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The Malabar dwarf puffer, Carinotetraodon travancoricus is the smallest known pufferfish (family Tetraodontidae) and one of the smallest freshwater fishes of the Indian subcontinent. Due to their miniature size, wacky behaviour and appearance, they are much preferred in the international aquarium fish trade, although little is known regarding their breeding activity in captivity and their embryonic development. The purpose of this study was to fill these knowledge gaps. Wild-caught Malabar dwarf puffers were acclimatised to conditions, and pairs were introduced to breeding tanks. Adult fishes were fed with live and frozen diets including Artemia nauplii, moina and bloodworm. During spawning seasons, adult fish displayed elaborate courtship behaviour around sunset. Carinotetraodon travancoricus is a batch spawner releasing 1 to 5 eggs per diem. The eggs were spherical, and non-sticky, with a diameter of 1.48 ± 0.1 mm, and hatching took place after 108 to 116 h post-incubation. The newly hatched larvae were 3.5 ± 0.2 mm in length, and weighed 2.9 ± 0.4 mg. The early larvae have substantial yolk and oil globules as an energy reserve. Histological studies on mature females suggested the batch spawning nature of the species and low fecundity. Given its unique reproductive behaviour and characters, in situ protected habitats are required to ensure their continued survival in the wild, apart from encouraging captive breeding to augment the demand in the international aquarium fish trade.

Although many Fertility Centers have adopted day 5 or 6 embryo transfer policy, yet, 30% of embryo transfers in the US are performed on day 3. This is mainly due to concerns related to longer embryo culture effect and higher rates of embryo transfer cancellation on day 5, with no effect on cumulative pregnancy rate. We conducted a retrospective cohort study comparing individual embryo transfer order rank, best embryo for fresh transfer and intention to freeze, of day-3 and day-5 embryos based on their morphology score. Day-3 embryos of each patient were ranked by embryologists for the order of transfer and intention to freeze, based on morphological score, blinded to actual blastulation outcome. The corresponding blastocysts were similarly ranked for the order of transfer and vitrification intention. Ranking was compared to test the predictive value of day-3 morphological assessment. Sixty patients with 784 day-3 embryos were included. There was only a moderate positive significant correlation between ranks on day-3 and ranks on day-5 [r = 0.662 95% CI (0.611-0.706, p < 0.001)]. Only 25% of the best embryos for transfer on day 3 (rank = 1) were chosen for fresh transfer on day 5. A total of 441 embryos were intended to be frozen on day 3. Of those, 201 were not transferred nor vitrified on day 5-6 (45%), 3.35 embryos per patient. No significant difference was found between average day-3 rank of embryos ranked 1, 2 (3.12 vs 4.12, p = 0.074) and 3 (3.12 vs 4.08, p = 0.082) on day-5-6. To conclude, this study brings a different perspective to the comparison of day 3 and day 5 by following each embryo's putative and actual designation. Day-3 ranking of embryo morphology did not provide a reliable prediction for blastocyst formation, transfer order and vitrification intention, and may support transfer or cryopreservation of blastocysts over cleavage stage embryos.

To date, implantation is the rate-limiting step for the success of in vitro fertilization (IVF) treatment. Accumulating evidence suggests that immune cells contribute to embryo implantation, and several therapeutic approaches have been proposed for the treatment of recurrent implantation failure (RIF). Endometrial immune modulation with autologous activated peripheral blood mononuclear cells (PBMCs) is one of the most widely used protocols. However, the effect of intrauterine insemination of mixed paternal and maternal-activated PBMCs has not yet been attempted and studied. The aim of our study is to test the effect of the addition of paternal lymphocytes on the implantation rate in RIF patients. Mononuclear cells were isolated from the peripheral blood of 98 RIF patients and cultured for 72 h before insemination into the endometrial cavity 48 h before embryo transfer. Our patients were divided into 4 groups according to the type and number of PBMCs inseminations. Our study shows that activated PBMCs promoted clinical pregnancy rates (CPR) in all groups. Moreover, we found that the groups injected with more than 2 million cells showed a better clinical outcome and, more interestingly, patients inseminated with both paternal and maternal activated PBMCs showed the highest CPR, reaching 47.2%, in addition to the highest implantation rate 31. 2% and the live birth rate 41.39%. Our work demonstrates the importance of administering a large number of activated PBMCs with the addition of paternal activated PBMCs to immunomodulate the endometrium for the success of in vitro fertilization in RIF patients.

Metabolite supplementation during in vitro embryo development improves blastocyst quality, however, our understanding of the incorporation of metabolites during in vitro maturation (IVM) is limited. Two important metabolites, follistatin and choline, have beneficial impacts during in vitro culture; however, effects of supplementation during IVM are unknown. The objective of this study was to investigate combining choline and follistatin during IVM on bovine oocytes and subsequent early embryonic development. We hypothesized that supplementation of choline with follistatin would synergistically improve oocyte quality and subsequent early embryonic development. Small follicles were aspirated from slaughterhouse ovaries to obtain cumulus oocyte complexes for IVM with choline (0, 1.3 or 1.8 mM) and follistatin (0 or 10 ng/mL) supplementation in a 3 × 2 design. A subset of oocytes underwent transcriptomic analysis, the remaining oocytes were used for IVF and in vitro culture (IVC). Transcript abundance of CEPT1 tended to be reduced in oocytes supplemented with 1.8 mM choline and follistatin compared to control oocytes (P = 0.07). Combination of follistatin with 1.8 mM choline supplementation during maturation, tended (P = 0.08) to reduce CPEB4 in oocytes. In the blastocysts, HDCA8, NANOG, SAV1 and SOX2 were increased with choline 1.8 mM supplementation without follistatin (P < 0.05), while HDCA8 and SOX2 were increased when follistatin was incorporated (P < 0.05). The combination of choline and follistatin during oocyte maturation may provide a beneficial impact on early embryonic development. Further research is warranted to investigate the interaction between these two metabolites during early embryonic development and long-term influence on fetal development.

We have established trophoblast cell lines, from parthenogenesis-derived buffalo blastocysts. The buffalo trophoblast cells were cultured continuously over 200 days and 21 passages. These cells were observed by phase-contrast microscopy for their morphology and characterized by reverse transcriptase polymerase chain reaction and immunofluorescence against trophoblast-specific markers and cytoskeletal proteins. Trophoblast cells showed positive staining for CDX2, a marker of these cells at both blastocyst and cell line levels. Epithelial morphology of these cells was revealed by positive staining against cytokeratins and tubulin but not against vimentin and dolichos biflorus agglutinin. Gene expression profiles of many important placenta-specific genes were studied in the primary trophectoderm outgrowths, which were collected on days 0, 5, 9, 12 and 15 of culture and trophoblast cell line at passages 12-15. Therefore, the trophoblast cell line derived can potentially be used for in vitro studies on buffalo embryonic development.

The intra-ovarian presence of ootids, i.e. female gametes that have completed meiosis, is considered exceptional in the animal kingdom. The present study explores the first such case to be reported in a sea cucumber (Echinodermata: Holothuroidea). In the overwhelming majority of animals, including holothuroids, oocytes (i.e. immature female gametes) that are developing in the ovary undergo a primary arrest at the prophase stage of meiosis, which may last from days to decades. In free-spawning taxa, this arrest is normally lifted only during or shortly before transit in the gonoduct, when gamete release (spawning) is imminent. However, oocytes of the holothuroid Chiridota laevis were discovered to have resumed the second meiotic division including the completion of germinal vesicle breakdown and polar-body expulsion inside the ovary, effectively reaching the ootid stage concomitantly with ovulation (i.e. escape from follicle cells) prior to spawning. The potential drivers and significance of this exceptionally rare case of full intra-ovarian oogenic maturation are discussed.

Spermatogenesis is a highly complex process through which mature sperms are produced, and it requires three important stages; mitosis, meiosis and sperm formation. The expression of genes regulated by transcription factors at specific stages exerts important regulatory effects on the development process of germ cells. Male mice with overexpressed programmed death ligand 1 (PD-L1) (B7 homolog1) in the testis have infertility and abnormal sperm development, thereby exhibiting severe malformation and sloughing throughout spermatid maturation and collapsed and disorganized seminiferous epithelium structure. Furthermore, PD-L1 overexpression causes overexpression of cysteine-rich secretory protein 1 (CRISP1) in the epididymis and adversely affects or precludes sperm energization, sperm-pellucida binding and sperm-oocyte fusion. These findings suggest that CRISP1 and PD-L1 can interact with each other to induce male infertility and germ-cell dissociation.

Testicular biopsies (9 mm³) from domestic cats (n = 10) submitted to orchiectomy were submitted to equilibrium vitrification in the presence of ethylene glycol (EG) alone or combined with dimethylsulfoxide (DMSO) as intracellular cryoprotectants, and sucrose or trehalose as extracellular cryoprotectants. The samples were vitrified with 40% EG or 20% EG + 20% DMSO, plus 0.1 M or 0.5 M of sucrose or trehalose. The study was divided into Step 1 and Step 2. In Step 1, intratubular cells (spermatogonia, spermatids, spermatocytes, and Sertoli cells) were quantified and classified as intact or degenerated (pyknotic and/or vacuolated cells). Cryodamage of seminiferous cords was determined by spermatogonia and Sertoli cell scoring of nuclei alterations, tubular basement membrane detachment, epithelium shrinkage, and tubular measures (total area, epithelium area, larger and smaller diameter, and height of the epithelium). In Step 2, Hoechst 33342 stain and propidium iodide (PI) fluorescent stain were used to assess the cell viability of the four best experimental groups in Step 1. The effect of treatments on all analyses was accessed using analysis of variance (ANOVA), and Fisher's post hoc test at P < 0.05 significance was considered. In Step 1, the mean percentage of spermatogonia and Sertoli cells morphological integrity did not show a difference when using both sugars at different concentrations, but their morphology was more affected when DMSO was used. EG use associated with 0.1 M of sucrose or trehalose positively affected spermatocyte and spermatid morphology, respectively. The larger diameter and epithelium height of seminiferous tubules were increased using DMSO plus 0.5 M sucrose and DMSO plus 0.1 M trehalose. The changes in spermatogonial/Sertoli nucleoli visualization were best scored in the EG groups, while the nuclei condensation was lower with sucrose. The basement membrane was satisfactorily preserved with 0.1 M sucrose. In Step 2, the percentage of cell viability was higher when EG plus 0.1 M sucrose was used. Therefore, DMSO's negative effect on the vitrification of testicular biopsies of adult domestic cats was evident. The EG plus 0.1 M of sucrose or trehalose associations are the most suitable CPAs to preserve the testicular histology structure of adult domestic cats in vitrification.

In vitro maturation of oocytes (IVM) represents an assisted reproductive technique that involves the minimal or absence of ovarian stimulation and is beneficial to specific groups of patients. These may include women with polycystic ovarian syndrome and/or patients who need a fertility preservation option before undergoing gonadotoxic treatment. However, when IVM is applied in cases where it is not recommended, it can be considered as an add-on technique, as described by the ESHRE Guideline Group on Female Fertility Preservation. Interestingly, IVM has not been proven yet to be as effective as conventional IVF in the laboratory, in terms of clinical pregnancy and live birth rates, while concerns have been raised for its long-term safety. As a result, both safety and efficacy of IVM remain still questionable and additional data are needed to draw conclusions.