This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO(np)+CUR) supplementation during the in vitro maturation (IVM) of bovine oocytes on the in vitro embryo production and the cellular antioxidant response. A total of 1,625 cumulus-oocyte complexes (COCs) were cultured in the maturation medium in the absence (0 µM - control) or presence of different concentrations of ZnO(np)+CUR (3 µM, 6 µM or 12 µM). After IVM, COCs were destined either to 1) in vitro embryo production or 2) analysis of reactive oxygen species production, superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidant capacity (FRAP). The results demonstrated that the addition of 6 and 12 µM ZnO(np)+CUR during in vitro maturation showed a higher rate of blastocyst production when compared to the control (p < 0.05). However, only 12 µM ZnO(np)+CUR treatment showed higher rates of embryo production when compared to 3µM ZnO(np)+CUR treatment. Supplementation of IVM medium with 6 µM ZnO(np)+CUR reduced ROS production (p < 0.05) compared to control and 12 µM ZnO(np)+CUR treatments. Also, the treatment containing ZnO(np)+CUR at 12 µM had lower SOD activity after IVM than control treatment. In conclusion, the best outcome for in vitro embryo production was obtained when 6 and 12 µM ZnO(np)+CUR was added during IVM of bovine oocytes. However, this improvement in in vitro embryo production was not associated with either the reduction of ROS production or SOD and CAT activities.

This study describes the embryonic and larval development of the Atabapo butterfly pleco Dekeyseria picta under captive conditions. A total of 19 wild-caught adults (male-to-female ratio of 9:10) were maintained together in a single 1000-L tank operated with a recirculating aquaculture system (RAS), at approximately 26°C, under a controlled 12:12 h light–dark cycle. A total of 21 natural spawning events produced an average of 66.5 ± 2.43 spherical eggs with a mean diameter of 3.896 ± 0.022 mm, characterized by a yellow yolk and a strongly adhesive chorion. Embryonic development lasted between three and seven days, and hatched larvae were classified into five developmental phases. Early phases exhibited limited differentiation and weak pigmentation, whereas advanced phases showed visible eyes, increased pigmentation, and progressive yolk absorption. Heartbeat activity and tail movements were observed throughout all phases. The hatching rate was below 40%, likely influenced by differences in chorion rupture efficiency among embryos at distinct developmental stages. Two developmental strategies potentially contributing to embryonic plasticity were identified: (i) a diapause-like state in which embryos temporarily arrested development at early phases without yolk utilization while remaining responsive to stimuli, and (ii) delayed hatching, in which embryos remained within the chorion for up to seven days, allowing further larval development in a protected environment. These strategies may represent adaptive responses to rapidly fluctuating hydrological and environmental conditions characteristic of the natural habitat of the species and emphasize the role of aquaculture in advancing reproductive knowledge and conservation.

The objective was to evaluate the use of resveratrol conjugated with silica nanoparticles during the in vitro maturation of bovine oocytes. The oocytes were divided into the following treatment groups during the maturation process: control (n = 159), resveratrol 0.5 μM (n = 158), resveratrol 1 μM (n = 155), nanoparticles conjugated with 0.5 μM resveratrol (n = 159), and nanoparticles conjugated with 1 μM resveratrol (n = 158). Several parameters were assessed, including cumulus oophorus size, reactive oxygen species (ROS) production, oocyte nuclear maturation, cell apoptosis, cleavage rates, and blastocyst production rates. Statistical analysis was conducted using Sigma Plot software (version 11) and SAS Studio, with statistical significance defined as P ≤ 0.05 for the main effects and interactions. The results indicated that the cumulus oophorus size was smaller in the resveratrol 1 μM treatment group, and the oocyte size was reduced in the nanoparticle 1 μM treatment group. No significant differences were detected between the treatment groups in terms of ROS production, oocyte maturation, or cell apoptosis. However, the resveratrol 1 μM treatment group exhibited decreased rates of cleavage and blastocyst formation. In contrast, the nanoparticles 0.5 μM and 1.0 μM treatments showed improved cleavage and blastocyst rates compared with the resveratrol 1.0 μM treatment group. In summary, while resveratrol alone at 1 μM concentration had a negative impact on cleavage and blastocyst rates, the use of silica nanoparticles conjugated with resveratrol (both 0.5 μM and 1 μM) enhanced these outcomes, suggesting a potential advantage in using nanoparticle-conjugated resveratrol for the in vitro maturation of bovine oocytes.

Obesity, a global health issue, is associated with numerous diseases and has been shown to affect male reproductive health by inducing endocrine hormonal changes, chronic inflammation, oxidative stress and epigenetic alterations in reproductive cells. This study investigates the impact of obesity on testicular gene expression across mice, monkeys and humans, identifying 730 conserved testis-specific genes. High-fat diet-induced obesity upregulates GNG5, INHA, MSH5, SLC30A8 and SLC7A4 in testes, suggesting their potential as regulatory targets in testicular damage associated with obesity. Single-cell analysis reveals species-conserved expression patterns of SLC7A4 in Sertoli cells and SLC30A8 in SPG cells. It also confirmed that SLC30A8 and SLC7A4 were significantly upregulated in the testes of spontaneously obese mice. The findings highlight the potential of these genes as regulatory targets in obesity-related testicular dysfunction, providing insights into male reproductive health impairments caused by obesity.

Intracytoplasmic sperm injection (ICSI) is a widely used assisted reproduction technique, but in cattle it faces major challenges due to inefficient oocyte activation after sperm microinjection. This study investigated different oocyte activation strategies and assessed the potential role of reducing agents glutathione (GSH), cysteamine (Cys) and dithiobutylamine (DTBA) to improve sperm head decondensation and embryo development following Piezo-ICSI. Haploid parthenogenetic activation using different ethanol concentrations (1%, 3%, 7% and 10%) failed to yield blastocysts, while diploid activation with ethanol or ionomycin combined with inhibitors significantly improved cleavage (43–55%) and blastocyst rates (14–27%), respectively. However, applying two ethanol pulses was detrimental, reducing both cleavage and blastocysts likely due to toxic overexposure. Sperm head decondensation compounds in Piezo-ICSI showed a high percentage of inactivated oocytes (75% GSH, 55% Cys and 40% DTBA). The highest male pronuclear formation rates were observed in the control without sperm head decondensation (21%) and with DTBA treatment (10%). Despite this, the treatment with Cys resulted in higher developmental potential to the blastocyst stage (22%) comparable to the control (24%). These data suggest that the inclusion of sperm head decondensing agents could represent a promising new strategy for enhancing the early in vitro development of ICSI-generated embryos. However, for this purpose, careful optimization of the concentration and incubation time of these decondensing compounds is essential.

The study documents the embryonic and larval development of Clarias dussumieri, a Near-Threatened, cultivable, and highly valued food fish endemic to the Western Ghats. Induced breeding facilitated detailed observation of developmental stages using light microscopy. Unfertilized eggs measured 1.5 ± 0.02 mm in diameter and enlarged to 1.7 ± 0.10 mm after fertilization (∼13% increase). The first cleavage occurred at 00:36 ± 00:02 h, reaching 64 cells by 01:35 ± 00:06 h. Epiboly progressed from 30% at 03:20 ± 00:10 h to 50% at 04:30 ± 00:05 h and 90% at 05:50 ± 00:10 h; neurulation was evident by 07:40 ± 01:10 h and somites by 11:08 ± 01:26 h. The first heartbeat appeared during the pharyngula period (17:25 ± 01:30 h), and hatching occurred at 25:30 ± 01:30 h at 26–27°C. Hatched larvae were 4.2 ± 0.02 mm total length, observed few melanophores by 12 h, conspicuous eye pigmentation by 24 h, and body pigmentation spreading by day 2 (5.90 ± 0.04 mm).The yolk sac was fully absorbed by 72 ± 2.50 h post-hatching, marking the onset of exogenous feeding. By day 4 (pre-flexion), larvae reached 7.1 ± 0.02 mm; initial notochord flexion began around day 7 and post-flexion from day 10, with juvenile-like morphology evident by day 25 (15.27–17.80 mm). The standardized timelines and measurements provide a baseline for hatchery practice and conservation aquaculture of C. dussumieri, supporting protocol refinement for improving survival and growth under controlled conditions

Immunological castration can be an alternative to traditional surgical castration. The active immunization against GnRH or kisspeptin has a castrating effect. To date, the fusion protein vaccine of combination with GnRH and kisspeptin have not been studied. Thus, the present study will develop a GnRH6-kisspeptin vaccine by genetic engineering method and investigate its immunocastration effect in male rats. Twenty 20-day-old male rats were randomly divided into two groups: the control group (n=10) and the immunization group (n=10). The initial immunization took place at week 0 followed by three booster doses administered intervals. The control group received an equivalent dose of white oil adjuvant. Orbital blood samples were collected at various time points following the initial immunization, at 0, 2, 4, 6, 8, 10 and 12 weeks, respectively. The entire left testis was weighed and its volume measured at week 12. Samples from the right testis were obtained for histological analysis. Serum levels of GnRH and kisspeptin antibodies, as well as testosterone levels were determined using ELISA. The results showed that the serum levels of GnRH and kisspeptin antibody titres of the immunized rats were significantly higher compared to the control group (P<0.05). Additionally, the testosterone concentration was effectively reduced following the intensified immunization. The testes of the immunized group exhibited a reduction in size and a significant decrease in the number of spermatogonia in the testicular tissue compared to the control group (P<0.05). These data indicate that the recombinant GnRH6-kisspeptin protein effectively induced immunological castration in rats.

This study aimed to investigate the efficacy of cell-free DNA (CF-DNA) in the spent cleavage and blastocyst medium versus blastomere biopsy for sex identification using short tandem repeat (STR) markers for the first time. In total, 39 samples of spent culture medium (SCM) from six couples were collected of which 28 samples were CF-DNA from blastocoel fluid + SCM (day 5) and 11 samples from SCM alone (day 3). The frequencies of allele dropout (ADO), fail rate and informativity markers were considered. The relationship between the morphology of embryos and ADO and the fail number of all markers was investigated. Sex identification rate between CF-DNA isolated from culture medium and fluorescence in situ hybridization (FISH) was then compared with measurement of Agreement Kappa (AK). The highest frequency of informative markers belonged to DXS6801 and HPRT. There was no relationship between the ADO number of all markers and embryo morphology. A significant difference was seen between embryo morphology and fail numbers. AK value between CF-DNA isolated from culture medium and FISH was 0.516, which is moderate. The ability of CF-DNA to detect the correct diagnosis of males and females showed that all values of specificity, sensitivity, positive predictive value, and negative predictive value were 100%. The presence of embryonic CF-DNA in the SCM on day 3 as well as blastocyst medium on day 5 using STR-based multiplex PCR is approximately consistent with FISH for sex identification. Advances in DNA extraction, amplification technique, and testing may allow for preimplantation genetic testing for aneuploidy (PGT-A) and monogenic/single-gene disorders (PGT-M) as a non-invasive approach without biopsy in the future either in sex determination or chromosomal abnormality.

In cattle, maternal metabolic health has been suggested to influence oocyte and embryo quality. Here, we examined whether maternal liver abnormalities affected in vitro oocyte maturation by screening meiotic maturation, spindle morphology, actin filaments, and lysosomes. In oocytes from the abnormal liver group, the maturation rate (80.2%) was significantly lower compared to a control group with healthy livers (90.8%; P < 0.05). Mean spindle area in oocytes of the abnormal group (50.4 ± 3.4 μm2) was significantly larger than in the control (40.8 ± 1.6 μm2; P < 0.05). Likewise, mean spindle width in the abnormal group (8.8 ± 0.3 μm) was significantly larger than in the control group (7.8 ± 0.2 μm; P < 0.05). The proportion of cells with correctly aligned chromosomes in the abnormal group (48.0%) was significantly lower than in the control (78.3%; P < 0.05). The number of cortical actin filaments in mature oocytes of the abnormal group (299.3 ± 3.7) was significantly lower than in the control (314.7 ± 3.2; P < 0.05). The number of lysosomes in mature oocytes of the abnormal group (1363.6 ± 39.0) was significantly higher than in the control (1123.4 ± 26.3; P < 0.05). In conclusion, our findings indicate that the quality of in vitro matured oocytes is lower in cattle with liver abnormalities than in healthy cattle.

A blastomere containing more than one nucleus is defined as a multinucleated blastomere. In our study, we aimed to investigate the relationship between serum anti-Mullerian hormone (AMH) levels and multinucleated (MLN) embryos, one of the parameters indicating embryo quality, in intracytoplasmic sperm injection (ICSI) cycles. The results of 888 ICSI cycles of patients aged 19–45 years attending an ART (assisted reproductive technology) clinic were retrospectively analysed. Cycles with at least one MLN embryo were defined as the study group (n = 237) and cycles without MLN embryos as the control group (n = 651). Univariate and multivariate logistic regression analyses were used to determine the risk factors affecting the dependent qualitative variables. The effect of AMH levels on multinucleation was found to be a significant risk factor (p < 0.001). One unit increase in AMH levels increases the risk of the presence of MLN embryos by 1.12 times. The mean MLN embryos/total embryo ratio in the group with clinical pregnancy was 0.34 ± 0.18, while the mean MLN embryos/total embryo ratio in the group that did not achieve clinical pregnancy was 0.47 ± 0.3 (p = 0.010). The presence of an MLN embryo has been associated with poor embryo development and ART outcomes. Parameters that can predict the formation of MLN embryos before treatment are crucial for the determination of the pregnancy rate. According to our results, serum AMH levels can be used as a predictive marker for the formation of MLN embryos.

Recently, the World Health Organization recommendation for abstinence time for semen analysis has been challenged in some studies and many of them have supported the advantages of a second short abstinence ejaculation. More evidence is needed to approve this for clinical use. This study aimed to compare the average routine abstinence time (2–7 days) with the short time (1–2 h) on sperm quality based on functional parameters in a population of oligo-astheno-teratozoospermia (OAT) men. The semen samples were retrieved from 50 men with OAT two times: one standard 2–7 days (long ejaculation) and short duration trimming (1–2 hours later the first ejaculation). All semen parameters as well as sperm DNA integrity were compared between groups. Results showed that mean sperm concentration (10.40 vs. 8.76), total sperm count (28.53 vs. 12.24) and mean semen volume (2.69 vs. 1.40) were higher in the first ejaculation (2–7 days of abstinence), while progressive motility (20.52 vs. 13.32), non-progressive motility (53.46 vs. 48.86), morphology (2.46 vs. 1.46) and viability (83.90 vs. 77.96) were significantly higher in the second ejaculation (P < 0.05). The second sample also showed lower immotile (26.82 vs. 38.02) and DNA fragmentation (19.5 vs. 26.96) (P < 0.05). Taking all data into account, an additional short abstinence period (AP) may be a simple and helpful strategy to obtain better sperm quality in couples with male infertility causes, especially in OAT patients. The recommended current guidelines regarding the AP may need to be revisited in severe male factors.

Dry incubators prevent bacterial growth and allow time-lapse imaging. However, they cause more significant osmotic pressure changes in the culture medium than in humidified incubators. Few reports have explored the effect of osmotic pressure on human embryos cultured under different conditions. This study examined how changes in osmotic pressure affect human embryos in a dry incubator. The study incubated embryos in culture mediums covered with mineral oil of varying viscosities and quantities. The osmotic pressure of the culture medium was measured daily for six-day period (Day 0–6) in four experimental groups established by varying the viscosity and volume of mineral oil: low viscosity (Light), 3.0 ml or 4.5 ml, and high viscosity (Heavy), 3.0 ml or 4.5 ml of mineral oil. The Light 3.0 ml and Heavy 4.5 ml groups, showing the greatest difference in the osmotic pressure, were used to culture of human embryos. After six days of incubation, the osmotic pressure increased the most in Light 3.0 ml group. Heavy 4.5 ml group had the smallest change. However, no significant differences were noted in the formation rates of blastocysts, good-quality blastocysts, or cell count between the two groups. The study suggests that even when the culture medium is covered with heavy mineral oil in a dry incubator, osmotic pressure increases after six days but does not significantly affect the formation of blastocysts. These findings provide valuable insights into the effects of varying osmotic pressure on embryonic development and may help in optimizing conditions for in vitro fertilization.

In Western diets, high consumption of meat and dairy products, known to be rich in branched-chain amino acids (BCAAs), including leucine (Leu), isoleucine (Ile), and valine (Val), as well as BCAAs supplementation itself, may have unforeseen consequences on sperm quality. In addition, bodybuilders are increasingly resorting to BCAA supplementation to build-up their muscle mass. This study aimed to assess the effect of dietary BCAAs, provided alone or in combination, on semen parameters, apoptotic gene expression, and blood amino acid (AA) profiles. To address this question and determine whether these different BCAAs have a distinct impact on sperm quality and testicular homeostasis, fifty NMRI mature male mice were exposed or not to BCAAs supplementations (control diet: CTR; CTR + Leu supplementation; CTR + Ile supplementation; CTR + Val supplementation; CTR + all three BCAA supplementation). Only valine supplementation resulted in a significant decrease in sperm concentration and viability. In addition, only valine supplementation was associated with a dramatic increase in sperm immotility. The Bax/Bcl2 ratio, an indicator of apoptosis, was found to be significantly higher in the testes of BCAA-supplemented animals when compared with the other groups. Caspase3 expression was also significantly higher in the testes of BCAA-supplemented and Val-supplemented animals. There were no significant differences in plasma AA profiles between groups. Thus, amongst BCAAs, valine supplementation appears to carry the greatest effect on sperm functional parameters and testicular apoptotic status.

Recently, mathematical and computational approaches have been incorporated into ICSI interventions as guiding tools. However, those tools carry no prognostic potential. Improving this capability may enhance ICSI attempts and assist clinicians working in infertility clinics. This study, thus aimed to investigate whether parental parameters could have predictive potential for the quality of resulting embryos with the ICSI approach using mathematical modelling techniques. Patient data including follicle number, MI and MII oocyte numbers, sperm number, sperm morphology and motility for 765 distinct couples attending British Cyprus IVF hospital was collected. Furthermore, morphological quality data as well as aneuploidy status for the 4123 resultant embryos were obtained. Regression analyses were conducted to observe the possible correlations between parental parameters and embryo quality and ploidy. Correlation analyses showed that follicle and oocyte numbers, as well as sperm parameters can be indicative of morphological quality of resulting embryos via ICSI (p values < 0.05). On the other hand, aneuploidy prediction remains too complicated to be predicted solely by these parameters (p values > 0.05). This study indicates a predictive potential of infertility measurements for male and female partners on ICSI success and is expected to act as a basis for the development of prognostic softwares to be used in IVF clinics.

During embryogenesis in Danio rerio (zebrafish), the earliest morphological patterning events are dependent on the precise temporal translation and/or localization of specific maternal mRNAs/proteins. Dorsoventral patterning in particular requires the translocation of maternal factors that are present in the Balbiani Body from the vegetal region of the unfertilized egg to the future dorsal side of the embryo (Fuentes et al., 2020), leading to the localized activation of the β-catenin pathway in the cells in that region. Since zebrafish are chordates, this dorsoventral patterning then leads to the formation of neural tissue on the dorsal side of the embryo. What is not yet clear is the identity of all maternal and zygotic factors that first establish dorsoventral patterning, and which factors lead to the establishment of neural versus non-neural tissue. Taking an evolutionary approach to this question, we investigated a gene in zebrafish, zsquidlike-A (hnrnpaba), that is homologous to a key dorsoventral patterning gene in fruit flies (Drosophila melanogaster) called squid (Kelley, 1993). While dorsoventral patterning in flies and fish looks quite different both morphologically and at the molecular level, we demonstrate that not only has a key dorsoventral patterning gene in flies been conserved in fish, maternal fish zsquidlike-A protein is synthesized precisely as dorsoventral patterning is unfolding in fish embryos, and in its absence, dorsoventral patterning is severely disrupted.

Sesquizygotic twinning (SZT) is one of the rarest events that can occur in multiple pregnancies. It has only been reported twice to date. Studies indicate that sesquizygotic twins result from the splitting of a chimeric embryo at an early stage. Theories and studies suggest that SZT represents a third unique twinning mechanism that is different from dizygotic and monozygotic twinning; however, the unique features of SZT have nothing to do with the twinning mechanism. Instead, it resembles more of an atypical monozygotic twinning form according to the twinning mechanism.