 38.3 µm for the cave populations. The oocyte chorion presents ridges and grooves that are arranged radially towards the micropyle. A plug in the micropyle zone was observed after fertilization, confirmed by the outer membrane of the chorion, which provides some weak adhesiveness to the substrate. We observed differences in chorion thickness between the contrasting environmental conditions. This is the first morphological characterization of the Sótanos Vázquez, Escondido and Tigre, which previous to this study were only known from speleological expeditions, with no previous biological information available.

Genomic constitution, allopolyploidy, and evolutionary proposal for Cynodon Rich. based on GISH.

Polyploidy is the main mechanism for chromosome number variation in Cynodon. Taxonomic boundaries are difficult to define and, although phylogenetic studies indicate that some species are closely related, the degree of genomic similarity remains unknown. Furthermore, the Cynodon species classification as auto or allopolyploids is still controversial. Thus, this study aimed to investigate the genomic constitution in diploid and polyploid species using different approaches of genomic in situ hybridization (GISH). To better understand the hybridization events, we also investigated the occurrence of unreduced gametes in C. dactylon diploid pollen grains. We suggest a genomic nomenclature of diploid species as DD, D1D1, and D2D2 for C. dactylon, C. incompletus, and C. nlemfuensis, and DDD2D2 and DD2D1D1 for the segmental allotetraploids of Cynodon dactylon and C. transvaalensis, respectively. Furthermore, an evolutionary proposal was built based on our results and previous data from other studies, showing possible crosses that may have occurred between Cynodon species.

Impact on Fertility Rate and Embryo-Larval Development Due to the Association Acidification, Ocean Warming and Lead Contamination of a Sea Urchin Echinometra lucunter (Echinodermata: Echinoidea).

Ocean warming and acidification can cause deleterious effects on marine biota, which may be potentialized when associated with metal pollution. Thus, the aim of this work was to evaluate the effects of pH decrease, temperature increase and lead contamination on fertility rate and embryo-larval development of Echinometra lucunter. Gametes and embryos were exposed at pH 8.2 (control) and 7.5; at 26°C (control) and 28°C; and at lead concentrations of 0 (control), 125, 250 and 500 µg/L. These conditions were tested individually and in combination. The fertilization rate of E. lucunter was only significantly reduced in the treatments where temperature was increased and in the treatment where pH decreased. However, the development rate of the pluteus larvae was significantly affected in the majority of treatments: metal contamination in the higher concentration; decreased pH in all metal concentrations; increased temperature in the highest metal concentration; decreased pH and increased temperature and all variables combined, which is decreased pH, increased temperature and metal contamination in relation to the control group (C). The development test was shown to be more sensitive than the fertilization test in all the studied scenarios. In general, the present study suggests that pH decrease, temperature increase and metal pollution may have a significant impact on E. lucunter reproductive cycle.

Photoperiod in aquaculture of the sea urchin Arbacia dufresnii (Echinodermata: Echinoidea): Effects on gamete production and maturity / Fotoperiodo en la acuicultura del erizo de mar Arbacia dufresnii (Echinodermata: Echinoidea): El efecto en la producción y madurez de gametas

Introduction: Photoperiod is, together with temperature and food availability, one of the main stimuli in the regulation of gametogenesis in a wide variety of species. Objective: To evaluate the effect of photoperiod on the production of mature gametes in cultured Arbacia dufresnii. Methods: An experiment was carried out with three varying light-dark regimes/treatments: constant light (24 h light), neutral photoperiod (12 h light, 12 h darkness), and constant darkness (24 h darkness). Twenty females were used in each treatment. All were induced to spawn and, ten randomly selected females from each treatment were induced to spawn again after 30 days. After 60 days, spawning was induced in the remaining females. The gametes were collected in filtered seawater, fixed in Davidson solution, quantified and measured per individual in triplicate in a Sedgewick-Rafter chamber. To determine maturation, fertilization success was evaluated 30 minutes after fertilization. Results: Our results showed that in the aquaculture system, after only two months, mature gametes were obtained, and in the neutral light regime there were 10 times more gametes than the number produced in wild sea urchins during the spawning period in question. We also found that with a greater exposure to light, a lower number of mature gametes was produced. Conclusions: This study suggests the viability of the production of mature gametes in a short period of time as regards Arbacia dufresnii.

Introducción: El fotoperiodo es, junto con la temperatura y la disponibilidad de alimentos, uno de los principales estímulos para el desarrollo de la gametogénesis en una amplia variedad de especies. Objetivo: Evaluar el efecto del fotoperiodo en la producción de gametas maduras de Arbacia dufresnii en un sistema de recirculación cerrado para determinar el mejor fotoperiodo para una acuicultura novedosa, enfocada en la producción de gametas con alta concentración de pigmentos para usos biotecnológicos. Métodos: Se realizó un experimento con tres regímenes/tratamientos diferentes de luz y oscuridad: luz constante (luz durante 24 h), fotoperiodo neutro (12 h de luz, 12 h de oscuridad) y oscuridad constante (oscuridad durante 24 h). Se utilizaron veinte hembras en cada tratamiento. Se indujo a todas las hembras a desovar al comienzo del experimento. Después de 30 días, diez hembras seleccionadas al azar de cada tratamiento fueron inducidas a desovar nuevamente. Al final del experimento, después de 60 días, se indujo el desove a las hembras restantes en cada tratamiento. Las gametas se recolectaron en agua de mar filtrada, se fijaron en solución de Davidson, se cuantificaron y midieron por triplicado en una cámara Sedgewick-Rafter. Para determinar la maduración, se evaluó el éxito de la fecundación después de 30 minutos de fertilización, calculando el porcentaje de huevos fertilizados. Resultados: Nuestros resultados muestran que, en el sistema acuícola, en solo dos meses se obtuvieron gametas maduras y casi 10 veces más la cantidad producida por los erizos de mar en su ambiente natural usando el fotoperiodo neutro (12 h luz:12 h oscuridad). También encontramos que la mayor exposición a la luz produce la menor cantidad de gametas maduras. Conclusión: Este estudio sugiere la viabilidad de la producción de gametos maduros en un corto período de tiempo en Arbacia dufresnii.

Maternal pineal melatonin in gestation and lactation physiology, and in fetal development and programming.

Pregnancy and lactation are reproductive processes that rely on physiological adaptations that should be timely and adequately triggered to guarantee both maternal and fetal health. Pineal melatonin is a hormone that presents daily and seasonal variations that synchronizes the organism's physiology to the different demands across time through its specific mechanisms and ways of action. The reproductive system is a notable target for melatonin as it actively participates on reproductive physiology and regulates the hypothalamus-pituitary-gonads axis, influencing gonadotropins and sexual hormones synthesis and release. For its antioxidant properties, melatonin is also vital for the oocytes and spermatozoa quality and viability, and for blastocyst development. Maternal pineal melatonin blood levels increase during pregnancy and triggers the maternal physiological alterations in energy metabolism both during pregnancy and lactation to cope with the energy demands of both periods and to promote adequate mammary gland development. Moreover, maternal melatonin freely crosses the placenta and is the only source of this hormone to the fetus. It importantly times the conceptus physiology and influences its development and programing of several functions that depend on neural and brain development, ultimately priming adult behavior and energy and glucose metabolism. The present review aims to explain the above listed melatonin functions, including the potential alterations observed in the progeny gestated under maternal chronodisruption and/or hypomelatoninemia.

Equine chorionic gonadotropin increases estradiol levels in the bovine oviduct and drives the transcription of genes related to fertilization in superstimulated cows.

In the bovine oviduct, estradiol (E2) stimulates secretion and cell proliferation, whereas progesterone (P4) suppresses them. In this study, we have evaluated the effect of two superstimulatory protocols (follicle-stimulating hormone [FSH] or FSH combined with equine chorionic gonadotropin [eCG]) on the oviductal levels of E2 and P4 and its outcome on oviductal cells. Compared with the control group (a single pre-ovulatory follicle), we have observed that the cows submitted to FSH/eCG treatment showed a higher concentration of E2 in the oviduct tissue, together with a higher abundance of messenger RNA encoding steroid receptors (ESR1 and progesterone receptor), and genes linked to gamete interactions and regulation of polyspermy (oviduct-specific glycoprotein 1, heat-shock protein family A member 5, α-l-fucosidase 1 [FUCA1], and FUCA2) in the infundibulum and ampulla segments of the oviduct. However, we did not observe any modulation of gene expression in the isthmus segment. Even though the FSH protocol upregulated some of the genes analyzed, we may infer that the steady effect of FSH combined with eCG on oviduct regulation might benefit fertilization and may potentially increase pregnancy rates.

S100 A9 is expressed and secreted by the oviduct epithelium, interacts with gametes and affects parameters of human sperm capacitation in vitro.

Our previous findings demonstrate that some oviductal secretion proteins bind to gametes and affect sperm physiology and gamete interaction. One of these proteins possesses an estimated molecular weight of 14 kDa. The objective of this study was to isolate and identify this 14 kDa protein, to localize it in the human oviduct, to detect gamete binding sites for the protein, and to evaluate its effects on sperm capacitation parameters and gamete interaction. Explants from the human oviductal tissues of premenopausal women were cultured in the presence of [35 S]-Methionine-proteins ([35S]-Met-proteins). De novo synthesized secreted [35 S]-Met-proteins were isolated from the culture media by affinity chromatography using their sperm membrane binding ability and analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using liquid chromatography-tandem mass spectrometry peptide sequencing, human S100 A9 was identified as one of the isolated proteins from the 14 kDa protein band. S100 A9 was detected in oviduct epithelium and oviduct secretion using immunohistochemistry and a Western blot. S100 A9 binding to human oocytes and spermatozoa was assessed by indirect immunofluorescence. The acrosome reaction (AR) affected S100 A9 ability to bind sperm cells. The presence of S100 A9 significantly increased both the induced AR and the sperm protein tyrosine phosphorylation, with respect to controls. However, the protein did not affect sperm-zona pellucida interaction. Results indicate that S100 A9 is present in the human oviduct and that it modulates parameters of sperm capacitation in vitro. Hence, the protein might contribute to the regulation of the reproductive process in the oviductal microenvironment.

Understanding the Role of Telomere Dynamics in Normal and Dysfunctional Human Reproduction.

In modern society, fertility problems and demand of treatment seem to be on the rise, which led to an increased interest in research regarding human reproduction. Among these efforts, the study of the molecular senescence process has gain notorious popularity as aging is one of the most important variables involved in reproductive capacity and since the comprehension of telomere dynamics has become an important and influential theme. This new knowledge regarding the reproductive aging process is expected to offer new tools to understand the acquisition, maintenance, and loss of fertility potential. Therefore, this review seeks to clarify the relevance of molecular aging (evaluated by telomere shortening) in human reproduction, showing that it is a dynamic and variable process modulated according to the specific tissue and stage of development. As well, it is discussed how telomere status influence the development and progression of some fertility pathologies, the outcome of assisted reproductive treatments, and programming of aging in the offspring.

Influence of growth differentiation factor 9 and bone morphogenetic protein 15 on in vitro maturation of canine oocytes.

Growth differentiation factor 9 (GDF-9) and bone morphogenetic protein 15 (BMP-15) have pivotal roles in oocyte development in many species, therefore the aim was to investigate these factors during in vitro maturation (IVM) of canine oocytes. Canine cumulus oocytes complexes (COCs) were cultured in six groups for 72 hr in a supplemented TCM199-Hepes medium as (a) Control group; (b) GDF-9 antibody (Ab); (c) BMP-15 Ab; (d) recombinant human (rh) GDF-9; (e) rh BMP-15 or (f) rh BMP-15 and GDF-9. Data were evaluated by ANOVA. The Abs against GDF-9 or BMP-15 had a negative impact on meiotic development. Higher (p < 0.05) number of oocytes was arrested at GVBD stage when they were incubated with either GDF-9 Ab (64.4 ± 2.1%) or BMP-15 Ab (67.2%± 4.9%) in comparison to those in control group (32.4 ± 7.8%). In contrast, more (p < 0.05) oocytes in control group reached MI (37.4 ± 1.3%) and MII stages (10.2 ± 2.1%) comparing to those groups with GDF-9 Ab (23.1 ± 4.7% MI; 0.0% MII) or BMP-15 Ab (16.4 ± 2.4%MI; 5.9% ± 2.1 MII). Higher rates (p < 0.05) of oocytes in control group stayed still arrested at GV (19.9 ± 8.6%) in comparison to those cultured with either rhGDF-9 (3.7 ± 0.4%) or rhBMP-15 (10.9 ± 0.7%). However, there were no differences in MII rates between oocytes cultured with GDF-9 (14.7 ± 3.1) and BMP-15 (7.8 ± 2.5) separately. But, more oocytes (p < 0.05) reached the MII stage (20.5 ± 3.8%) compared to those exposed to each protein separately and to the control group. These results suggest that these proteins likely contribute to the meiotic development in dogs.

Chilling sensitivity of Steindachneridion parahybae (Siluriformes: Pimelodidae) oocytes in different cryoprotectants.

The viability of post-thaw fish oocytes can be affected by different stages of the freezing process, such as cryoprotectant toxicity, cold sensitivity, freezing curves and thawing. Therefore, these steps need to be investigated for the development of a protocol. In the present study, the aim was to investigate chilling sensitivity at different oocyte stages of Steindachneridion parahybae. Immature and mature oocytes were incubated in Hanks' or 90% L15 solutions containing different CPAs (cryoprotectant solutions) per experiment: (1) 0.1-0.4 M sucrose + 1-2 M methanol and (2) 1-4 M methanol X 1-4 M propylene glycol X 1-4 M DMSO for mature oocytes; (3) 0.5 M sucrose or fructose + 2 M methanol or PG or DMSO and (4) 0.25-1 M fructose + 1-4 M DMSO for immature oocytes. All treatments were kept for 120 min at -5.9 ±â€¯2.8°C. For the control treatment, only Hanks' or 90% L15 solutions were carried out. Evaluations were made by viability tests: membrane integrity staining in 0.4% Trypan blue (TB) and fertilization rate (%F) sole for mature oocytes. Results presented that mature oocytes were the most sensitive to lower temperatures, because there was no %F. All cryoprotectants tested in the different concentrations can be used for immature oocytes, however the statistically superior cryoprotectant was CPA with fructose and DMSO, with the low concentration of this CPA being was the best statistically. This may indicate that for this species the immature stages have presented a lower chilling sensitivity than the mature stages.

Biotecnologias da reprodução animal: de Aristóteles à edição gênica / Biotechnologies of animal reproduction: from Aristotle to gene editing

Atualmente, com a rapidez e a facilidade da difusão da informação, os jovens acadêmicos têm percepção de que o conhecimento é um produto pronto, onde as características podem ser manipuladas e empregadas como um objeto oriundo de uma fábrica. A compreensão da realidade que nos cerca é um desejo desde os primórdios da presença do homem sobre a terra e os conceitos se modificam na medida em que o conhecimento avança. As tecnologias reprodutivas surgiram e evoluíram ao longo dos tempos, levando a aplicações com diferentes impactos na produção e seleção animal, com repercussões na saúde animal, na saúde humana, na preservação de espécies em extinção e em diferentes cenários da atividade humana. Este texto traça uma linha de tempo das ações em reprodução animal, sob a ótica dos autores, dando ênfase ao gradual desenvolvimento das biotécnicas como ferramenta auxiliar na rotina experimental e nos diversos sistemas de produção animal.(AU)

Today the rapid and ease information diffusion makes young academics have a perception that knowledge is a ready product, where characteristics could be manipulated and used as an object from a factory. The understanding of the reality that surrounds us is a desire from the earliest days of man's presence on earth and the concepts change as knowledge advances. Reproductive technologies have emerged and evolved over time, leading to applications with different impacts on animal production and selection, with repercussions on animal and human health, on the preservation of endangered species and on different scenarios of human activity. This text summarizes the evolution of thought, knowledge and actions in animal reproduction from the perspective of the authors, emphasizing the gradual development of biotechnology as an auxiliary tool in the experimental routine and in the various systems of animal production.(AU)

Biotecnologias da reprodução animal: de Aristóteles à edição gênica / Biotechnologies of animal reproduction: from Aristotle to gene editing

Atualmente, com a rapidez e a facilidade da difusão da informação, os jovens acadêmicos têm percepção de que o conhecimento é um produto pronto, onde as características podem ser manipuladas e empregadas como um objeto oriundo de uma fábrica. A compreensão da realidade que nos cerca é um desejo desde os primórdios da presença do homem sobre a terra e os conceitos se modificam na medida em que o conhecimento avança. As tecnologias reprodutivas surgiram e evoluíram ao longo dos tempos, levando a aplicações com diferentes impactos na produção e seleção animal, com repercussões na saúde animal, na saúde humana, na preservação de espécies em extinção e em diferentes cenários da atividade humana. Este texto traça uma linha de tempo das ações em reprodução animal, sob a ótica dos autores, dando ênfase ao gradual desenvolvimento das biotécnicas como ferramenta auxiliar na rotina experimental e nos diversos sistemas de produção animal.

Today the rapid and ease information diffusion makes young academics have a perception that knowledge is a ready product, where characteristics could be manipulated and used as an object from a factory. The understanding of the reality that surrounds us is a desire from the earliest days of man's presence on earth and the concepts change as knowledge advances. Reproductive technologies have emerged and evolved over time, leading to applications with different impacts on animal production and selection, with repercussions on animal and human health, on the preservation of endangered species and on different scenarios of human activity. This text summarizes the evolution of thought, knowledge and actions in animal reproduction from the perspective of the authors, emphasizing the gradual development of biotechnology as an auxiliary tool in the experimental routine and in the various systems of animal production.

Screening the Pathogen Box for Molecules Active against Plasmodium Sexual Stages Using a New Nanoluciferase-Based Transgenic Line of P. berghei Identifies Transmission-Blocking Compounds.

Malaria remains an important parasitic disease with a large morbidity and mortality burden. Plasmodium transmission-blocking (TB) compounds are essential for achieving malaria elimination efforts. Recent efforts to develop high-throughput screening (HTS) methods to identify compounds that inhibit or kill gametocytes, the Plasmodium sexual stage infectious to mosquitoes, have yielded insight into new TB compounds. However, the activities of these compounds against gametes, formed in the first minutes of mosquito infection, are typically not assessed, unless screened in a standard membrane feeding assay, a labor-intensive assay. We demonstrate here the generation of a Plasmodium model for drug screens against gametes and fertilization. The new P. berghei line, named Ookluc, was genetically and pharmacologically validated and scalable for HTS. Screening the Pathogen Box from the Medicines for Malaria Venture using the new model identified promising TB compounds. The use of Ookluc in different libraries of compounds may aid in the identification of transmission-blocking drugs not assessed in screens against asexual stages or gametocytes.

Different protocols using PGF2α as ovulation inducer in Nelore cows subjected to estradiol-progesterone timed AI based protocols.

The objective of this study was to evaluate the effect of a PGF2α-analogue (PGF) on ovulation and pregnancy rates after timed artificial insemination (TAI) in cattle. In Experiment 1 cows received an intravaginal progesterone-releasing device (CIDR) plus 2 mg im of estradiol benzoate (EB) on Day 0. The CIDR devices were removed on Day 8, and all cows received 150 µg im of d-cloprostenol (PGF2α-analogue), 300 IU of eCG and 1 mg of estradiol cypionate (ECP) im. On Day 9, cows were randomly assigned into two groups: 1) ECP Group (n = 17), that did not receive any further treatment; and 2) ECP-PG Group (n = 14) that were given 150 µg of d-cloprostenol (PGF) as adjuvant stimulus for ovulation. No difference between groups was detected in interval for ovulation (P = 0.5), and in the proportion of cows ovulating (P = 0.09). In Experiment 2, multiparous suckling crossbred Aberdeen Angus cows (n = 260), were treated into two groups, similarly as Experiment 1; ECP group (n = 122), and ECP-PG group (n = 138). All females were TAI on Day 10. The proportion of cows treated with ECP that became pregnant was 54.9% (67/122), and cows treated with ECP plus PGF was 55.1% (76/138; P = 0.9). In Experiment 3, 686 Nelore cows, 40 to 50 days postpartum, were treated as Experiment 1 (ECP group), however, on Day 8 cows were divided into 3 groups: ECP Group (n = 216); PGF-SC Group (n = 228), in which cows did not receive ECP and were given an additional subcutaneous injection of PGF on Day 8; and PGF-IM Group (n = 242), in which cows also did not receive ECP on Day 8 and were given an additional injection of PGF im on Day 9. On Day 10, estrus was evaluated at timed AI (TAI). There was no difference in the diameter of the dominant follicle at CIDR removal and at TAI, and pregnancy per AI among groups (P > 0.05). However, the proportion of cows that displayed estrus between CIDR removal and TAI was higher in ECP group than in PGF-SC and PGF-IM groups (P < 0.001). Cows that displayed estrus has higher P/AI than cows that did not (P = 0.008). In conclusion, these results suggested that intramuscular or subcutaneous injection of PGF2α could be successfully used to induce ovulation in cattle undergoing TAI, with similar pregnancy rates when compared with ECP. The subcutaneous injection of PGF on the same day of CIDR removal could be an interesting alternative due it reduces cattle management to obtain similar results.

Short-term storage of the oocytes affects the ploidy status in the yellowtail tetra Astyanax altiparanae.

In fish, many factors can affect reproduction during in vitro fertilization, therefore determination of the factors that affect affecting gamete quality is needed. However, few studies have focused on gamete quality and the ploidy status. This study was conducted to elucidate whether oocyte storage can affect ploidy status, survival, and embryo viability in the characid species Astyanax altiparanae. Oocytes were stored in Dulbecco's phosphate-buffered saline (PBS) at 26°C, then aliquots were fertilized immediately after extrusion (control) and also after 60, 120, 180, and 240 min of storage. Fertilization and hatching rates were measured, and the developmental stages were analyzed at each stage before describing the main abnormalities. Ploidy status was analyzed by flow cytometry and blood smear. In the control group, 100% of the samples were diploid. After treatment for 60 min, 95.56 ± 4.44% samples were diploid and 4.44 ± 4.44% were triploid. After 120 min, 94.44 ± 9.62% of the samples was diploid and 5.56 ± 5.56% were triploid; 100% of the samples were diploid after 180 min and, after 240 min, there was no survival. In other treatments, the highest percentage of hatching was after 60 min (88.93 ± 5.15%; P = 0.015), and treatment with 180 min storage resulted in the highest percentage of abnormal larvae (95.76 ± 12.67%; P = 0.012). These results show that oocyte storage can affect ploidy status and may be an interesting parameter for analysis in studies on chromosome set manipulation and micromanipulation.