Reproductive Biology Research Articles

Reproductive performance of Jersey heifers submitted for first insemination based on age vs. body weight – a randomized controlled trial

Raising replacement heifers represents a major cost on a dairy farm. The ideal time to submit a heifer to insemination is a balance between reproductive biology, growth, and econom­ics. While getting heifers pregnant sooner may reduce raising costs, it may compromise their future productive performance. The objective of this study is to compare the reproductive per­formance of Jersey heifers submitted for first insemination (AI) based on age vs. body weight (BW). We hypothesize that the re­productive performance of heifers moved to the breeding pen (BP) based on a minimum BW would not be inferior to that of heifers moved based on minimum age.

Puzzling parrotfishes: Radiocarbon age validation and updated longevity estimates for western Atlantic species in support of sustainable fisheries management

For management efforts to succeed in Caribbean fisheries, local fishers must support and be willing to comply with fishing regulations. This is more likely when fishers are included in a stock assessment process that utilizes robust scientific evidence, collected in collaboration with fishers, to evaluate the health of fish stocks. Caribbean parrotfishes are important contributors to coral reef ecosystem health while also contributing to local fisheries. Scientifically robust stock assessments require regional species-specific information on age-based key life history parameters, derived from fish age estimates. Evaluation of the accuracy of age estimation methods for fish species is a critical initial step in managing species for long-term sustainable harvest. The current study resulted from a collaborative research program between fish biologists and local fishers investigating age, growth, and reproductive biology of the seven parrotfish species landed in U.S. Caribbean fisheries; specifically, we validated age estimation for stoplight parrotfish Sparisoma viride and queen parrotfish Scarus vetula. This is the first study to directly validate age estimation for any parrotfish species through analysis of Δ14C from eye lens cores. Our age estimation validation results show that enumeration of opaque zones from thin sections of sagittal otoliths for a Sparisoma and a Scarus species provides accurate age estimates. The oldest stoplight parrotfish and queen parrotfish in the Δ14C age estimation validation series were 14 y and 16 y; while the oldest stoplight parrotfish and queen parrotfish we aged to-date using the Δ14C validated age estimation method were 20 y and 21 y, respectively. Fish longevity (maximum age attained/life span) is a key life history parameter used for estimation of natural mortality, survivorship, and lifetime reproductive output. Past reviews on parrotfishes from the Pacific and Atlantic concluded that most Caribbean/western Atlantic parrotfish species are relatively short-lived with estimated maximum ages ranging from 3–9 y. However, information from our collaborative research in the U.S. Caribbean combined with recently published age estimates for Brazilian parrotfish species indicate that many western Atlantic parrotfishes are relatively long-lived with several species attaining maximum ages in excess of 20 y.

Deciphering male influence in gynogenetic Pengze crucian carp (Carassius auratus var. pengsenensis): insights from Nanopore sequencing of structural variations

In this study, we investigate gynogenetic reproduction in Pengze Crucian Carp (Carassius auratus var. pengsenensis) using third-generation Nanopore sequencing to uncover structural variations (SVs) in offspring. Our objective was to understand the role of male genetic material in gynogenesis by examining the genomes of both parents and their offspring. We discovered a notable number of male-specific structural variations (MSSVs): 1,195 to 1,709 MSSVs in homologous offspring, accounting for approximately 0.52%–0.60% of their detected SVs, and 236 to 350 MSSVs in heterologous offspring, making up about 0.10%–0.13%. These results highlight the significant influence of male genetic material on the genetic composition of offspring, particularly in homologous pairs, challenging the traditional view of asexual reproduction. The gene annotation of MSSVs revealed their presence in critical gene regions, indicating potential functional impacts. Specifically, we found 5 MSSVs in the exonic regions of protein-coding genes in homologous offspring, suggesting possible direct effects on protein structure and function. Validation of an MSSV in the exonic region of the polyunsaturated fatty acid 5-lipoxygenase gene confirmed male genetic material transmission in some offspring. This study underscores the importance of further research on the genetic diversity and gynogenesis mechanisms, providing valuable insights for reproductive biology, aquaculture, and fostering innovation in biological research and aquaculture practices.

Ecological perspectives on female and male reproductive success with competition in two Serapias species.

The deceptive strategies by which orchids are pollinated and how these are capable of attracting pollinators remain understudied with regard to their implications on plant fitness. Despite its ecological importance, limited investigations have been conducted on sexual deception and shelter mimicry in orchid species, making it a compelling avenue in orchid biology research. To expand the knowledge of these reproductive mechanisms, we studied the pollination of Serapias lingua and S. parviflora, in co-occurring and isolated sites in Balearic Islands (Spain), further accentuated by the presence of a hybrid, indicating shared pollinators. We employed bagging and hand pollination experiments to examine their reproductive biology. Furthermore, we evaluated the influence of phenotypical and ecological factors on reproductive success, including biometric measurements, reproductive performance and neighborhood diversity (co-flowering and pollinator communities). Reproductive mechanisms between these two orchid species exhibit substantial disparities. Serapias lingua relies primarily on insect-mediated pollination, while S. parviflora demonstrates self-reproduction capacity. Although events of open pollination are rare, hybridization occurs predominantly when S. lingua is the pollen donor. Fruit set in S. parviflora was positively correlated with plant height, while in S. lingua it was negatively associated with flower size. The coexistence of both species positively affected pollinia removal in S. parviflora, but did not exert an influence on reproductive traits in S. lingua. Overall, biometric parameters were diminished in isolated compared to co-occurring sites. At the community level, the increased diversity of co-flowering species in the vicinity exhibited an inhibitory effect on pollinia removal in S. parviflora. Under a context of pollinator loss or phenological mismatch between pollinator presence and flowering, the selfing capacity of S. parviflora would guarantee reproduction whereas S. lingua survival would be compromised. Furthers studies are needed to assess the effects of phenotypical and ecological factors on reproductive success of S. lingua in pollinator-decline scenarios.

Sexual dimorphism and reproductive biology of commercially harvested oriental rat snakes (Ptyas mucosa: Colubridae) from West Java

Large (>3 m) slender-bodied rat snakes are abundant in agroecosystems of southern Asia and are heavily exploited for their skins and meat. We examined 216 specimens killed at commercial facilities in Cirebon, West Java, to quantify morphological and reproductive traits and evaluate harvest sustainability by comparing this sample to one taken 25 years previously. The snakes we examined were mostly adults, and mostly male. Females were less heavy-bodied than males of the same body length, matured at a larger size than males, but attained smaller maximum sizes. Reproduction and energy (fatbody) storage was seasonal in both sexes, with females containing oviductal eggs primarily during the annual wet season. Clutch size averaged 12.6 eggs and increased with maternal body size. Comparison of the two samples taken 25 years apart revealed strong similarity in sex ratio, the numerical preponderance of adults, body sizes at sexual maturation and mean adult body sizes, sexual dimorphism in body length and mass, reproductive seasonality in females, mean fecundity, and the relationship between fecundity and maternal body size. That consistency suggests that commercial harvesting over the intervening period has not affected the biological traits of rat snake populations, consistent with a sustainable level of offtake.

Experimental heatwaves reduce the effectiveness of ejaculates at occupying female reproductive tracts in a model insect.

Globally, heatwaves have become more common with hazardous consequences on biological processes. Research using a model insect (Tribolium castaneum) found that 5-day experimental heatwave conditions damaged several aspects of male reproductive biology, while females remained unaffected. However, females' reproductive fitness may still be impacted, as insects typically store sperm from multiple males in specialized organs for prolonged periods. Consequently, using males which produce sperm with green fluorescent protein (GFP)-tagged sperm nuclei, we visualized in vivo whether thermal stress affects the ejaculate occupancy across female storage sites under two scenarios; (i) increasing time since insemination and (ii) in the presence of defending competitor sperm. We reconfirmed that sperm from heatwave-exposed males sired fewer offspring with previously mated females and provided new scenarios for in vivo distributions of heat-stress-exposed males' sperm. Sperm from heatwave-exposed males occupied a smaller area and were at lower densities across the females' storage sites. Generally, sperm occupancy decreased with time since insemination, and sperm from the first male to mate dominated the long-term storage site. Reassuringly, although heated males' ejaculate was less successful in occupying female tracts, they were not lost from female storage at a faster rate and were no worse than control males in their offensive ability to enter storage sites occupied by competitor sperm. Future work should consider the potential site-specificity of factors influencing sperm storage where amenable.

The role of kisspeptin in female and male reproduction

Kisspeptin is a neuropeptide responsible for controlling the synthesis of gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH). The G-protein-coupled receptor 54/kisspeptin 1 receptor (GPR54/KISS1R) is involved in its action. The comprehension of kisspeptin and its actions represents a significant breakthrough in the field of reproductive biology. Kisspeptins play a crucial role in the development and optimal functioning of the reproductive system in both female and male. Additionally, it contributes to the onset of puberty, regulating feedback processes, and influencing sexual desire and arousal. It regulates a number of reproductive functions in women, including ovulation, lactation, ovarian development, follicle development, oocyte maturation, and pregnancy via the hypothalamic-pituitary-gonadal (HPG) axis. Spermatogenesis, sperm function, Leydig cells, and reproductive behaviour are all impacted by it in men. Infertility and polycystic ovarian syndrome (PCOS) are among the diseases linked to kisspeptin dysregulation, according to the research. For potential future use in diagnosing and treating problems, it may be helpful to understand the mechanisms behind kisspeptin's effects on the reproductive system. This review focuses on the regulatory function of kisspeptin on the HPG axis and the impact of kisspeptin on reproductive processes in both female and male.

Unraveling the spawning and reproductive patterns of tomato hind grouper, Cephalopholis sonnerati (Valenciennes, 1828) from south Kerala waters.

The objective of this study is to provide information on the reproductive biology of tomato hind grouper, Cephalopholis sonnerati (Valenciennes, 1828) for conservation and management purposes. Fish caught by artisanal fishermen from September 2019 to August 2021 were analysed. A total of 280 females, 31 males, and 4 transitional and 178 sex-undetermined fish were analysed. The female to male sex proportion was 9:1, and the fish reached a maximum total body length of 38.5 and 54.5 cm for females and males, respectively. The following microscopic stages were identified: immature, developing, ripe, running ripe/releasing, and spent in both males and females. Several asynchronous development patterns were observed in the studied gonads, including multiple oocyte stages and early and advanced stages of sexual transition. High gonadosomatic index (GSI) for both males and females was recorded in March, May, and November. Running ripe and releasing stages in females were identified in the months from March to June, which indicates the spawning season. The absolute and relative fecundity of the species ranged from 162,723 ± 207,267 and 239 ± 285, respectively. An exponential relationship was found between fecundity and total body length (TL), fecundity and total body weight (TW), and fecundity and gonad weight (GW).

Olive (Olea europaea L.) reproductive biology: implications for yield, compatibility conundrum, environmental constraints.

Olive (Olea europaea L.) is an important Mediterranean tree species with a longstanding history of cultivation, boasting a diverse array of local cultivars. While traditional olive orchards are valued for their cultural and aesthetic significance, they often face economic sustainability challenges in the modern context. The success of both traditional and newly introduced cultivars (e.g. those obtained by crossbreeding) is hindered by self-incompatibility, a prevalent issue for this species that results in low fruit set when limited genetic diversity is present. Further, biological, environmental, and agronomic factors have been shown to interlink in shaping fertilization patterns, hence impacting on the final yield. Climatic conditions during pollination, such as excessive rainfall or high temperatures, can further exacerbate the problem. In this work, we provide an overview of the various complex and multifaceted factors that trigger the phenomenon of sub-optimal fruit set in olive trees. Through this work, we aim to provide a comprehensive understanding of the interplay among these factors, shedding light on potential mechanisms and pathways that contribute to the observed outcomes in the context of self-incompatibility of olive. This review aims to contribute to the development of sustainable olive production systems and the preservation of this vital component of Mediterranean culture.

Establishment of an immortalized yak granulosa cell line: in vitro tool for understanding the molecular processes of ovarian follicle development.

The yak, a unique species of cattle found exclusively on the western plateau of China, is a valuable source of livelihood for local residents. However, their low fecundity restricts the expansion of yak farming, whereas regional factors limit studies on yak breeding. Granulosa cells (GCs), which provide essential steroid hormones and growth factors for oocytes, have been the focus of many studies on the mechanisms of follicular growth and atresia. This study aimed to establish an immortalized cell line model that could serve as a tool for future studies on the mechanisms of ovarian follicle development in yaks. First, we isolated primary yak granulosa cells (yGCs) and evaluated their replicative senescence after continuous in vitro subculturing. Subsequently, an immortalized culture method for primary yGC was explored, and a new cell line model was established to study the mechanism of follicular development in vitro. We used a mammalian gene expression lentivirus vector to transfer the simian virus 40 large T antigen (SV40T) into primary yGC to obtain an immortalized cell line. The immortalized yGCs were morphologically identical to the primary yGCs, and cell proliferation and growth were normal within a limited number of generations. Follicle-stimulating hormone receptor (FSHR), a specific marker for GCs, was positively expressed in immortalized yGCs. Furthermore, the immortalized yGCs retained the ability of GCs to synthesize estradiol and progesterone and expressed genes related to steroid synthesis. The establishment of immortalized yGC opens up a myriad of possibilities for advancing our understanding of yak reproductive biology and improving yak breeding strategies.

Single-Cell Transcriptome Sequencing Reveals Molecular Expression Differences and Marker Genes in Testes during the Sexual Maturation of Mongolian Horses.

This study aimed to investigate differences in testicular tissue morphology, gene expression, and marker genes between sexually immature (1-year-old) and sexually mature (10-year-old) Mongolian horses. The purposes of our research were to provide insights into the reproductive physiology of male Mongolian horses and to identify potential markers for sexual maturity. The methods we applied included the transcriptomic profiling of testicular cells using single-cell sequencing techniques. Our results revealed significant differences in tissue morphology and gene expression patterns between the two age groups. Specifically, 25 cell clusters and 10 cell types were identified, including spermatogonial and somatic cells. Differential gene expression analysis highlighted distinct patterns related to cellular infrastructure in sexually immature horses and spermatogenesis in sexually mature horses. Marker genes specific to each stage were also identified, including APOA1, AMH, TAC3, INHA, SPARC, and SOX9 for the sexually immature stage, and PRM1, PRM2, LOC100051500, PRSS37, HMGB4, and H1-9 for the sexually mature stage. These findings contribute to a deeper understanding of testicular development and spermatogenesis in Mongolian horses and have potential applications in equine reproductive biology and breeding programs. In conclusion, this study provides valuable insights into the molecular mechanisms underlying sexual maturity in Mongolian horses.

Viewing early life without labels: optical approaches for imaging the early embryo.

Embryo quality is an important determinant of successful implantation and a resultant live birth. Current clinical approaches for evaluating embryo quality rely on subjective morphology assessments or an invasive biopsy for genetic testing. However, both approaches can be inherently inaccurate and crucially, fail to improve the live birth rate following the transfer of in vitro produced embryos. Optical imaging offers a potential non-invasive and accurate avenue for assessing embryo viability. Recent advances in various label-free optical imaging approaches have garnered increased interest in the field of reproductive biology due to their ability to rapidly capture images at high-resolution, delivering both morphological and molecular information. This burgeoning field holds immense potential for further development, with profound implications for clinical translation. Here, our review aims to: 1) describe the principles of various imaging systems, distinguishing between approaches that capture morphological and molecular information, 2) highlight the recent application of these technologies in the field of reproductive biology, and 3) assess their respective merits and limitations concerning the capacity to evaluate embryo quality. Additionally, the review summarizes challenges in the translation of optical imaging systems into routine clinical practice, providing recommendations for their future development. Finally, we identify suitable imaging approaches for interrogating the mechanisms underpinning successful embryo development.

The reproductive biology of <em>Marathrum rubrum Novelo & C. T. Philbrick</em>: morphological and anatomical changes during flower development

Background: Podostemaceae is the largest family of strictly aquatic angiosperms. Species of this family possess unusual morphological and reproductive characters, grow attached to rocks in rivers with strong currents, and have a life cycle tightly linked to seasonality. Due to its reduced floral structures, which characterizes most species, the analysis of developmental stages and events such as anthesis or stigma receptivity has proven difficult.
 Question: How floral morphology, floral development and stigma receptivity of different stages of flower development of Marathrum rubrum Novelo & C. T. Philbrick are related among them?
 Studied species: Marathrum rubrum Novelo & C. T. Philbrick.
 Study site and dates: Horcones River, Jalisco, Mexico. January, 2014 and 2015.
 Methods: We made an in situ and lab analyses of flower morphology to assess the relationships between flower morphology, stages of development, reproductive structures, and stigma receptivity, during different stages of flower development.
 Results: When flowers emerge from the spathella, both the ovules and pollen grains are fully developed, implying that sporogenesis and gametogenesis take place within the stem while the plant is still submerged.
 Conclusions: The morphological changes observed in the flowers of M. rubrum during the analyzed stages are related only to events associated with fertilization and fruit development. The time-course of morphological changes in the flowers, from anthesis to fruit formation, lasts 20 to 30 days. The detailed observation of the aerial reproductive biology of M. rubrum bears important implications to the genetic structure of populations, plant fitness and conservation biology in threatened environments.

Reproductive biologies of Psammogobius knysnaensis and Caffrogobius gilchristi in a warm-temperate South African estuary

Reproductive biologies (spawning period, length at maturity and fecundity) of the Knysna sand goby Psammogobius knysnaensis and the Prison goby Caffrogobius gilchristi were investigated between September 2018 and August 2019 in the warm-temperate Sundays Estuary, South Africa. The two species reproduced during the same period (spring and summer) with peak reproduction occurring in January. P. knysnaensis egg production was higher than C. gilchristi and was not affected by fish size. Although C. gilchristi egg production increased with fish size, this was not significant. P. knysnaensis reached LM50 at smaller sizes than C. gilchristi. Reproductive periods were likely coupled with prey availability, as phytoplankton and zooplankton abundances are highest in warm-temperate estuaries during this period. The ability of these gobies to reproduce at small sizes (< 40 mm TL), which is an adaptive trait of many r-selected species, underpins their high abundance in South African estuaries as they are well adapted to stochastic environments.

Gene expression profiles of Japanese precious coral Corallium japonicum during gametogenesis.

Corallium japonicum, a prized resource in Japan, plays a vital role in traditional arts and fishing industries. Because of diminished stock due to overexploitation, ongoing efforts are focused on restoration through transplantation. This study aimed to enhance our understanding of the reproductive biology of these valuable corals and find more efficient methods for sex determination, which may significantly contribute to conservation initiatives. We used 12 three-month aquarium reared C. japonicum colony fragments, conducted histological analysis for maturity and sex verification, and performed transcriptome analysis via de novo assembly and mapping using the C. rubrum transcriptome to explore gene expression differences between female and male C.japonicum. Our histological observations enabled sex identification in 33% of incompletely mature samples. However, the sex of the remaining 67% of samples, classified as immature, could not be identified. RNA-seq yielded approximately 21-31 million short reads from 12 samples. De novo assembly yielded 404,439 highly expressed transcripts. Among them, 855 showed significant differential expression, with 786 differentially expressed transcripts between females and males. Heatmap analysis highlighted 283 female-specific and 525 male-specific upregulated transcripts. Transcriptome assembly mapped to C. rubrum yielded 28,092 contigs, leading to the identification of 190 highly differentially expressed genes, with 113 upregulated exclusively in females and 70 upregulated exclusively in males. Blastp analysis provided putative protein annotations for 83 female and 72 male transcripts. Annotation analysis revealed that female biological processes were related to oocyte proliferation and reproduction, whereas those in males were associated with cell adhesion. Transcriptome analysis revealed sex-specific gene upregulation in incompletely mature C. japonicum and shared transcripts with C. rubrum, providing insight into its gene expression patterns. This study highlights the importance of using both de novo and reference-based assembly methods. Functional enrichment analysis showed that females exhibited enrichment in cell proliferation and reproduction pathways, while males exhibited enrichment in cell adhesion pathways. To the best of our knowledge, this is the first report on the gene expressions of each sex during the spawning season. Our findings offer valuable insights into the physiological ecology of incompletely mature red Japanese precious corals and suggest a method for identifying sex using various genes expressed in female and male individuals. In the future, techniques such as transplantation, artificial fertilization, and larval rearing may involve sex determination methods based on differences in gene expression to help conserve precious coral resources and ecosystems.

Soybean (Glycine max L. Merrill): A Multipurpose Legume Shaping Our World

Soybean (Glycine max L. Merrill), indigenous to East Asia, has assumed a pivotal role as a global substantial crop, substantially contributing to food security, a wide spectrum of product lines and rigorous scientific exploration. This unassuming legume, bearing the scientific nomenclature Glycine max L. Merrill, has transcended its East Asian origins to become a cornerstone in supplying 25% of the world's edible oil and approximately two-thirds of the global livestock protein concentrate. The significance of soybeans extends beyond their utility in direct human consumption; they serve as an indispensable component in crop rotation, thereby fostering soil health and averting erosion. This comprehensive review embarks on a profound exploration of soybeans, encompassing their historical significance, botanical attributes, geographical distribution, climatic influence and reproductive biology. Furthermore, it provides a detailed account of advancements in genetic enhancement and biotechnological applications within soybean breeding, emphasizing the importance of transgenic soybean production. Disease resistance efforts, including the identification of resistance loci, markers and mutations, are thoroughly discussed, with a specific focus on the improvement of Indian soybean varieties.

High-Quality, Chromosome-Level Reference Genomes of the Viviparous Caribbean Skinks Spondylurus nitidus and S. culebrae.

New World mabuyine skinks are a diverse radiation of morphologically cryptic lizards with unique reproductive biologies. Recent studies examining population-level data (morphological, ecological, and genomic) have uncovered novel biodiversity and phenotypes, including the description of dozens of new species and insights into the evolution of their highly complex placental structures. Beyond the potential for this diverse group to serve as a model for the evolution of viviparity in lizards, much of the taxonomic diversity is concentrated in regions experiencing increasing environmental instability from climate and anthropogenic change. Consequently, a better understanding of genome structure and diversity will be an important tool in the adaptive management and conservation of this group. Skinks endemic to Caribbean islands are particularly vulnerable to global change with several species already considered likely extinct and several remaining species either endangered or threatened. Combining PacBio long-read sequencing, Hi-C, and RNAseq data, here we present the first genomic resources for this group by describing new chromosome-level reference genomes for the Puerto Rican Skink Spondylurus nitidus and the Culebra Skink S. culebrae. Results indicate two high quality genomes, both ∼1.4 Gb, assembled nearly telomere to telomere with complete mitochondrion assembly and annotation.

The Mortiño (Vaccinium floribundum Kunth): a review of its suitability as a promissory crop in the Ecuadorian Paramo and its potential uses, environmental role, and health benefits

AbstractThe mortiño (Vaccinium floribundum Kunth), belonging to the Ericaceae family, is a native species of the Ecuadorian paramos. It has a shrub-like habit and produces edible fruits. In the Neotropics, it is primarily found in the Andes of Colombia and Ecuador, thriving in humid environments up to an elevation of 3700 m above sea level. In this review, a selection of studies was carried out that evaluated the taxonomy, reproduction biology, and nutraceutical, environmental properties, and industrial use of the mortiño (V. floribundum). The data gathered from various bioassays were essential in determining the appropriate techniques for tissue differentiation and assessing the quality of resulting plants. This work aims to generate a deep knowledge of the cultivation of mortiño, as well as the properties of its fruits and the benefits they provide for health. These are rich in compounds with antioxidant activity, so the consumption of V. floribundum fruits is related to health benefits. Besides, the environmental role of V. floribundum and its applications in various industries, especially in the development of nanoparticles contributes to the valorization of this plant. Overall, this research contributes to establishing sustainable methods for the propagation of Vaccinium floribundum, ensuring its successful cultivation and utilization for both commercial purposes and ecological preservation.

Assessing the role of EDTA and SA in mustard under Cd and Pb stress

Phytoextraction is an economically viable and environmentally sustainable method for approaching heavy metal-polluted soils. Pollen grains are notably more susceptible to pollutants compared to other parts of the plant. However, in polluted environments, certain metals like lead (Pb) and cadmium (Cd) may have limited bioavailability, posing significant challenges to the phytoextraction process. One way to overcome this limitation is by employing chelators. The experiment was conducted in a randomized block design to investigate the effects of two chelators, EDTA and salicylic acid (SA), in enhancing the plant water status, biochemicals and reproductive parameters of Brassica juncea seedlings. These seedlings were sown in pots and subjected to various treatments of Cd and Pb of concentrations 10 and 20 mM, either alone or in combination with 0.5 mM EDTA and SA. The toxicity induced by Pb and Cd in B. juncea was recorded through decreased levels of osmotic potential in leaves and roots and decreased leaf chlorophyll content. Additionally, various reproductive parameters, including pollen viability, pollen germination, and in-vitro pollen germination, were highly affected by stress. Conversely, the supplementation of EDTA and SA to Cd, and Pb-treated plants effectively mitigated the negative impact of heavy metal stress by increasing osmotic potential, chlorophyll content, and reproductive parameters. Notably, EDTA and SA also significantly increased Pb and Cd accumulation. The result demonstrates the potential of EDTA and SA in improving plant reproductive biology.

A hundred-year-old mystery—the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae)

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group’s reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).