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Background: Chromatin configuration of the germinal vesicle (GV) and quality of the cytoplasm are critical factors in achieving oocyte meiotic and developmental capacity during folliculogenesis. Besides gaining new insights into the timing and cellular mechanisms associated with the acquisition and regulation of GV oocyte competence, the domestic cat model was used to examine (i) the relation between GV chromatin configuration and oocyte functionality during folliculogenesis and (ii) the role of the cytoplasmic environment on the GV competence and stability.Methods: Structural and functional properties of GV oocytes were characterized after isolation from different follicle stages of non-stimulated cat ovaries. GV transfers, artificial chromatin compaction and oocyte vitrification were used to demonstrate the respective roles of GV and cytoplasm on the oocyte functionality.Results: GVs acquired the intrinsic capability to resume meiosis during the pre-antral follicle stage, whereas the capacity to support embryo development occurred while the antrum started to form. Chromatin configuration of the GV did not undergo extensive modification during the acquisition of competence or during the arrest of transcriptional activity at the large antral follicle stage. However, the quality and quantity of the cytoplasm regulated and enhanced GV functionality. This finding also held for GVs transferred from incompetent or subpar oocytes into the cytoplasm of good quality oocytes or when chromatin was artificially modified or vitrified.Conclusions: The cat model provides a new insight into GV oocyte structure and function during folliculogenesis while challenging current concepts about oocyte quality criteria based on the GV morphology. This suggests alternative evaluative approaches for oocytes from other species too, including humans. Cat GVs also appear competent at an early follicle stage and are resilient to perturbations which designate this organelle as an attractive target for developing novel fertility preservation tactics. [ABSTRACT FROM AUTHOR]

It has long been thought that exocytosis was driven exclusively by well-studied fusion proteins. Some decades ago, the role of lipids became evident and escalated interest in the field. Our laboratory chose a particular cell to face this issue: the human sperm. What makes this cell special? Sperm, as terminal cells, are characterized by their scarcity of organelles and the complete absence of transcriptional and translational activities. They are specialized for a singular membrane fusion occurrence: the exocytosis of the acrosome. This unique trait makes them invaluable for the study of exocytosis in isolation. We will discuss the lipids' role in human sperm acrosome exocytosis from various perspectives, with a primary emphasis on our contributions to the field. Sperm cells have a unique lipid composition, very rare and not observed in many cell types, comprising a high content of plasmalogens, long-chain, and very-long-chain polyunsaturated fatty acids that are particular constituents of some sphingolipids. This review endeavors to unravel the impact of membrane lipid composition on the proper functioning of the exocytic pathway in human sperm and how this lipid dynamic influences its fertilizing capability. Evidence from our and other laboratories allowed unveiling the role and importance of multiple lipids that drive exocytosis. This review highlights the role of cholesterol, diacylglycerol, and particular phospholipids like phosphatidic acid, phosphatidylinositol 4,5-bisphosphate, and sphingolipids in driving sperm acrosome exocytosis. Furthermore, we provide a comprehensive overview of the factors and enzymes that regulate lipid turnover during the exocytic course. A more thorough grasp of the role played by lipids transferred from sperm can provide insights into certain causes of male infertility. It may lead to enhancements in diagnosing infertility and techniques like assisted reproductive technology (ART). [ABSTRACT FROM AUTHOR]

The North American cheetah (Acinonyx jubatus) population serves as both an insurance population for their rapidly decreasing wild cohorts as well as a research population to understand the unique biology of this species. This review focus on the complexity of the female cheetah reproductive system and the recent advances that have been made towards understanding basic biology and reproductive function, and application of assisted breeding technologies to enhance reproduction and maintain genetic diversity of this species in human care. Cheetah females are non-seasonal breeders that exhibit lengthy periods of anestrus that are not associated with age, environment, or reproductive potential. It is possible to collect good quality oocytes, that support fertilisation and successful early embryonic development, regardless of female age (from 2 to 12 yr old). However, the prevalence of uterine pathologies increases with age and prevents middle to advanced age females from establishing pregnancy. Pregnancy can be diagnosed in non-sedated cheetah females via ultrasonography (first month), steroid hormone analysis (second/third month) or radiography (third month). Fecal biomarkers, such as Immunoglobulin J, show great promise for diagnosing pregnancy at an early stage as well as other physiological states. Several decades of basic research have led to efficient management of natural breeding and recent successes in assisted reproduction. • Cheetah females are non-seasonal breeders and exhibit lengthy periods of anestrus that are not associated with age, environment, or reproductive potential. • Good quality oocytes, that support fertilisation and successful early embryonic development, can be harvested from females regardless of age (from 2 to 12 yr old). • Pregnancy can be diagnosed in cheetah females via ultrasonography (first trimester), steroid hormone analysis (second/third) or radiography (third trimester). • Fecal biomarkers, such as Immunoglobulin J, show promise for diagnosing pregnancy at an early stage as well as other physiological states. [ABSTRACT FROM AUTHOR]

Simple Summary: Gonadal tissue transplantation as a fertility preservation technique is highly conditioned by our current knowledge of functionality following transplantation, tissue cryodamage, and ischemia–reperfusion injury. This paper presents an updated review of the literature on mitochondrial dysfunction and oxidative stress in the context of gonadal tissue cryopreservation and transplantation. Advances in tissue preservation techniques have allowed reproductive medicine and assisted reproductive technologies (ARTs) to flourish in recent years. Because radio- and chemotherapy procedures are often gonadotoxic, irreversible damage can preclude future gamete production and endocrine support. Accordingly, in recent years, the freezing and storage of gonadal tissue fragments prior to the first oncological treatment appointment and autologous transplantation post-recovery have been considered improved solutions for fertility recovery in cancer survivors. Nevertheless, the cryopreservation and transplantation of thawed tissues is still very limited, and positive outcomes are relatively low. This review aims to discuss the limitations of oncofertility protocols with a focus on the impacts of mitochondrial dysfunction, oxidative stress, and the loss of antioxidant defense in graft integrity. [ABSTRACT FROM AUTHOR]

Simple Summary: Sperm acquire the ability to fertilize the egg during their transit through the female reproductive tract. This process, known as sperm capacitation, is well recognized in mammals and can be accomplished under laboratory conditions using specialized media. However, it remains unknown whether this process occurs in lizards. In this study, we investigated sperm incubation under conditions that promote capacitation to determine if similar changes occurred in their sperm. Our sperm assessment revealed functional changes, such as modifications in movement and staining patterns, commonly observed in mammals. This suggests that sperm capacitation may occur in this group of animals. Understanding sperm physiology is crucial for developing assisted reproduction technologies to aid conservation efforts for threatened species. Sperm capacitation involves biochemical and physiological changes that enable sperm to fertilize the oocyte. It can be induced in vitro under controlled conditions that simulate the environment of the oviduct. While extensively studied in mammals, its approach in lizards remains absent. Understanding the mechanisms that ensure reproduction is essential for advancing the implementation of assisted reproductive technologies in this group. We aimed to perform a sperm analysis to determine if capacitation-related changes were induced after incubation with capacitating media. Fifteen males of Sceloporus torquatus were collected during the early stage of the reproductive season. The sperm were isolated from the seminal plasma and then diluted up to a volume of 150 μL using BWW medium to incubate with 5% CO2 at 30 °C for a maximum duration of 3 h. A fraction was retrieved hourly for ongoing sperm assessment. The sperm analysis included assessments of its motility, viability, the capacitation status using the chlortetracycline (CTC) assay, and the acrosome integrity with the lectin binding assay to detect changes during incubation. We found that total motility was maintained up to 2 h post incubation, after which it decreased. However, sperm viability remained constant. From that moment on, we observed a transition to a deeper and less symmetrical flagellar bending in many spermatozoa. The CTC assay indicated a reduction in the percentage of sperm showing the full (F) pattern and an increase in those exhibiting the capacitated (B) and reactive (RA) patterns, accompanied by an elevation in the percentage of damaged acrosomes as revealed by the lectin binding assay. In mammals, these changes are often associated with sperm capacitation. Our observations support the notion that this process may also occur in saurian. While sperm analysis is a valuable method for assessing certain functional changes, additional approaches are required to validate this process. [ABSTRACT FROM AUTHOR]

The application of reproductive technologies to amphibian conservation breeding programs is gaining momentum and the field is poised to contribute significantly toward amphibian species recovery. We briefly discuss the opportunities for reproductive technologies to enhance conservation breeding outcomes, including their potential to enhance the genetic management, and in turn, the fitness of threatened species. Despite this potential, an important consideration that is not yet well understood is the degree to which specific reproductive technologies might influence (either enhance, or in some instances potentially decrease) individual fitness and lead to shifts in population viability. The development of a standardised approach to monitoring offspring throughout life-stages to detect changes to morphology, behaviour, physiology, survivorship, and developmental trajectories is essential. The primary focus of this review is to provide a 'best-practise' framework for quantifying key fitness determining traits expected to contribute to the fitness of individuals and long-term viability of populations, which will ultimately allow us to progress the field of amphibian reproductive technologies and assess the impact of protocol refinement. [ABSTRACT FROM AUTHOR]

The term 'mitochondrial vesicle' was first used in 2003 in a description of anuran sperm and persists to this day throughout the literature on assisted reproductive technologies (ART) for amphibians. In the present paper, we argue that the term is inappropriate because the widely accepted definition of a 'vesicle' refers to an integral structure with an enclosing lipid bilayer/membrane. Moreover, there are no electron micrographs that show a vesicular structure encapsulating mitochondria on amphibian sperm heads in the literature. In fact, in 1993, the mitochondria in the anuran sperm head had been described as positioned in 'mitochondrial collars ' or 'mitochondrial sheaths' surrounded by the plasma membrane of the sperm head. On the other hand, mitochondrial-derived vesicles are defined as vesicles shed from mitochondria surfaces, potentially creating confusion. Therefore, our view is that the term 'mitochondrial vesicle' should be avoided in describing the positioning of mitochondria on sperm. The term 'mitochondrial vesicle' was first used in 2003 in a description of anuran sperm and persists to this day in the literature on assisted reproductive technologies (ART) for amphibians. In the present paper, we argue that the term 'mitochondrial vesicle' is inappropriate because the widely accepted definition of a 'vesicle' refers to an integral structure with an enclosing lipid bilayer/membrane. Photograph by Robert Browne. This article belongs to the Collection Dedication to Jim Cummins. [ABSTRACT FROM AUTHOR]

The utilization of assisted reproductive technologies (ART) is on the rise, resulting in a growing population of ART-conceived offspring. The health concerns of this unique population have attracted significant attention. During ART procedures, gametes and early-stage embryos are exposed to various non-physiological conditions, such as manipulation, culture media, and cryopreservation, which may disrupt embryonic development and potentially impact the health of offspring. Notably, the potential impact of ART on neurodevelopment and its association with an increased risk of neurodevelopmental disorders (NDD) later in life remains a subject of debate. This review aims to summarize the current research advancements concerning the effects of ART on neurodevelopment, specifically focusing on the evidence of the relationship between ART, epigenetic modifications, and NDD, including autism spectrum disorder, intellectual disability, attention deficit hyperactivity disorder, and cerebral palsy. Future studies should prioritize large sample sizes, rigorous adjustment for confounding factors, and the use of interdisciplinary approaches to effectively monitor the neurodevelopmental outcomes of ARTconceived children. [ABSTRACT FROM AUTHOR]

This review aims to explore advanced reproductive technologies for male fertility preservation, underscoring the essential role that animal models have played in shaping these techniques through historical contexts and into modern applications. Rising infertility concerns have become more prevalent in human populations recently. The surge in male fertility issues has prompted advanced reproductive technologies, with animal models playing a pivotal role in their evolution. Historically, animal models have aided our understanding in the field, from early reproductive basic research to developing techniques like artificial insemination, multiple ovulation, and in vitro fertilization. The contemporary landscape of male fertility preservation encompasses techniques such as sperm cryopreservation, testicular sperm extraction, and intracytoplasmic sperm injection, among others. The relevance of animal models will undoubtedly bridge the gap between traditional methods and revolutionary next-generation reproductive techniques, fortifying our collective efforts in enhancing male fertility preservation strategies. While we possess extensive knowledge about spermatogenesis and its regulation, largely thanks to insights from animal models that paved the way for human infertility treatments, a pressing need remains to further understand specific infertility issues unique to humans. The primary aim of this review is to provide a comprehensive analysis of how animal models have influenced the development and refinement of advanced reproductive technologies for male fertility preservation, and to assess their future potential in bridging the gap between current practices and cutting-edge fertility techniques, particularly in addressing unique human male factor infertility. [ABSTRACT FROM AUTHOR]

Collagen-based biomaterials (CBB) are highly esteemed by researchers in materials science and biomedicine due to their extensive applications across various biomedical disciplines. In recent years, owing to advancements in developmental biology techniques, this superior biomaterial has seen increasing utilization in 3D in vitro tissue culture. Three-dimensional cell cultures, often referred to as organoids, have emerged in response to technological advancements in biomaterials and the growing need in the field of medical research. They serve as important models for simulating normal physiological activities in vivo, addressing limitations in experimental material sources, and resolving ethical issues. In this review, we discuss the material characteristics of CBBs commonly used for organoid culture, integrating aspects such as Matrigel and decellularized ECM as culture matrices. We also analyzed the development prospects and directions of various materials in the context of biology, clinical medicine, and particularly reproductive medicine. Currently, despite the FDA approval and clinical research incorporating numerous CBBs, existing challenges in multiple studies indicate a significant unmet need in the development of key tissue models for both medical research and clinical applications. In summary, CBBs are swiftly broadening their applicability in the realms of organoid nature and medical research, serving as a versatile and high-performing material for 3D in vitro tissue culture. [ABSTRACT FROM AUTHOR]

Fertility preservation technologies have existed for decades, and the field is rapidly advancing; limited data exist regarding the use of these technologies by transgender patients. Many options are available for transgender patients who wish to preserve fertility before transitioning. These options include the cryopreservation of gametes, embryos, or ovarian tissue. Currently, ejaculated, or testicular sperm, immature oocytes, and ovarian tissue can be preserved for later use, but no such use option exists for immature testicular tissue. Many financial, sociological, and legal barriers and a lack of awareness among physicians and patients also hinders the utilization of these fertility preservation services. While options are abundant, usage rates are relatively low. The initial data regarding the successful use of preserved tissues appears promising, with birth rates not dissimilar to non-transgender patients. Further investigations into this area are needed. In addition, counseling regarding fertility preservation options should become a significant part of the provider-patient conversation before transitioning therapies. [ABSTRACT FROM AUTHOR]

The present study aimed to test the efficiency of transcervical artificial insemination techniques with cervical immobilization (TCAI-CI) or cervical traction (TCAI-CT), associated or not with the use of oxytocin (OT) as a protocol for cervical dilation, in the brown brocket deer (Subulo gouazoubira). The study was carried out in a crossover design using four adult females in two replicates with an interval of 60 days. Estrus was synchronized with oral melengestrol acetate (MGA) associated with estradiol benzoate and sodium cloprostenol. TCAI techniques were performed from 18 to 24 h after estrus onset. All females received either an i.v. application of 50 IU of OT (G-OT, n = 4) or 1 mL of saline solution (G-Control, n = 4) 20 min before the TCAI procedure. The TCAIs were performed using frozen-thawed semen motility 40%, vigor 3, acrosome integrity 87%, membrane integrity of 95% and 13% of total post-thaw defects from the same batch. Behavioral estrus was observed in 100% of the females, in both replicates. It was achieved a 50% (4/8) success of cervical transposition with semen deposition in the uterine. Regarding inseminations, most of them (87.5%) were performed using the TCAI-CT technique, and the overall conception rate was 50%. Cervical transposition times (< 1 min) and TCAI procedures (~ 17 min) were considered satisfactory. Thus, the performance of the TCAI-CI and TCAI-CT techniques was successful, regardless of using OT as a cervical dilation protocol. This procedure is proposed as a method of choice for artificial insemination with greater applicability in different conservation centers, compared to more advanced reproductive biotechniques, and with a favorable impact on the conservation of deer species. [ABSTRACT FROM AUTHOR]

Sperm DNA fragmentation (SDF) that occurs during the freezing–thawing of sperm may negatively impact the treatment outcomes of assisted reproductive technologies (ART). In a previous study, we developed a human sperm cryopreservation reagent containing carboxylated poly-L-lysine (CPLL) that reduced SDF after freeze-thawing compared with clinically popular cryopreservation reagents containing human serum albumin. However, it is unclear whether CPLL reduces SDF, as it differed from the constituents of the commercial cryopreservation reagents used for comparison. Therefore, here, we examined whether CPLL reduces the SDF of human sperm and evaluated reactive oxygen species (ROS) levels and lipid peroxidation (LPO), which are the causes of SDF; mitochondrial injury, ROS production; and impaired sperm motility. Furthermore, optimal antioxidants and their concentrations that could further enhance the reduction in SDF were determined for future clinical application in ART and underwent the same functional evaluations. CPLL can reduce SDF via inhibition of intracytoplasmic ROS and LPO. Furthermore, the addition of 0.1 mM resveratrol avoided the enhancement of SDF, which potentially affects mitochondrial and cytoplasmic ROS and LPO. This novel human sperm cryopreservation reagent containing CPLL and resveratrol has the potential to improve treatment outcomes in ART using frozen sperm. [ABSTRACT FROM AUTHOR]

The northern white rhinoceros is functionally extinct with only two females left. Establishing methods to culture ovarian tissues, follicles, and oocytes to generate eggs will support conservation efforts using in vitro embryo production. To the best of our knowledge, this is the first description of the structure and molecular signature of any rhinoceros, more specifically, we describe the neonatal and adult southern white rhinoceros (Ceratotherium simum simum) ovary; the closest relation of the northern white rhinoceros. Interestingly, all ovaries contain follicles despite advanced age. Analysis of the neonate reveals a population of cells molecularly characterised as mitotically active, pluripotent with germ cell properties. These results indicate that unusually, the neonatal ovary still contains oogonia in germ cell nests at birth, providing an opportunity for fertility preservation. Therefore, utilising ovaries from stillborn and adult rhinoceros can provide cells for advanced assisted reproductive technologies and investigating the neonatal ovaries of other endangered species is crucial for conservation. A comprehensive histological assessment of southern white rhinoceros ovaries from both mature females and a neonate provides insight into follicular development in this species. [ABSTRACT FROM AUTHOR]

Simple Summary: The development of reproductive technologies for amphibian species has accelerated over the last decade to the point that the industry implementation of these methodologies to assist breeding and genetic management is underway globally. A conservation breeding program was established at Taronga Conservation Society (Australia) in 2019 for the critically endangered Booroolong frog (Litoria booroolongensis) following an emergency extraction from the wild. Shortly thereafter, a trial was undertaken to compare the efficacy of two sperm cryopreservation protocols with a view to creating a "sperm bank" from these genetically important founder individuals. Spermic urine samples from L. booroolongensis were successfully cryopreserved using both methods tested; however, higher numbers of sperm retained motility post-thaw when using a faster cooling method for cryopreservation. Moreover, the two cryoprotectants tested were equally effective at supporting sperm survival and function post-thaw, regardless of cooling speed. We utilized the method outlined in this paper to collect and cryopreserve spermic urine samples across three breeding seasons from all (n = 28) L. booroolongensis founder males at Taronga and are now investigating the use of these samples to create offspring via assisted fertilization. The cryopreservation and storage of gametes (biobanking) can provide a long-term, low-cost option for the preservation of population genetic diversity and is particularly impactful when applied to manage selective breeding within conservation breeding programs (CBPs). This study aimed to develop a sperm cryopreservation protocol for the critically endangered Booroolong frog (Litoria booroolongensis) to capture founder genetics within the recently established (est. 2019) CBP for this species. Hormone-induced sperm release was achieved using established protocols, and spermic urine samples were collected over a 6-h period. Pooled spermic urine samples (n = 3 males) were divided equally between two cryoprotectant (CPA) treatments and diluted by 1:5 (sperm:CPA) with either 15% (v/v) dimethyl sulfoxide + 1% (w/v) sucrose in simplified amphibian Ringer's (SAR; CPAA) or 10% (v/v) dimethylformamide + 10% (w/v) trehalose dihydrate in SAR (CPAB). The samples were cryopreserved in 0.25 mL straws using either a programmable freezer (FrA) or an adapted dry shipper method (FrB). The thawed samples were activated via dilution in water and assessed for viability and motility using both manual assessment and computer-assisted sperm analysis (CASA; 0 h, 0.5 h post-thaw). Upon activation, the survival and recovery of motility (total motility, forward progression and velocity) of cryopreserved sperm suspensions were higher for sperm preserved using FrB than FrA, regardless of CPA composition. This work supports our long-term goal to pioneer the integration of biobanked cryopreserved sperm with population genetic management to maximize restoration program outcomes for Australian amphibian species. [ABSTRACT FROM AUTHOR]

Simple Summary: Recurrent successful breeding of the critically endangered Vanishing Rainfrog (Craugastor evanesco) in captivity has been challenging. Therefore, the objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this species in the near future. The Gonadotropin-Releasing Hormone agonist, at a concentration of 4 micrograms per gram of body weight, stimulated the production of high-quantity and quality spermatozoa. This study also described the morphology of C. evanesco spermatozoa for the first time. These results contributed new and valuable knowledge on the reproductive biology of C. evanesco and for the development of other assisted reproductive technologies that might be applied to this and other species' conservation programs. The Vanishing Rainfrog (Craugastor evanesco) is an endemic and critically endangered frog species of Panama. It is suspected that 90% of the population has disappeared from the wild. Frogs were collected from the wild and brought to a Captive Breeding Program; however, accomplishing regular reproductive events for this species has been difficult. The objective of this study was to determine the effect of hormonal stimulation on the production and quality of C. evanesco spermatozoa, aiming to develop an efficient and safe sperm collection protocol as a tool to help reproduce this endangered species. Mature males received intra-peritoneal injections with one of six hormone treatments, including des-Gly10, D-Ala6, Pro-NHEt9—GnRH-A, Amphiplex or hCG. Urine samples were collected at 10 different time points post-injection. Quality assessments included sperm concentration, percentage motility, percentage forward progressive motility (FPM), osmolality, pH and morphology analysis. Our results indicate that the optimal treatment for the collection of highly concentrated sperm samples of C. evanesco is 4 µg/gbw GnRH, followed by Amphiplex and 2 µg/gbw GnRH as sub-optimal treatments and finally, 6 µg/gbw GnRH and 5 and 10 IU/gbw hCG as non-optimal treatments. GnRH-A at 4 μg/gbw and Amphiplex stimulated the production of samples with the highest sperm concentrations and quality, despite Amphiplex producing lower percentages of intact acrosome and tail. In contrast, hCG concentrations were not reliable inducers of sperm production, consistently showing lower concentrations, higher percentages of sperm abnormalities and more acidic spermic urine than that induced by Amphiplex and GnRH-A. Morphological assessments revealed that C. evanesco spermatozoa have a filiform shape with a large acrosome on the anterior part of an elongated head, a small midpiece and a long tail with two filaments joined together by an undulating membrane. [ABSTRACT FROM AUTHOR]

Simple Summary: Oxidatively stressed sperm can fertilise oocytes and, hence, transfer its damaged genome to a progeny. Sperm cannot repair its own genome, therefore the oocyte's capacity to deal with damaged sperm influences the outcome of a fertilisation. We distinguished bovine oocytes of different qualities in vitro using Brilliant Cresyl Blue (BCB) staining and in vitro fertilised them with non-oxidised and oxidised bovine semen. BCB-negative oocytes were less able to support embryo development after fertilisation with oxidatively stressed bovine semen, demonstrated by lower cleavage and blastocyst rates, a delay in development and lower blastocyst quality. These results support a further description of the oocytes' quality in regard of their capacity to deal with damaged sperm, which will help in selecting the best oocytes when damaged sperm have to be used in assisted reproduction. The extent of oxidative damage transferred by the damaged sperm to the progeny is likely to be limited by the oocyte's repair and antioxidative capacity. We aimed to assess the association between Brilliant Cresyl Blue (BCB) staining in oocytes and their competence for embryo development after in vitro fertilisation (IVF) with damaged sperm. For this purpose, bovine sperm were incubated without (non-oxidised sperm, NOX S) or with 100 µM H2O2 (oxidised sperm, OX S) and were used to fertilise in-vitro-matured bovine oocytes (BCB-pos./BCB-neg.). Unstained oocytes served as controls (US). Development was assessed at 30, 46, 60 h and on Days (D) 7 and 8 after IVF. Total cell number and apoptotic index were analysed in D7 blastocysts. BCB-neg. oocytes showed lower cleavage rates and blastocyst rates than unstained oocytes after IVF with NOX S (p < 0.05). They showed the highest reduction in D7 blastocyst rate upon fertilisation with OX S and showed a delayed embryo development at 46 and 60 h after IVF compared to embryos produced with NOX S (p < 0.05). Total cell number in blastocysts produced with BCB-neg. oocytes was lower (p < 0.05) in the embryos produced with OX S than in embryos after IVF with NOX S. In conclusion, BCB-neg. oocytes have a lower competence to support embryo development after in vitro fertilisation with oxidised sperm. [ABSTRACT FROM AUTHOR]

Summary: Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed. [ABSTRACT FROM AUTHOR]

The primary role of semen processing and preservation is to maintain a high proportion of structurally and functionally competent and mature spermatozoa, that may be used for the purposes of artificial reproduction when needed, whilst minimizing any potential causes of sperm deterioration during ex vivo semen handling. Out of a multitude of variables determining the success of sperm preservation, bacterial contamination has been acknowledged with an increased interest because of its often unpredictable and complex effects on semen quality. Whilst antibiotics are usually the most straight-forward option to prevent the bacterial contamination of semen, antimicrobial resistance has become a serious threat requiring widespread attention. As such, besides discussing the consequences of bacteriospermia on the sperm vitality and the risks of antibiotic overuse in andrology, this paper summarizes the currently available evidence on alternative strategies to prevent bacterial contamination of semen prior to, during, and following sperm processing, selection, and preservation. Alternative antibacterial supplements are reviewed, and emphasis is given to modern methods of sperm selection that may be combined by the physical removal of bacteria prior to sperm preservation or by use in assisted reproductive technologies. [ABSTRACT FROM AUTHOR]

Simple Summary: The integration of reproductive technologies into conservation breeding programs can contribute to the propagation and genetic management of threatened amphibian species. Here, we report on the first application of reproductive technologies to the critically endangered Baw Baw frog, Philoria frosti. We present the results of the successful use of protocols for hormone-induced spawning in male–female pairs, hormone-induced sperm-release in isolated males, and sperm cryopreservation. Reproductive technologies (RTs) can assist integrated conservation breeding programs to attain propagation targets and manage genetic diversity more effectively. While the application of RTs to enhance the conservation management of threatened amphibians has lagged behind that of other taxonomic groups, a recent surge in research is narrowing the divide. The present study reports on the first application of RTs (hormone-induced spawning, hormone-induced sperm-release, and sperm cryopreservation) to the critically endangered Baw Baw frog, Philoria frosti. To determine the effect of hormone therapy on spawning success, male–female pairs were administered either 0 μg/g gonadotropin-releasing hormone agonist (GnRHa), 0.5 μg/g GnRHa, or 0.5 μg/g GnRHa + 10 μg/g metoclopramide (MET) (n = 6–7 pairs/treatment), and the number of pairs ovipositing, total eggs, and percent fertilisation success were quantified. To determine the effect of hormone therapy on sperm-release and to establish the peak time to collect sperm post-hormone administration, males were administered 0 IU/g (n = 4), or 20 IU/g hCG (n = 16). Total sperm, sperm concentration, and percent viability were quantified at 0, 2, 4, 6, 8, 10, and 12 h post-hormone administration. Overall, the percentage of pairs ovipositing was highest in the GnRHa + MET treatment, with 71% of pairs ovipositing, compared to 57% and 33% of pairs in the GnRHa and control treatments, respectively. The quantity of sperm released from males in response to hCG peaked at 4 h post-hormone administration, though it remained high up to 12 h. The percent sperm viability also peaked at 4 h post-administration (94.5%), exhibiting a steady decline thereafter, though viability remained above 77% throughout the 12 h collection period. The remaining sperm samples (n = 22) were cryopreserved using established protocols and biobanked for long-term storage and future conservation applications. The mean post-thaw sperm viability was 59%, and the percent total motility was 17%. The results from this preliminary study will direct further applications of RTs to the critically endangered Baw Baw frog to assist with species recovery. [ABSTRACT FROM AUTHOR]

Simple Summary: Integrating reproductive technologies into amphibian conservation breeding programs can enhance the propagation and the genetic management of threatened species. Advances in cold storage and cryopreservation of amphibian sperm, coupled with assisted fertilisation technologies, are valuable tools for long-term genetic management and are being applied to an increasing number of threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and long-term cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring. Multidisciplinary approaches to conserve threatened species are required to curb biodiversity loss. Globally, amphibians are facing the most severe declines of any vertebrate class. In response, conservation breeding programs have been established in a growing number of amphibian species as a safeguard against further extinction. One of the main challenges to the long-term success of conservation breeding programs is the maintenance of genetic diversity, which, if lost, poses threats to the viability and adaptive potential of at-risk populations. Integrating reproductive technologies into conservation breeding programs can greatly assist genetic management and facilitate genetic exchange between captive and wild populations, as well as reinvigorate genetic diversity from expired genotypes. The generation of offspring produced via assisted fertilisation using frozen–thawed sperm has been achieved in a small but growing number of amphibian species and is poised to be a valuable tool for the genetic management of many more threatened species globally. This review discusses the role of sperm storage in amphibian conservation, presents the state of current technologies for the short-term cold storage and cryopreservation of amphibian sperm, and discusses the generation of cryo-derived offspring. [ABSTRACT FROM AUTHOR]

Ovarian tissue cryopreservation (OTC) or testicular tissue cryopreservation (TTC) are effective and often the only options for fertility preservation in female or male patients due to oncological, medical, or social aspects. While TTC and resumption of spermatogenesis, either in vivo or in vitro, has still be considered an experimental approach in humans, OTC and autotransplantation has been applied increasingly to preserve fertility, with more than 200 live births worldwide. However, the cryopreservation of reproductive cells followed by the resumption of gametogenesis, either in vivo or in vitro, may interfere with sensitive and highly regulated cellular processes. In particular, the epigenetic profile, which includes not just reversible modifications of the DNA itself but also post-translational histone modifications, small non-coding RNAs, gene expression and availability, and storage of related proteins or transcripts, have to be considered in this context. Due to complex reprogramming and maintenance mechanisms of the epigenome in germ cells, growing embryos, and offspring, OTC and TTC are carried out at very critical moments early in the life cycle. Given this background, the safety of OTC and TTC, taking into account the epigenetic profile, has to be clarified. Cryopreservation of mature germ cells (including metaphase II oocytes and mature spermatozoa collected via ejaculation or more invasively after testicular biopsy) or embryos has been used successfully for many years in medically assisted reproduction (MAR). However, tissue freezing followed by in vitro or in vivo gametogenesis has become more attractive in the past, while few human studies have analysed the epigenetic effects, with most data deriving from animal studies. In this review, we highlight the potential influence of the cryopreservation of immature germ cells and subsequent in vivo or in vitro growth and differentiation on the epigenetic profile (including DNA methylation, post-translational histone modifications, and the abundance and availability of relevant transcripts and proteins) in humans and animals. [ABSTRACT FROM AUTHOR]

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The ongoing rapid climate change, combined with the disturbance of fish breeding grounds, may impact reproduction by endangering successful breeding and survival, and thus affect the viable sustainability in aquaculture systems as well as in the sea. In this study we focus on the biocomplexity of fish reproduction in response to climate change. Further, we propose adaptive strategies, including technological advancements, using a noninvasive and non-lethal approach, and we outline an assisted reproduction and nutrigenomics approach to mitigating fish reproductive risks posed by climate change. This was done in an effort to monitor fish aquaculture and ensure that, as a livelihood, it may provide a useful source of nutrition for our society. [ABSTRACT FROM AUTHOR]

Simple Summary: Constant threats to wildlife have led countless researchers to develop or improve conservation strategies, among which the formation of biobanks stands out. The systematic storage of male gametes in freezing temperatures is useful for future use in assisted reproduction and for improving the reproductive management of rare species. In this study, the effect of including Equex STM® paste or different concentrations of sodium dodecyl sulfate (SDS) on various functional metrics of frozen and thawed peccary semen, such as sperm kinetic parameters, membrane functionality and integrity, mitochondrial activity and binding capacity, was verified. In parallel, sperm longevity was evaluated through a thermal resistance test. In general, it was demonstrated that the addition of 0.1% SDS to the Tris–egg yolk–glycerol diluent favored the maintenance of the sperm kinetic parameters of peccaries during freezing and thawing procedures. We evaluated the effects of detergents based on sodium dodecyl sulfoxide (SDS) on the functional parameters of collared peccary frozen–thawed sperm. Semen aliquots from ten individuals were diluted in a Tris–egg yolk–glycerol extender alone or with 0.5% Equex STM® paste or SDS (at 0.1%, 0.3% or 0.5% (v/v) concentration). Samples were fast frozen in liquid nitrogen with a post-thaw evaluation of motility, membrane functionality and integrity, mitochondrial activity, sperm binding ability and thermal resistance. The treatments without SDS (41.8 ± 3.5%) and those containing Equex (41.8 ± 4.4%) or 0.1% SDS (41.2 ± 5.5%) provided greater sperm motility (p < 0.05) than those containing SDS 0.3% (30.5 ± 4.7%) and 0.5% (31.2 ± 6.3%). Immediately after thawing, only treatments containing 0.1% SDS effectively preserved sperm straightness (STR) when compared to the negative control. All treatments preserved the amplitude of lateral head (ALH) and straightness (STR) during a thermal resistance test (p > 0.05), but SDS 0.5% impaired the membrane functionality and mitochondrial activity after thawing (p < 0.05). All treatments provided a similar recovery of sperm binding ability after thawing (p < 0.05). Our results showed that the addition of 0.1% SDS to the Tris–yolk–glycerol extender optimized the freeze–thaw recovery of peccary semen. [ABSTRACT FROM AUTHOR]

Simple Summary: This study was conducted to compare the minimum volume vitrification method using material from female felids to protect them in wild felid conservation programs. In the experiment, the three most popular techniques for oocyte vitrification were compared in terms of their survival and developmental competence. The results show that using this method as an emergency protocol is essential to protecting the gametes of wild felids threatened with extinction in conservation programs based on assisted reproductive techniques (ART). This project aimed to compare the three most popular commercial oocyte vitrification techniques to determine their suitability for the vitrification of felid germlines in rescue and conservation programs. The present study aimed to determine the viability and developmental competence of feline oocytes after IVM and vitrification using a commercial vitrification method. In the first experiment, oocytes were vitrified after in vitro maturation (IVM) using the Kitazato, Cryotech, and Vitrolife methods. The oocytes were stained with fluorescein diacetate and ethidium bromide to evaluate their viability. The differences between Vitrolife and the control, Cryotech and Kitazato were statistically significant (p < 0.05), and between the control and Kitazato, were highly significant (p < 0.01). There were no significant differences between the control and Cryotech, Vitrolife and Cryotech, or Kitazato and Vitrolife. In the second part of the experiment, oocytes, after IVM and vitrification using three commercial methods, were subjected to fertilization. After vitrification, IVF was performed. We observed 35% of embryonic divisions in the group where Vitrolife and Kitazato media were used and 45% in the control group. In the presented experiment, vitrification with Vitrolife media gave slightly better results for survival and fertilization, while in the case of emergency protocol vitrification, all of the above methods may be useful to protect material derived from valuable wild felids. [ABSTRACT FROM AUTHOR]

The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first cell cycle indicated that progression through the pronuclear stages of SER-generated embryos is advanced in comparison with control-generated embryos. These findings prompted further analysis of potential effects of SER on pronuclear chromatin dynamics, focusing on the key H3K4me3 and H3K27ac histone modifications. Nearly all the SER-generated embryos displayed H3K4me3 in the male pronuclei at 12 h post-insemination (HPI), while a subset of the control-generated embryos did not. Additionally, SER-generated embryos displayed a more homogenous intensity of H3K27ac at 8 and 12 HPI compared to control embryos. These changes in histone modifications during the first cell cycle were accompanied by differences in gene expression at the two-cell stage; both of these changes in early embryos could potentially play a role in the improved developmental outcomes of these embryos later in development. Our results indicate that sperm incubation conditions have an impact on early embryo development and can be useful for the improvement of assisted reproductive technology outcomes. [ABSTRACT FROM AUTHOR]

The current paper presents efforts undertaken to generate biorepositories of cryogenically protected male gametes, embryos, and somatic cell lines aimed at their subsequent use for a broad spectrum of the strategies related to experimental and applied embryology. Creating repositories of biological materials derived from rare indigenous breeds of selected livestock species (sheep, goats, cattle and pigs) seems to be highly justified in view of the pivotal role of the Department of Reproductive Biotechnology and Cryoconservation (DRBC), the National Research Institute of Animal Production (NRIAP). This role is reflected in the maintenance of biodiversity as a result of NRIAP activities focused on restoration and stabilization of the effective population size of critically endangered or endangered autochthonic livestock breeds that are characterized by extremely high or relatively high inbreeding rates. Moreover, this paper also seeks to provide the state-of-the-art method and an overview of applying biorepositories with cryopreserved germplasm-carrier biological materials and somatic cell lines to a wide variety of in vitro embryo production (IVP) and assisted reproductive technologies (ARTs) targeted at not only standard and microsurgical in vitro fertilization (IVF), but also cloning by somatic cell nuclear transfer (SCNT). [ABSTRACT FROM AUTHOR]

Wild ungulates are of fundamental importance for balancing ecosystems, as well as being the species of economic interest. Increasing concern over the accelerated population reduction of these species has resulted in the development of assisted reproduction techniques, such as in vitro fertilization (IVF), as a tool for conservation and multiplication. In the present scenario, IVF protocols were developed based on the methodologies used for domestic ungulates. Nevertheless, owing to the physiological and reproductive differences among the species, several factors associated with IVF and its relationship with the characteristics of the species of interest require clarification. In vitro conditions for the collection and selection of female and male gametes, oocyte maturation, sperm capacitation, co-incubation of gametes, and embryonic development can influence IVF results. Therefore, the present review considers the main advances in the methodologies already used for wild ungulates, emphasizing the strategies for improving the protocols to obtain better efficiency rates. Additionally, we discuss the conditions of each IVF stage, with emphasis on aspects related to in vitro manipulation and comparability with the protocols for domestic ungulates. [ABSTRACT FROM AUTHOR]

Preservation of human spermatozoa in vitro at normothermia or hypothermia maintaining their functions and fertility for several days plays a significant role in reproductive biology and medicine. However, it is well known that human spermatozoa left in vitro deteriorate over time irreversibly as the consequence of various stresses such as the change of osmolarity, energy deficiency, and oxidative damage, leading to substantial limitations including the need for semen examinations, fertility preservation, and assisted reproductive technology. These problems may be addressed with the aid of non-freezing storage techniques. The main and most effective preservation strategies are the partial or total replacement of seminal plasma with culture medium, named as extenders, and temperature-induced metabolic restriction. Semen extenders consist of buffers, osmolytes, and antioxidants, etc. to protect spermatozoa against the above-mentioned adverse factors. Extended preservation of human spermatozoa in vitro has a negative effect on sperm parameters, whereas its effect on ART outcomes remains inconsistent. The storage duration, temperature, and pre-treatment of semen should be determined according to the aims of preservation. Advanced techniques such as nanotechnology and omics have been introduced and show great potential in the lifespan extension of human sperm. It is certain that more patients will benefit from it in the near future. This review provided an overview of the current knowledge and prospects of prolonged non-freezing storage of human sperm in vitro. [ABSTRACT FROM AUTHOR]

Context: With global amphibian biodiversity rapidly declining, improving reproductive technology outcomes has become essential. Captive breeding programs have struggled because amphibian breeding physiology often requires specific environmental cues that reproductive technologies can circumvent. Aims: This study tests the efficiency of hormonal induction by evaluating sperm quality in the endangered Litoria verreauxii alpina. Methods: We assessed the effects of exogenous hormones – gonadotrophin-releasing hormone agonist (GnRH-a, Lucrin), and human chorionic gonadotrophin (hCG, Chorulon) – on sperm quality. Key results: Hormone induction with hCG showed high efficacy while GnRH-a yielded a low response in producing sperm. Sperm quantity was affected by time post injection, with the greatest quantity at 1 h post injection. Sperm quality was also affected by time, where the sperm head size decreased by 11% at 7 h post injection. Conclusions: Based on the results from this study, we recommend that that sperm be collected soon after induction, and not more than 4 h post induction in L. v. alpina. More work needs to be completed before recommending an optimal hormone induction method and dose, but 120 IU of hCG per male was successful for inducing spermiation. Implications: This study represents a useful starting point for developing assisted reproductive techniques for non-model organisms. Amphibian biodiversity is rapidly declining and we must improve our ability to captive breed animals. However, artificial reproductive techniques are often species specific and different species require specific protocols. We tested two hormone treatments on sperm induction in an endangered frog and found that only one hormone was successful, and sperm quantity and quality decreased rapidly after treatment. This study represents a useful starting point for developing artificial reproductive techniques for non-model organisms to improve captive breeding strategies. [ABSTRACT FROM AUTHOR]

The objective of this review is to ask whether, and how, principles in conservation biology may need to be revisited in light of new knowledge about the power of epigenetics to alter developmental pathways. Importantly, conservation breeding programmes, used widely by zoological parks and aquariums, may appear in some cases to reduce fitness by decreasing animals' abilities to cope when confronted with the 'wild side' of their natural habitats. Would less comfortable captive conditions lead to the selection of individuals that, despite being adapted to life in a captive environment, be better able to thrive if relocated to a more natural environment? While threatened populations may benefit from advanced reproductive technologies, these may actually induce undesirable epigenetic changes. Thus, there may be inherent risks to the health and welfare of offspring (as is suspected in humans). Advanced breeding technologies, especially those that aim to regenerate the rarest species using stem cell reprogramming and artificial gametes, may also lead to unwanted epigenetic modifications. Current knowledge is still incomplete, and therefore ethical decisions about novel breeding methods remain controversial and difficult to resolve. [ABSTRACT FROM AUTHOR]

The European mink (Mustela lutreola) is one of Europe's most endangered species, and it is on the brink of extinction in the Iberian Peninsula. The species' precarious situation requires the application of new ex situ conservation methodologies that complement the existing ex situ and in situ conservation measures. Here, we report for the first time the establishment of a biobank for European mink mesenchymal stem cells (emMSC) and oocytes from specimens found dead in the Iberian Peninsula, either free or in captivity. New emMSC lines were isolated from different tissues: bone marrow (emBM-MSC), oral mucosa (emOM-MSc), dermal skin (emDS-MSC), oviduct (emO-MSc), endometrium (emE-MSC), testicular (emT-MSC), and adipose tissue from two different adipose depots: subcutaneous (emSCA-MSC) and ovarian (emOA-MSC). All eight emMSC lines showed plastic adhesion, a detectable expression of characteristic markers of MSCs, and, when cultured under osteogenic and adipogenic conditions, differentiation capacity to these lineages. Additionally, we were able to keep 227 Cumulus-oocyte complexes (COCs) in the biobank, 97 of which are grade I or II. The European mink MSC and oocyte biobank will allow for the conservation of the species' genetic variability, the application of assisted reproduction techniques, and the development of in vitro models for studying the molecular mechanisms of infectious diseases that threaten the species' precarious situation. [ABSTRACT FROM AUTHOR]

Simple Summary: Many cat species reproduce less successfully in human care than in nature. For this reason, maintaining a viable and healthy zoo population is challenging but urgently needed as a back-up for threatened cat species in the wild. Methods of assisted reproduction have gained significance to improve reproduction in captivity. These methods include, for example, egg collection and fertilization outside the body (IVF) and the transfer of embryos back to a recipient female. For the domestic cat, IVF is well-established, and also the transfer of embryos has already resulted in healthy offspring. In this study, the IVF system developed for the domestic cat was tested on the African lion. Sperm and eggs were collected from the gonads of lions that had been euthanized for age or population management reasons. The eggs were fertilized in a laboratory with fresh or frozen–thawed sperm. Embryos were generated, demonstrating that the domestic cat IVF system is also suitable for lions. Unlike in domestic cats, the lion samples had to be transported to the laboratory from zoos distributed all over Europe. The prolonged transportation led to a loss of quality of the eggs and sperm and contributed to lower overall fertilization success. In the last 30–40 years, in vitro maturation (IVM) and fertilization (IVF) of domestic cat oocytes have been established as part of the panel of assisted reproduction technologies. As a representative of wild felids, the African lion is not yet considered endangered. Nevertheless, the zoo population management of the African lion itself as well as other closely related felids would benefit from the establishment of an IVF system. Here, we aimed to investigate the transferability of domestic cat IVF technology to the African lion. From the ovaries of 42 lionesses aged between 0.75 and 15 years, a total of 933 IVF-suitable oocytes were retrieved and subjected to IVM and IVF. The overall maturation rate was 40.6% and 18.9% of these oocytes cleaved after fertilization, respectively. Embryos were generated by intracytoplasmic sperm cell injection as well as co-culture with epididymal sperm. Improvements in the model system also led to an improved outcome with in vitro produced embryos in the lion. Compared to domestic cats, the transportation of gonads to a specialized laboratory was time-consuming and influenced oocyte quality negatively. In conclusion, the domestic cat IVF system is adoptable for the African lion, although success rates are still lower. [ABSTRACT FROM AUTHOR]

In order to preserve endangered psittacine species, more basic and applied research in reproductive biology is required. Assisted reproductive technologies such as artificial insemination play an important role in parrots species conservation programs to overcome the problem of infertile eggs and male infertility. The aim of this study was to define an effective in vitro protocol in order to standardize the sperm quality evaluation in psittacines, studying Melopsittacus undulatus as model species. Semen was collected from twenty adult males by massage technique from May to June. Sperm concentration was measured by the spectrophotometric method. Sperm quality (sperm membrane integrity (SMI), motility, and kinetic parameters) was assessed on fresh semen. Three different experimental protocols were performed to compare the effects of various processing conditions on SMI, motility, and kinetic parameters. In protocol 1, test was performed by Lake extender with three different pH, 7.4 versus 8.2 versus 8.4, and two different equilibration temperatures after dilution of fresh semen (4°C versus 25°C). In protocol 2, two dilution rates of semen after collection were valuated, 1 : 3 versus 1 : 4, as well as three different semen storage temperatures (4°C versus 25°C versus 38°C) before sperm motility analysis with the computer-assisted sperm analysis (CASA). In protocol 3, two different Makler chamber temperatures (38 versus 41°C) during motility analysis were tested. A significant progressive improvement in spermatozoa motility and kinetic parameters was registered with pH 8.4. Progressive motility and all kinetic parameters were higher at 4°C equilibration temperature. Straightness (STR) kinetic parameter was better with 1 : 4 dilution rate. Total motile sperm was higher in 41°C Makler chamber. In this study, for the first time, the effects of different processing protocols on psittacines seminal quality analysis were investigated. Significant differences conditioning the effectiveness of analysis protocols have been described. [ABSTRACT FROM AUTHOR]

Amidst a global amphibian decline, captive breeding has become an important component of many conservation management programs. Some species fail to readily reproduce in captivity, including leiopelmatid frogs, an archaic lineage endemic to New Zealand. Assisted reproductive technologies can improve the reproductive potential and genetic management of captive programs. Herein we describe efforts to induce spermiation and mating in the threatened Hamilton's frog Leiopelma hamiltoni using a gonadotrophin-releasing hormone agonist and human chorionic gonadotrophin. The spermiation response was poor, with few individuals releasing spermatozoa and a low sperm concentration (mean (±s.e.) 27.4 ± 8.5 × 103 spermatozoa mL−1) when spermiation was stimulated. The sperm concentration peaked 12–24 h after hormone administration, suggesting this species may be slower to react than other anurans. Spermic urine was present in five males before hormone treatment. The sperm concentration was higher in this group (128.7 ± 54.9 × 103 spermatozoa mL−1) than in the hormone-induced males. All spermatozoa observed exhibited no motility or, at most, a limited swaying movement with no forward progression. In separate hormone induction trials with 60 individuals (two males to one female per trial), no mating was exhibited during the 12 h after treatment. Further studies to hormonally stimulate gamete release and mating are needed in leiopelmatids. Assisted reproductive technologies are useful tools to improve captive breeding programs for threatened species. The frogs native to Aotearoa New Zealand are all threatened with extinction and captive breeding programs to save this archaic lineage of frogs has failed. The goal of this study was to use hormones (gonadotrophin-releasing hormone agonist and human chorionic gonadotrophin) to attempt to induce mating and sperm collection in the threatened Hamilton's frog. The hormone doses trialled in this study resulted in poor spermiation responses for this species and no mating. [ABSTRACT FROM AUTHOR]

Actual loss of lizard biodiversity continues, even with the implementation of conventional conservation programs. An approach including assisted reproductive techniques such as sperm cryopreservation may contribute to the management of endangered species. We developed a method for sperm cryopreservation in sceloporine lizards and compared the response among the studied species. Prior to the mating season, we obtained semen from adult males of Sceloporus aeneus (n  = 21), Sceloporus grammicus (n  = 20) and Sceloporus torquatus (n  = 21) via pressure of the genital papilla. Volume and sperm concentration were measured before semen dilution in a Tris–egg yolk (TEY) medium to evaluate progressive motility, sperm viability, morphology, plasma membrane and acrosome integrity. Then, we cooled the remaining volumes to 5°C at a rate of 0.1°C per minute to incorporate glycerol (8% v/v) in two fractions. Immediately afterwards, we placed 40 μl of the mix on solid CO2 to form pellets and immersed them in liquid nitrogen for storage. We thawed the pellets at 29°C for 3 minutes and diluted them 1:1 (v/v) in TEY medium to assess sperm quality. We found a positive relationship between body weight and seminal volume in S. grammicus and S. torquatus and a negative correlation with sperm concentration in S. grammicus (P  < 0.05). Moreover, we observed that the freezing–thawing process decreased sperm quality in the three species, mostly affecting motility and viability. However, S. torquatus and S. aeneus showed a higher sperm tolerance than S. grammicus. [ABSTRACT FROM AUTHOR]

Sperm cryopreservation is a vital tool in amphibian assisted reproductive technologies that aids in genetic and population management, specifically for at-risk species. Significant advancements have been made in the cryopreservation of amphibian sperm, yet there is little information on how the cryopreservation process influences fertilization and embryonic development. In this study, we tested several cryoprotective agents (CPAs) and freezing rates on sperm recovery, fertilization potential and embryo development using Fowler's toads (Anaxyrus fowleri) as a model amphibian species for application to at-risk anurans. Three cryoprotectant treatments were tested, which included 10% trehalose + 0.25% bovine serum albumin with (1) 5% N,N-dimethylformamide (DMFA); (2) 10% DMFA; or (3) 10% dimethyl sulfoxide (DMSO). Additionally, sperm in each cryoprotectant was frozen at two different rates, −32 to −45°C/min and −20 to −29°C/min. Post-thaw sperm analysis included motility, morphology, viability, fertilization success and embryo development. Results show that 10% DMFA produced significantly higher (P =  0.005) post-thaw sperm motility than 5% DMFA and was similar to 10% DMSO. Furthermore, sperm frozen at −32 to −45°C/min had significantly higher post-thaw motility (P  < 0.001) compared to sperm frozen at −20 to −29°C/min. We also found that embryos fertilized with sperm frozen with 5% DMFA resulted in significantly higher (P  = 0.02) cleavage than 10% DMSO, yet there was no other effect of CPA on fertilization or embryo development. Furthermore, embryos fertilized with sperm frozen at −32 to −45°C/min resulted in significantly higher cleavage (P  = 0.001), neurulation (P  = 0.001) and hatching (P  = 0.002) numbers than sperm frozen at a rate of −20 to −29°C/min. Overall, eggs fertilized with frozen–thawed sperm produced 1327 tadpoles. These results provide insight towards a biobanking strategy that can be applied to imperilled species to preserve genetic lineages and bolster offspring genetic diversity for reintroduction. [ABSTRACT FROM AUTHOR]

Objective: To describe the reproductive characteristics of female jaguars and assisted reproduction techniques. Methodology: A literature review about reproductive characteristics and assisted reproduction techniques was done to document information on this subject. Results: Jaguars are the largest feline native to America; females sizes range from 1.57 to 2.19 m in length and weigh 45 to 82 kg. Jaguar females are ready for mating at about 2 years old and have an estrous cycle every 37 days. The reproductive behaviour patterns of the jaguar in captivity are grouped into four categories: 1) sexual, 2) affiliative, 3) agonists and 4) individual. The gestation period lasts between 91 to 111 days, and usually give birth to one to four young. For assisted reproduction, artificial insemination contributes to the genetic distribution or variability of germplasm. Limitations on study: The destruction of forests and jungles, habitat fragmentation, indiscriminate hunting, the lack of prey and food sources and the conflict with ranchers put Jaguars on the brink of extinction. Also, there is few information about their reproductive characteristics, which is why in Mexico, it is imperative to generate this information, mainly for free-living jaguars. Conclusions: The reproductive characteristics and assisted reproductive techniques in the female jaguar were documented. However, work is needed on assisted reproduction techniques in this species, which will contribute to the conservation of oocytes or embryos. It is advisable to design protocols to control and induce ovulation in the female Panthera onca. [ABSTRACT FROM AUTHOR]

Summary: Sperm morphometric and morphologic data have been shown to represent useful tools for monitoring fertility, improving assisted reproduction techniques and conservation of genetic material as well as detecting inbreeding of endangered primates. We provide here for the first time sperm morphologic and morphometric data from Cercopithecus neglectus, Cercopithecus cephus, Papio papio and critically endangered Cercopithecus roloway, as well as comparative data from other Cercopithecinae species, i.e. Allochrocebus lhoesti, Mandrillus sphinx and Papio anubis. Following collection from the epididymis, spermatozoa were measured for each species for the following parameters: head length, head width, head perimeter, head area, midpiece length and total flagellum length, and the head volume, ellipticity, elongation, roughness and regularity were then calculated. Our data are consistent with both the general morphology and the morphometric proportions of Cercopithecinae sperm. Some specificities were observed, with C. cephus displaying a narrow head (width = 2.76 ± 0.26 µM) and C. roloway displaying a short midpiece (6.65 ± 0.61 µM). This data set represents an important contribution, especially for Cercopithecus roloway, one of the most endangered monkeys in the world, and further data on additional specimens coupled to data on mating systems and reproductive ecology should allow a better understanding of the mechanisms underlying these morphological differences across primate species. [ABSTRACT FROM AUTHOR]

The proboscis monkey (Nasalis larvatus) is an endangered species with a declining population. This article describes the first successful attempt at sperm collection and evaluation, and the testicular and sperm morphometries of the wild proboscis monkey in Sabah, Malaysia. Eight semen collection procedures using electro-ejaculation and digital manipulation were conducted in three wild adult male proboscis monkeys. A total of 21 ejaculates were collected. The testicular biometry was measured with the aid of ultrasonography. Sample evaluation included semen volume and pH and sperm concentration, viability, and abnormality. The sperm morphometry was undertaken using phase contrast microscopy. The mean (±SD) total testicular volume of these animals was 5.77 cm3 (±1.58). Semen collection by electro-ejaculation resulted in an 84% success rate, while digital manipulation did not result in any ejaculation. Each animal showed different semen characteristics, where the volume was 5–540 μL, pH 8–9, and sperm concentration 0.041–83.00 ×106/mL. The percentage of abnormal sperm was high at 76.8% (±89.60), largely due to midpiece abnormality. Normal sperm had a spherical head and long tail with a head : midpiece : tail length ratio of 1 : 2: 8. The social status of these animals may contribute to the generally low quality of the semen. The techniques and data from this study are useful for future conservation and application of assisted reproductive technology in this species. [ABSTRACT FROM AUTHOR]

Assisted reproductive technologies (ARTs) have a significant role to play in reptile conservation, yet are severely lacking. Previous attempts to cryopreserve spermatozoa in the threatened lizard Varanus panoptes achieved approximately 48% motile sperm post-thaw for samples frozen immediately after collection. However, the feasibility of extended cold storage before cryopreservation has not been tested. We held V. panoptes spermatozoa at either 258Cor 48C for 8 days, assessing sperm motility at days 1, 2, 4 and 8. Subsamples were cryopreserved on days 1 and 4 following the previously reported protocol for this species. Percentage motility decreased rapidly at 258C, but did not decrease significantly until 4 days after collection at 48C, with .30% motility maintained after 8 days. There was no significant difference in post-thaw motility or viability of samples cryopreserved after 1 or 4 days storage at 48C, yielding substantial results for both parameters (mean motility 23.8% and 28.1% and mean viability 50.1% and 57.5% after 1 and 4 days respectively). We demonstrate the capacity to extend sperm viability for up to 8 days in unfrozen samples and to produce acceptable post-thaw motility in samples frozen after 4 days of storage, contributing to the development of valuable ARTs for lizards and other reptiles. [ABSTRACT FROM AUTHOR]

Despite the growing roles of assisted reproduction and captive breeding in reintroductions, we have a limited understanding of the viability of these animals once released. We radiotracked endangered dusky gopher frogs Lithobates sevosus for up to 24 days after they were released into the wild to (1) assess the viability of juvenile frogs produced using artificial fertilization and raised in captivity until four weeks post‐metamorphosis, (2) investigate survival rates, movement patterns, and habitat use and (3) examine whether it is more beneficial to release frogs at the pond edge or into burrows in the uplands. Released frogs had a relatively high survival rate (76% overall, with mortality ceasing after 14 days) and behaved as expected, moving fairly long distances (up to 153 m daily and 426 m total) and locating suitable shelters, including burrows and stump holes. Frogs that sheltered underground had a 22% higher survival rate than frogs that did not, highlighting the importance of these habitat features. Compared to frogs released into burrows, frogs released at the pond had a 33% lower survival rate, spent less time underground and moved farther and more often, likely while searching for shelter. Frogs selected habitat characteristics that are associated with effects of fire, including an open canopy and abundant ground vegetation. Our study demonstrates that juvenile dusky gopher frogs successfully transitioned to their natural habitat, and that any effects of artificial fertilization or captivity on juvenile viability appear to be minimal. Releasing frogs into areas with abundant underground refuges and using frequent prescribed fire to maintain the habitat should benefit dusky gopher frog populations and improve the success of reintroductions. [ABSTRACT FROM AUTHOR]

Captive breeding and reintroduction programs have been established for several threatened amphibian species globally, but with varied success. This reflects our relatively poor understanding of the hormonal control of amphibian reproduction and the stimuli required to initiate and complete reproductive events. While the amphibian hypothalamo–pituitary–gonadal (HPG) axis shares fundamental similarities with both teleosts and tetrapods, there are more species differences than previously assumed. As a result, many amphibian captive breeding programs fail to reliably initiate breeding behaviour, achieve high rates of fertilization or generate large numbers of healthy, genetically diverse offspring. Reproductive technologies have the potential to overcome these challenges but should be used in concert with traditional methods that manipulate environmental conditions (including temperature, nutrition and social environment). Species-dependent methods for handling, restraint and hormone administration (including route and frequency) are discussed to ensure optimal welfare of captive breeding stock. We summarize advances in hormone therapies and discuss two case studies that illustrate some of the challenges and successes with amphibian reproductive technologies: the mountain yellow-legged frog (Rana muscosa ; USA) and the northern corroboree frog (Pseudophryne pengilleyi ; Australia). Further research is required to develop hormone therapies for a greater number of species to boost global conservation efforts. [ABSTRACT FROM AUTHOR]

The domestic ferret (Mustela putorius furo) provides a good model for developing new reproductive technologies for use with threatened related species. Such technologies could also be used in the reproductive management of this pet species. The present work reports an improved freezing protocol for ferret sperm. Semen was collected by electroejaculation plus rectal massage (in an attempt to reduce the electrical stimulation necessary) from five adult male ferrets, and then subjected to one of two freezing protocols: (a) from 5 to −35°C at 40°C/min, then from −35 to −65°C at 17°C/min, and finally from −65 to −85°C at 3°C/min—a decelerating freezing rate; and (b) from 5 to − 10°C at 5°C/min, and then from −10 to −130°C at 60°C/min—an accelerating freezing rate. After thawing, the viability and acrosomal integrity of the sperm frozen via the two‐step accelerating method were better than those frozen via the three‐step decelerating method (43.3 ± 3.5% and 71.2 ± 3.4% compared with 29.7 ± 3.7% and 58.8 ± 3.4% respectively; p <.05). No differences were seen between the methods with respect to sperm motility variables; most sperm (>90%) remained static with both freezing methods. In conclusion, although the method with accelerating freezing rate was associated with better post‐thaw sperm viability and acrosome integrity values, neither of the two freezing methods tested provided adequate motility results after thawing. Combining rectal massage with electrical stimuli seemed to reduce the number of the latter required for successful sperm collection. [ABSTRACT FROM AUTHOR]

Contents: Cryopreservation of in vitro‐derived bovine embryos is a crucial step for the widespread reproduction and conservation of valuable high‐merit animals. Given the current popularity of bovine in vitro embryo production (IVP), there is a demand for a highly efficient ultra‐low temperature storage method in order to maximize donor ovum pickup (OPU) turn‐over, recipient availability/utilization and domestic/overseas commercial trading opportunities. However, IVP bovine embryos are still very sensitive to chilling and cryopreservation, and despite recent progress, a convenient (simple and robust) protocol has not yet been developed. At the moment, there are two methods for bovine IVP embryo cryopreservation: slow programmable freezing and vitrification. Both of the aforementioned techniques have pros and cons. While controlled‐rate slow cooling can easily be adapted for direct transfer (DT), ice crystal formation remains an issue. On the other hand, vitrification solved this problem but the possibility of successful DT commercial incorporation remains to be determined. Moreover, simplification of the vitrification protocol (including warming) through the use of an in‐straw dilution without the use of a microscope is a prerequisite for its use under farm conditions. This review summarizes the bovine IVP embryo cryopreservation achievements, strengths and limitations of both freezing systems and prospective improvements to enhance cryosurvival, as well as perspectives on future directions of this assisted reproductive technology. [ABSTRACT FROM AUTHOR]

In this review the advantages of the gonadotropin-releasing hormone agonist (GnRHa) trigger are discussed beyond those immediately associated with ovarian hyperstimulation syndrome (OHSS) prevention. The GnRHa trigger concept has sparked the development of novel protocols, enriching the assisted reproductive technology (ART) armamentarium for the benefit of present and future patients. Thus, GnRHa trigger already has a pivotal role, not only for the standard in vitro fertilisation (IVF) patient, but also for patient groups like oocyte donors, cancer patients, patients with poor ovarian reserve, and patients with immature oocyte syndrome and empty follicle syndrome. Herein, we discuss the importance of the GnRHa-elicited midcycle FSH surge and the potential improvement in oocyte yield and embryo competence. [ABSTRACT FROM AUTHOR]

Procuring high-quality oocytes is the rate-limiting step for assisted reproduction technologies intended for embryo production. Although much is known about the intraovarian processes that dictate oocyte growth and maturation, subtleties in the process of oogenesis have yet to be replicated in in vitro systems. In contrast with the mouse, in which functional oocytes have been derived from stem cells under ex vivo conditions, the generation of developmentally competent oocytes in other species has yet to be achieved. This paper reviews the principles and practices based on stem cell and organ culture strategies that hold promise for developing a technological base upon which future efforts to recapitulate or augment oogenesis in mammals could be realised. [ABSTRACT FROM AUTHOR]

Reproductive technologies such as genome storage and assisted reproduction have a significant role to play in ending or reversing species extinctions. However, such technologies for non-model organisms (i.e. non-mammalian species) are poorly developed. This is particularly true for the reptiles, in which there is a dearth of successful protocols for cryopreserving reptile spermatozoa, despite limited attempts. We investigated sperm cryopreservation in the Australian lizard Varanus panoptes with the objective of addressing the unmet need for an optimized cryopreservation protocol for the spermatozoa of squamate reptiles. We tested the efficacy of two cryoprotectants [dimethyl sulfoxide (DMSO) and glycerol] as well supplementation with a phosphodiesterase inhibitor (caffeine) to promote post-thaw motility. For cryopreservation, sperm were cooled in straws suspended in liquid nitrogen vapour for 5 minutes (approximately −135°C), before being plunged into liquid nitrogen (approximately −196°C), and later thawed in a water bath at 35°C. Samples were incubated post-thaw for 10 minutes in the presence or absence of 10 mM of caffeine. Both cryoprotectant type and concentration significantly affected percent sperm motility pre-freezing, with DMSO being less cytotoxic than glycerol and motility decreasing at higher concentrations of both cryoprotectant types. While cold shock did not significantly affect sperm motility, both cryoprotectant type and concentration did significantly impact the motility of post-thawed spermatozoa. Thus, mid-range concentrations (10% v/v) of DMSO and glycerol yielded a greater post-thaw motility compared with 5 and 20% v/v, while DMSO proved superior to glycerol. The addition of caffeine resulted in a significant recovery of post-thaw motility for both cryoprotectants, with higher rates of motility being associated with higher cryoprotectant concentrations. These protocols provide a significant step forward for in situ and ex situ management of threatened reptiles and add to recent evidence that reptilian sperm may have the full range of phosphorylation-mediated cellular mechanisms associated with capacitation, motility and metabolic regulation found in mammalian sperm. [ABSTRACT FROM AUTHOR]

Background: Amphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians. Methods: The aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined. Results: Hormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials. Conclusions: Overall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species. [ABSTRACT FROM AUTHOR]