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Objective: The aim of the current work is studying the effect of antioxidants and nano-antioxidants on in vitro development and mitochondrial function of buffalo oocytes. Materials and Methods: Good and excellent Buffalo oocytes were in vitro matured: (1) tissue culture medium-199 (control group), (2) TCM-199 + melatonin (Mel) 10−9 M (Mel group), (3) TCM-199 + zinc 10−6 M (Zn group), (4) TCM-199 + nano- Mel 10−6 M (N-Mel group), and (5) TCM-199 + nano-zinc-oxide 10−6 M (N-ZnO group) and incubated with CO2 5% and 38.5°C for 22 hr. In vitro-matured oocytes were either stained for mitochondrial function or cultured for detection of embryo development. Results: The maturation rate of buffalo oocytes in the N-Mel and N-ZnO groups had a significant (p < 0.05) increase (91.89% and 93.64%, respectively) compared to the Mel group (85.78%) and Zn group (81.37%), and all groups were significantly higher than the control (73.16%). Mitochondrial intensity was significantly elevated (p < 0.05) in the N-Mel and N-ZnO groups than in oocytes matured in the Mel, Zn, or control groups. Rates of fertilization, cleavage, and transferable embryos of buffalo oocytes matured in vitro were significantly raised in the N-ZnO group (88.35%, 85.93%, and 30.71%, respectively) and the N-Mel group (86.74%, 82.75%, and 28.32%, respectively) (p < 0.05) when compared with the Mel group (82.46%, 77.25%, and 21.29%, respectively) and the Zn group (79.98%, 75.19%, and 19.68%, respectively), and all were increased significantly (p < 0.05) compared to the control group (71.76%, 68.7%, and 11.98%, respectively). Conclusion: Supplementation of maturation medium with Mel 10−9 M and zinc sulfate 10−6 M and nano-Mel 10−6 M and nano-zinc oxide 10−6 M improves buffalo oocyte maturation rates, mitochondrial function, and embryo development.

Background: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10 −9 M melatonin for 22 h (at 38.5°C in 5% CO 2 ). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10 −9 M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Increasing evidence has demonstrated that obesity impairs female fertility and negatively affects human reproductive outcome following medically assisted reproduction (MAR) treatment. In the United States, 36.5% of women of reproductive age are obese. Obesity results not only in metabolic disorders including type II diabetes and cardiovascular disease, but might also be responsible for chronic inflammation and oxidative stress. Several studies have demonstrated that inflammation and reactive oxygen species (ROS) in the ovary modify steroidogenesis and might induce anovulation, as well as affecting oocyte meiotic maturation, leading to impaired oocyte quality and embryo developmental competence. Although the adverse effect of female obesity on human reproduction has been an object of debate in the past, there is growing evidence showing a link between female obesity and increased risk of infertility. However, further studies need to clarify some gaps in knowledge. We reviewed the recent evidence on the association between female obesity and infertility. In particular, we highlight the association between fat distribution and reproductive outcome, and how the inflammation and oxidative stress mechanisms might reduce ovarian function and oocyte quality. Finally, we evaluate the connection between female obesity and endometrial receptivity.

To investigate the effects of dietary quercetin on the retrieved mouse oocytes and IVF outcomes. Female mice were divided into two groups. Mice were given 0.2 mL water without (control group) or with quercetin 30 mg/kg (quercetin group) orally via gavage, once a day for 21 consecutive days. After that female mice were superovulated for in vitro fertilization. We observed the effect of dietary quercetin on the number of retrieved oocytes, oocyte degeneration rate, fertilization rate, blastocyst formation rate, and blastocyst cell numbers. There was no difference in the number of retrieved oocytes per mouse (27.3±6.7 and 27.2±5.8), oocyte fragmentation rate (28.4% and 25.0%), fertilization rate (47.4% and 50.6%) and blastocyst formation rate (34.8% and 34.7%) in the quercetin group compared to the control group. The proportion of hatching and hatched blastocyst was significantly lower in the quercetin group (17.2% and 27.8%, p=0.004) and significantly lower numbers of cells in TE (47.4±15.3 and 57.2±17.7) and total cells (66.2±18.5 and 77.5±20.7) compared to the control group (p=0.001). Dietary quercetin supplementation has a detrimental effect on mouse embryo quality. Moreover, it did not show any beneficial effect on the ovary in both quantity and quality. This finding raises awareness of the general use of dietary quercetin supplements in infertile females.

Advanced paternal age has been overlooked, and its effect on fertility remains controversial. Previous studies have focused mainly on intracytoplasmic sperm injection (ICSI) cycles in men with oligozoospermia. However, few studies have reported on men with semen parameters within reference ranges. Therefore, we conducted a retrospective cohort study analyzing the reproductive outcomes of couples with non-male-factor infertility undergoing in vitro fertilization (IVF) cycles. In total, 381 cycles included were subgrouped according to paternal age (<35-year-old, 35-39-year-old, or ≥40-year-old), and maternal age was limited to under 35 years. Data on embryo quality and clinical outcomes were analyzed. The results showed that fertilization and high-quality embryo rates were not significantly different (all P > 0.05). The pregnancy rate was not significantly different in the 35-39-year-old group (42.0%; P > 0.05), but was significantly lower in the ≥40-year-old group (26.1%; P < 0.05) than that in the <35-year-old group (40.3%). Similarly, the implantation rate significantly decreased in the ≥40-year-old group (18.8%) compared with that in the <35-year-old group (31.1%) and 35-39-year-old group (30.0%) (both P < 0.05). The live birth rate (30.6%, 21.7%, and 19.6%) was not significantly different across the paternal age subgroups (<35-year-old, 35-39-year-old, and ≥40-year-old, respectively; all P > 0.05), but showed a declining trend. The miscarriage rate significantly increased in the 35-39-year-old group (44.8%) compared with that in the <35-year-old group (21.0%; P < 0.05). No abnormality in newborn birth weight was found. The results indicated that paternal age over 40 years is a key risk factor that influences the assisted reproductive technology success rate even with good semen parameters, although it has no impact on embryo development.

The aim of this study was to investigate the effects of acylated ghrelin supplementation during in vitro maturation (IVM) of bovine oocytes. IVM medium was supplemented with 20, 40 or 60 pM acylated ghrelin concentrations. Cumulus expansion area and oocyte nuclear maturation were studied as maturation parameters. Cumulus–oocyte complexes (COC) were assessed with the comet, apoptosis and viability assays. The in vitro effects of acylated ghrelin on embryo developmental capacity and embryo quality were also evaluated. Results demonstrated that acylated ghrelin did not affect oocyte nuclear maturation and cumulus expansion area. However, it induced cumulus cell (CC) death, apoptosis and DNA damage. The damage increased as a function of the concentration employed. Additionally, the percentages of blastocyst yield, hatching and embryo quality decreased with all acylated ghrelin concentrations tested. Our study highlights the importance of acylated ghrelin in bovine reproduction, suggesting that this metabolic hormone could function as a signal that prevents the progress to reproductive processes.

The endoplasmic reticulum (ER) is a multifunctional organelle in the cytoplasm that plays important roles in female mammalian reproduction. The endoplasmic reticulum and mitochondria interact to maintain the normal function of cells by maintaining intracellular calcium homeostasis. As proven by previous research, glycine (Gly) can regulate the intracellular free calcium concentration ([Ca 2+ ] i ) and enhance mitochondrial function to improve oocyte maturation in vitro . The effect of Gly on ER function during oocyte in vitro maturation (IVM) is not clear. In this study, we induced an ER stress model with thapsigargin (TG) to explore whether Gly can reverse the ER stress induced by TG treatment and whether it is associated with calcium regulation. The results showed that the addition of Gly could improve the decrease in the average cumulus diameter, the first polar body excretion rate caused by TG-induced ER stress, the cleavage rate and the blastocyst rate. Gly supplementation could reduce the ER stress induced by TG by significantly improving the ER levels and significantly downregulating the expression of genes related to ER stress (Xbp1, ATF4, and ATF6). Moreover, Gly also significantly alleviated the increase in reactive oxygen species (ROS) levels and the decrease in mitochondrial membrane potential (ΔΨ m) to improve mitochondrial function in porcine oocytes exposed to TG. Furthermore, Gly reduced the [Ca 2+ ] i and mitochondrial Ca 2+ ([Ca 2+ ] m ) levels and restored the ER Ca 2+ ([Ca 2+ ] ER ) levels in TG-exposed porcine oocytes. Moreover, we found that the increase in [Ca 2+ ] i may be caused by changes in the distribution and expression of inositol 1,4,5-triphosphate receptor (IP 3 R1) and voltage-dependent anion channel 1 (VDAC1), while Gly can restore the distribution and expression of IP 3 R1 and VDAC1 to normal levels. Apoptosis-related indexes (Caspase 3 activity and Annexin-V) and gene expression Bax, Cyto C, and Caspase 3) were significantly increased in the TG group, but they could be restored by adding Gly. Our results suggest that Gly can ameliorate ER stress and apoptosis in TG-exposed porcine oocytes and can further enhance the developmental potential of porcine oocytes in vitro .

Interspecific hybridization was carried out between Lilium longiflorum and L. lophophorum var. linearifolium by using the cut style method of pollination, as a contrast, intraspecific hybridization between L. longiflorum 'Gelria' and L. longiflorum was also made, but no mature seeds and offspring were obtained from the two combinations under in vivo condition. Ovules excised from each carpel 5-35 days after pollination (DAP) were cultured on B5 or half-strength B5 medium containing sucrose at different concentrations in vitro. In L. longiflorum x L. lophophorum var. linearifolium, only 1.17% of ovules excised at 10 DAP developed into seedlings, and in L. longiflorum 'Gelria' x L. longiflorum, only 0.99% of ovules excised at 25 DAP developed into seedlings; none of the ovules excised at other different DAP in the two cross combinations produced any seedlings. The results showed that interspecific hybridization had a more serious post-fertilization barrier than the intraspecific hybridization, and that a lower concentration (3%) of sucrose led to better embryo development and higher percentage of seedlings in ovule cultures. All hybrid seedlings obtained were successfully transplanted to soil and grew normally. The progenies investigated were identified as true hybrids based on inter-simple sequence repeat (ISSR) analysis.

The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.

In this study we examined the biosynthesis of abscisic acid (ABA) by developing corn (Zea mays L.) embryos. Three comparisons were made: ABA biosynthesis in embryos isolated from kernels grown in vitro with those grown in the field; the developmental profile of ABA content with that of biosynthesis; and ABA biosynthesis in corn embryos lacking carotenoid precursors with ABA biosynthesis in normal embryos. Embryos were harvested at various times during seed development and divided into two groups. Endogenous levels of ABA were measured in one group of embryos and ABA biosynthetic capacity was measured in the other group. The ABA biosynthetic capacity was measured with and without tetcyclacis (an inhibitor of ABA degradation) in embryos from both field-grown and in-vitro-grown corn kernels. Reduced-carotenoid (either fluridone-treated or genetically viviparous) embryos were also included in the study. Corn kernels developing under field and in-vitro conditions differed from each other in their responses to tetcyclacis and in their profiles of ABA biosynthesis during development. Therefore, in-vitro kernel culture may not be an appropriate substitute for field conditions for studies of embryo development. The developmental profiles of endogenous ABA content differed from those of ABA biosynthesis in isolated embryos of both in-vitro- and field-grown kernels. This indicated that ABA levels in the developing embryos were determined by import from the maternal tissues available to the embryos rather than by in-situ biosynthesis. In embryos with reduced levels of carotenoids, either fluridone-treated or genetically viviparous embryos, ABA biosynthesis was low or nonexistent. This result is expected for the presence of an indirect pathway of ABA biosynthesis and in the absence of ABA precursors.