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Oviduct fluid extracellular vesicles (oEV) are essential for periconceptional events. The presence of EV has already been identified in the oviduct fluid (OF) from mammalian species, except in caprine. Therefore, this study aimed to isolate and characterize the caprine oEV (coEV). Initially, in Experiment 1, coEV were isolated from the OF of either each animal individually or from a pool of three animals. In experiment 2, coEV were isolated during the follicular or luteal phases of the estrous cycle. The coEV were characterized by size distribution, polydispersity index (PDI), and zeta potential using dynamic light scattering (DLS) analysis, as well as, by transmission electron microscopy (TEM) and dot blotting (DB). Our results indicated that the physicochemical characteristics of the coEV were similar (P > 0.05), regardless of the isolation method (individual or pool). However, coEV collected during the luteal phase were larger (P < 0.05) than those during the follicular phase. The TEM showed spherical and cup-shaped particles, characteristic of exosomes. The DB revealed the presence of exosomal proteins involved in the biogenesis of coEV. In conclusion, it is possible to isolate and characterize coEV from a single caprine female and the estrous cycle phase influences the vesicles average size and PDI., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

Anticancer therapy often leads to premature ovarian insufficiency (POI) and infertility due to the extreme sensitivity of the ovarian follicle reserve to the effects of chemotherapy. Withanolides are known for their cytotoxic effect on cancer cells and low cytotoxicity on non-malignant or healthy cells. Therefore, this study aimed to investigate the in vivo effects of three withanolides derivatives: 27-dehydroxy-24,25-epoxywithaferin A (WT1), 27-dehydroxywithaferin A (WT2), and withaferin A (WTA) on fertility, and the ovarian preantral follicles of young female mice. To achieve this, mice received 7 intraperitoneal doses of WT1, WT2, or WTA at a concentration of 2 mg/kg (Experiment I) and 5 or 10 mg/kg (Experiment II) over 15 alternate days. In experiment I , two days after administration of the last dose, half of the mice were mated to evaluate the effects of withanolides on fertility. The other half of the mice, as well as all mice from experiment II , were sacrificed for histological, inflammation, senescence, and immunohistochemical analyses of the follicles present in the ovary. Regardless of the administered withanolide, the concentration of 2 mg/kg did not show toxicity on the follicular morphology, ovarian function, or fertility of the mice. However, at concentrations of 5 and 10 mg/kg, the three derivatives (WT1, WT2, and WTA) increased follicular activation, cell proliferation, and ovarian senescence without affecting inflammatory cells. Furthermore, at a concentration of 10 mg/kg, the three withanolides showed intensified toxic effects, leading to DNA damage as evidenced by the labeling of γH2AX, activated Caspase 3, and TUNEL. We conclude that the cytotoxic effect of the tested withanolide derivatives (WT1, WT2, and WTA) in the concentration of 2 mg/kg did not show toxicity on the ovary. However, in higher concentrations, such as 10 mg/kg, toxic effects are potentiated, causing DNA damage., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare., (Copyright © The Author(s).)

The present study aims to establish the morphological, morphometric, and immunostaining patterns of the steroidogenic enzymes 17β-HSD and 5α-reductase and androgen receptors (AR) during the prenatal development of the male gonad and epididymis of Cavia porcellus. Fetuses at 22, 25, 30, 40, 45, 50, and 60 days of gestation (DG) were used. Specimens were dissected and subjected to macroscopic, histological, histomorphometric, and immunohistochemical analyses. Genital and scrotal protrusions were identified in 22 DG embryos. Gonocytes were identified at 25 DG and the formation of primary testicular cords was observed at 30 DG. Through anatomical evaluation, we observed differentiation of the epididymis into the head, body, and tail at 45 DG. During development, there is a progressive decrease in the diameters of the testicular cords and epididymal ducts. 17β-HSD enzyme immunostaining was observed in Leydig cells at all ages, while 5α-reductase was observed in Leydig cell cytoplasm and gonocytes at 40, 50, and 60 DG. AR shows gonocyte labeling at 30 DG. Thus, from the second trimester of pregnancy, it is possible to observe patterns of anatomical development, such as genital and scrotal prominence (22 DG), the appearance of gonocytes in the testicular cords at 25 DG, and the beginning of the organization of primary testicular cords at 30 DG, suggesting sexual differentiation. The 17β-HSD, 5α-reductase, and ARs play an essential role in sexual development and differentiation, presenting immunostaining at different reproductive process times., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Due to the great interest in ovarian cryopreservation and, consequently conservation and restoration of female fertility in the last decades, different vitrification procedures (vitrification devices or solutions) have been developed, patented, and used both for academic research purposes and for clinical use. Therefore, the present study aimed to provide a systematic review and meta-analysis of data obtained from the application of different patented and non-patented vitrification devices and solutions in different countries. For this purpose, relevant observational studies published between the years 2000 to 2021 were selected to verify the efficiency of ovarian vitrification processes on parameters such as morphology, viability, and apoptosis in preantral ovarian follicles after transplantation or in vitro culture. Our research revealed that, although several countries were considered in the study, the United States and Japan were the countries that registered the most processes, and 22 and 16 vitrification devices and solutions out of a total of 51, respectively were patented. Sixty-two non-patented processes were also considered in the study in all countries. We also observed that transplantation and in vitro ovarian culture were the techniques predominantly used to evaluate the efficiency of the devices and vitrification solutions, respectively. In conclusion, this review showed that patented or non-patented protocols available in the literature are able to successfully preserve preantral follicles present in ovarian tissue. Despite the satisfactory results reported so far, adjustments in ovarian vitrification protocols in order to minimize cryoinjuries to the follicles remain one of the goals of cryopreservation and preservation of the female reproductive function. We found that vitrification alters the morphology and viability, and offers risks leading in some cases to follicular apoptosis. However, adjustments to current protocols to develop an optimal procedure can minimize damage by not compromising follicular development after vitrification/warming., Competing Interests: Conflicts of interest: The authors have no conflict of interest to declare.

This study aims to define the best method (slow freezing or vitrification) and fragment size (1, 5, or 9 mm³) for prepubertal goat testis cryopreservation, as well as to evaluate testicular morphological integrity after cryopreservation and in vitro culture (IVC). Initially (experiment I), 1, 5, or 9 mm³ testis fragments were cryopreserved by slow freezing using a Mr. Frosty container with 20% Dimethylsulfoxide (DMSO) or vitrified using the Ovarian Tissue Cryosystem (OTC) device, (Equilibration solution - ES: 10% DMSO and 10% ethylene glycol - EG; Vitrification solution - VS: 20% DMSO and 20% EG) and then subjected to morphological analysis, type I and III collagen quantification and gene expression (Oct4, C-kit, Bax, and Bcl-2). Subsequently, (experiment II), fresh or cryopreserved by slow freezing testis fragments were cultured in vitro and submitted to morphological analysis by scanning electron microscopy. The data from the experiment I revealed fewer morphological alterations in 1 and 5 mm³ fragments after vitrification and slow freezing, respectively. The percentage of type I collagen fibers in 5 and 9 mm³ frozen was higher than in fresh or vitrified fragments. For type III collagen, fresh or frozen fragments of 1 and 5 mm 3 showed a higher percentage than fragments of 9 mm 3 . Gene expression for Oct4 and C-kit after slow freezing or vitrification in the 5 mm 3 fragments was lower than that observed in the fresh fragments. The Bax:Bcl-2 ratio in the 1 and 9 mm³ fragments was lower than in the 5 mm³ fragments for fresh fragments or after freezing. In experiment II, fragments cultured in vitro, previously frozen or not, showed more morphological alterations than fresh or frozen fragments. We concluded that slow freezing of 5 mm³ fragments was the best protocol for cryopreserving prepubertal goat testis and although the results of IVC are encouraging, it still needs improvement to restore testicular function after cryopreservation., Competing Interests: Declaration of competing interest The authors have no conflict of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

This work investigated the effect of zinc oxide nanoparticles functionalized with curcumin (ZnO (np)  + CUR) supplementation during the in vitro embryo culture (IVC) on the bovine in vitro embryo production, and the cellular antioxidant response. The cumulus-oocyte complexes (COCs) were matured, fertilized and then the presumptive zygotes were cultured in the medium in the absence (0 μM-control) or presence of different concentrations of ZnO (np)  + CUR (3, 6 or 12 μM). After IVC, the embryos were destined either to assay intracellular ROS levels and mitochondrial membrane potential. The results demonstrated that only the addition of 12 μM ZnO (np)  + CUR during IVC decreased intracellular ROS production and the rate of blastocyst production when compared to the control (p < .05). In conclusion, ZnO (np)  + CUR addition during the IVC impaired in concentration-dependent-manner bovine in vitro embryo production., (© 2023 Wiley-VCH GmbH. Published by John Wiley & Sons Ltd.)

Background: We evaluated pathological findings in targeted biopsies of PI-RADS4 and PI-RADS5 lesions, and clinical data that could predict those patients with benign findings., Materials and Methods: A retrospective study was conducted to summarize the experience from a single nonacademic center using cognitive fusion and a 1.5 or 3.0 Tesla scanner., Results: We found a false positive rate of 29 and 3.7% for any cancer in PI-RADS 4 and 5 lesions, respectively. Diverse histologic patterns were observed among target biopsies. At multivariate analysis, size ≤ 6 mm and previous negative biopsy were independent predictors of false positive PI-RADS4 lesions. The small number of false PI-RADS5 lesions precluded further analyses., Conclusion: Benign findings are common in PI-RADS4 lesions and most of them do not show obvious glandular or stromal hypercellularity as expected in hyperplastic nodules. Size ≤ 6 mm and previous negative biopsy predict a higher probability of false positive results in patients with PI-RADS 4 lesions., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Discarding the first ejaculate is recommended as an alternative for improving seminal quality after long sexual resting, especially when semen should be used for cryopreservation. However, when the males are not in sexual resting the necessity to discarding the first ejaculate is still unknown. Therefore, this study aimed to compare by flow cytometry the quality of the first and second ejaculates. Ten kids and uniform goats between 5 and 6 months of age were used in a completely randomized design. Semen collection was carried out every 4 days, until a total of five ejaculates per animal in each treatment was completed. The fresh and frozen semen collected were processed and analyzed using macroscopic and microscopic parameters, resistance test, hypo-osmotic medium test, and flow cytometry (FC). The FC parameters were production of reactive oxygen species, plasma and acrosomal membrane integrity, and lipid peroxidation of the plasma membrane. The ejaculates did not differ for the resistance test, the reactivity in the hypo-osmotic medium and for the macroscopic and microscopic seminal parameters, except for sperm volume and concentration. The first ejaculate had a higher percentage of minor and total defects. None of the FC parameters analyzed differed between the first and second ejaculates. The first and second ejaculates demonstrated similar seminal qualities, so for Alpine kid goats without a sexual resting period, discarding the first ejaculate it is not recommended.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Objectives: To date, it is unknown whether systematic biopsies can be safely omitted in patients with unsuspicious MRI findings or if systematic biopsies should be required when targeting focal lesions (PI-RADS 3-5)., Methods: A series of 366 patients (249 without a previous biopsy) were examined in a 1.5 Tesla MRI scanner. All patients were submitted to systematic biopsies (12-14 regions) with additional targeted biopsies (by cognitive fusion) of focal PI-RADS lesions (PI-RADS 3-5)., Results: In our series, patients with PI-RADS 1/2 findings had rates of adenocarcinoma of any grade, >GG1 and GG4/5 of 34%, 14% and 3%, respectively. The use of MRI prior to biopsy in our series increased the detection of clinically significant prostate cancer (CSPCa) in 28% of patients with focal lesions, and focal lesions were present in 293/366 (80%) of all patients. For CSPCa (>GG1), targeted biopsies improved the diagnosis in 28% of patients, while systematic biopsies resulted in an additional 19% of cancer cases in the series., Conclusion: Systematic biopsies should still be considered in patients with PI-RADS 1/2 findings. Our findings also suggest a stronger benefit of the combined strategy of targeted and systematic biopsies than the findings of previous studies concerning the detection of CSPCa in biopsy-naïve patients.

This study aimed to evaluate the replacement of soybean meal by detoxified castor bean meal on the intake and digestibility of nutrients, body weight gain, carcass yield, physiological and urinary parameters, and creep-feeding method effectiveness. For this trial, 43 male lambs were used, Dorper × Santa Inês, with an average initial body weight of 7.95 kg, 15 days old. Lambs were distributed in a randomized block design. Five experimental diets were provided: Without supplementation, with supplementation but without the use of castor bean meal, and with gradual levels of replacement of soybean meal by detoxified castor bean meal (33%, 67%, and 100%). Higher values of DMI and nutrient digestibility ( p < 0.05) were observed for animals that received supplements. The milk intake did not differ among the diets. The DMD showed a linear effect, while NDFD had a quadratic effect, depending on castor bean meal inclusion. The carcass yield did not differ between experimental diets. There was no significant effect on the urinary volume and metabolites investigated. In conclusion, the replacement of soybean meal by detoxified castor bean meal does not compromise animals' development. Besides, the creep-feeding method positively affects lamb development, with higher body weight gain for supplemented animals.

Excessive intake of nutrients can result in decreases in the reproductive efficiency of goat bucks and increase production costs in goat e nterprises. In this study, the aim was to compare the reproductive features of young bucks fed diets for maintenance of growth or were fed ad libitum. Ten bucks were used (n = 5/group), with a completely randomized design. Blood and semen were collected from each animal every 2 weeks for nine collections. Semen was frozen and later analyzed using flow cytometry to compare reactive oxygen species, plasma membrane integrity, acrosome integrity, and membrane lipid peroxidation. Body condition score, sexual behavior, testicular biometry and histomorphometry of the testis, as well as oxidative enzymes and carbonylated proteins, were also evaluated. Animals fed ad libitum had a greater body condition score (3.32 + 0.08 × 2.88 + 0.05), sperm volume (0.68 + 0.04 × 0.52 + 0.03 mL), and testis circumference (25.2 + 0.21 × 24.5 + 0.24 cm) and volume (121 + 5.83 × 107 + 4.38 cm³, left testicle; 116 + 5.26 × 100 + 3.75 cm³, right testicle). Ejaculates of the bucks fed diets to maintain growth rate had a larger number of minor and total sperm morphology defects. There were no differences in testosterone concentrations and sexual behavior. In conclusion, there can be feeding for growth rate maintenance instead of ad libitum feeding, for the reduction in production costs of young bucks without compromising semen quality and reproductive functions., (Copyright © 2020 Elsevier B.V. All rights reserved.)

To determine the effects of maternal nutrition on modifications of foetal development of the skeletal muscle and possible increase in the potential of skeletal muscle growth in cattle, gestating cows were either fed 190% NRC recommendations (overnourished; ON) or 100% NRC recommendation (control; CO). Interaction between maternal nutrition (MN) and the foetal sex (FS) was also investigated. Foetuses were necropsied at four different time points throughout gestation (139, 199, 241 and 268 days of gestation) to assess the mRNA expression of myogenic, adipogenic and fibrogenic markers in skeletal muscle. Phenotypic indicators of the development of skeletal muscle fibres, intramuscular lipogenesis and collagen development were also evaluated. Modifications in mRNA expression of skeletal muscle of foetuses were observed in function of MN and FS despite the lack of effect of MN and FS on foetal weight at necropsy. Maternal ON increased the mRNA expression of the myogenic marker Cadherin-associated protein, beta 1 (CTNNB1) and adipogenic markers Peroxissome proliferator-activated receptor gamma (PPARG) and Zinc finger protein 423 (ZNF423) at midgestation. However, no differences on foetal skeletal muscle development were observed between treatments at late gestation indicating that a compensatory development may have occurred on CO foetuses making the effect of MN on skeletal muscle development not significant at late gestation. Moreover, our data have shown an evidence of sexual dimorphism during foetal stage with a greater skeletal muscle development in male than in female foetuses. In conclusion, providing a higher nutritional level to pregnant cows changes the trajectory of the development of skeletal muscle during midgestation, but apparently does not change the potential of post-natal growth of muscle mass of the offspring, as no differences in skeletal muscle development were observed in late gestation., (© 2017 Blackwell Verlag GmbH.)