Pollen production in olive cultivars and its interannual variability.

BACKGROUND AND AIMS: Olive (Olea europaea subsp. europaea var. europaea) is the most extensively cultivated fruit crop worldwide. It is considered a wind-pollinated and strictly outcrossing crop. Thus, elevated pollen production is crucial to guarantee optimum fruit set and yield. Despite these facts, the variability of pollen production within the cultivated olive has been scarcely studied. This study aimed to characterize this feature by analysing a representative set of worldwide olive cultivars. METHODS: We evaluated the average number of pollen grains per anther in 57 principal cultivars over three consecutive years. We applied a standard generalized linear model (GLM) approach to study the influence of cultivar, year and the previous year's fruit load on the amount of pollen per anther. Additionally, the K-means method was used for cluster analysis to group cultivars based on their pollen production capacity. KEY RESULTS: Pollen production per anther was highly variable among olive cultivars. The cultivar significantly accounted for 51.3 % of the variance in pollen production and the year for 0.3 %. The interaction between the two factors explained 8.4 % of the variance, indicating that not all cultivars were equally stable in producing pollen across the years. The previous year's fruit load and its interaction with the year were significant, but barely accounted for 1.5 % of the variance. Olive cultivars were classified into four clusters according to their capacity to produce pollen. Interestingly, the fourth cluster was composed of male-sterile cultivars, which presumably share this character by inheritance. CONCLUSIONS: Pollen production per anther varied extensively within the cultivated olive. This variation was mainly driven by the cultivar and its interaction with the year. The differential capacity of olive cultivars to produce pollen should be considered not only for designing new orchards but also gardens where this species is used as an ornamental.

Rumen biohydrogenation and milk fatty acid profile in dairy ewes divergent for feed efficiency.

A sustainable increase in livestock production would require selection for improved feed efficiency, but the mechanisms underlying this trait and explaining its large individual variation in dairy ruminants remain unclear. This study was conducted in lactating ewes to test the hypothesis that rumen biohydrogenation (BH) would differ between high- and low-efficiency animals, and these differences would be reflected in rumen fatty acid (FA) profile and affect milk FA composition. A second aim was to identify differences in FA that may serve as biomarkers of feed efficiency. Data of daily feed intake and milk yield and composition, as well as body weight, were collected individually over a 3-wk period in 40 ewes. The difference between the mean actual and predicted feed intake (estimated through metabolizable energy requirements for maintenance, production, and body weight change) over the period was used as the feed efficiency index (FEI) to select 8 of the highest feed efficiency (H-FE) and 8 of the lowest feed efficiency (L-FE) animals. In addition, residual feed intake (RFI) was estimated as the residual term from the regression of feed intake on various energy sinks. Rumen and milk FA composition were characterized by using gas chromatography, and results were analyzed using a statistical model that included the fixed effect of the group (H-FE vs. L-FE). The FEI averaged -0.29 ± 0.046 and 0.81 ± 0.084 in H-FE and L-FE, respectively, whereas RFI averaged -0.16 ± 0.084 and 0.18 ± 0.082, respectively. The correlation coefficient between both metrics was 0.69. Feed intake was similar in both groups, but H-FE showed greater milk yield, with increases in lactose content and yield, and in milk protein and fat production. Results from rumen FA profiles included a lower proportion of 18:2n-6, cis-9 18:1, and of several of their BH metabolites, and a greater concentration of 18:0, which may indicate that the apparent BH would be more complete in more efficient sheep. Milk FA analysis suggested that the greater fat yield in the H-FE group was mostly explained by increased de novo FA synthesis, whereas their milk would have lower proportions of cis-9 18:1 and C20 to 22n-6 polyunsaturated FA than L-FE. Stepwise multiple linear regression suggested that milk C20 to 22n-6 PUFA might be convenient biomarkers to discriminate more efficient dairy sheep. Further research is needed to validate these findings (e.g., under different dietary conditions).

Impact of sperm DNA damage and oocyte-repairing capacity on trout development.

Zygotic repair of paternal DNA is essential during embryo development. In spite of the interest devoted to sperm DNA damage, its combined effect with defect-repairing oocytes has not been analyzed. Modification of the breeding season is a common practice in aquaculture. This practice reduces developmental success and could affect the both factors: sperm DNA integrity and oocyte repair capacity. To evaluate the maternal role, we analyzed the progeny outcome after fertilizing in-season trout oocytes with untreated and with UV-irradiated sperm. We also analyzed the offspring obtained out of season with untreated sperm. The analysis of the number of lesions in 4 sperm nuclear genes revealed an increase of 1.22-11.18 lesions/10 kb in out-of-season sperm, similar to that obtained after sperm UV irradiation (400 µW/cm(2)5 min). Gene expression showed in out-of-season oocytes the overexpression of repair genes (ogg1, ung, lig3, rad1) and downregulation of tp53, indicating an enhanced repairing activity and reduced capacity to arrest development upon damage. The analysis of the progeny in out-of-season embryos revealed a similar profile tolerant to DNA damage, leading to a much lower apoptotic activity at organogenesis, lower hatching rates and increased rate of malformations. The effects were milder in descendants from in-season-irradiated sperm, showing an enhanced repairing activity at epibolia. Results point out the importance of the repairing machinery provided by the oocyte and show how susceptible it is to environmental changes. Transcripts related to DNA damage signalization and repair could be used as markers of oocyte quality.

Inhibition of zygotic DNA repair: transcriptome analysis of the offspring in trout (Oncorhynchus mykiss).

Zygotic repair of the paternal genome is a key event after fertilization. Spermatozoa accumulate DNA strand breaks during spermatogenesis and can suffer additional damage by different factors, including cryopreservation. Fertilization with DNA-damaged spermatozoa (DDS) is considered to promote implantation failures and abortions, but also long-term effects on the progeny that could be related with a defective repair. Base excision repair (BER) pathway is considered the most active in zygotic DNA repair, but healthy oocytes contain enzymes for all repairing pathways. In this study, the effects of the inhibition of the BER pathway in the zygote were analyzed on the progeny obtained after fertilization with differentially DDS. Massive gene expression (GE; 61 657 unique probes) was analyzed after hatching using microarrays. Trout oocytes are easily fertilized with DDS and the high prolificacy allows live progeny to be obtained even with a high rate of abortions. Nevertheless, the zygotic inhibition of Poly (ADP-ribose) polymerase, upstream of BER pathway, resulted in 810 differentially expressed genes (DEGs) after hatching. DEGs are related with DNA repair, apoptosis, telomere maintenance, or growth and development, revealing a scenario of impaired DNA damage signalization and repair. Downregulation of the apoptotic cascade was noticed, suggesting a selection of embryos tolerant to residual DNA damage during embryo development. Our results reveal changes in the progeny from defective repairing zygotes including higher malformations rate, weight gain, longer telomeres, and lower caspase 3/7 activity, whose long-term consequences should be analyzed in depth.

Association between IL7RA polymorphisms and the successful therapy against HCV in HIV/HCV-coinfected patients.

Interleukin-7 (IL-7) is a critical factor in maintaining or inducing effective antiviral CD4+ and CD8+ T-cell responses. The aim of this study was to examine the association of interleukin-7 receptor-α (IL7RA) polymorphisms with a sustained virologic response (SVR) after hepatitis C virus (HCV) therapy with pegylated interferon-alpha plus ribavirin (pegIFNα/ribavirin) in 177 human immunodeficiency virus (HIV)/HCV-coinfected patients. We performed a retrospective study in 177 naïve patients who started HCV treatment. The IL7RA rs6897932, rs987106, and rs3194051 polymorphisms were genotyped by the GoldenGate® assay. An SVR was defined as undetectable HCV viral load through 24 weeks after the end of HCV treatment. The highest SVR rate was found in patients with the rs6897932 CC (p = 0.029) and rs3194051 GG (p = 0.002) genotypes, and HCV genotypes 2/3 (GT2/3) infected patients with the rs987106 AA genotype (p = 0.048). Additionally, carriers of the rs3194051 GG genotype had a higher likelihood of achieving an SVR [adjusted odds ratio (aOR) = 5.32; 95 % confidence interval (CI) = 1.07-26.94; p = 0.040] than patients with the rs3194051 AA/AG genotype, while rs6897932 CC (aOR = 0.63; p = 0.205) and rs987106 AA (aOR = 0.60; p = 0.213) were not significant. Moreover, three major haplotypes were found: 46.6 % for CTA, 32.4 % for CAG, and 20.7 % for TAA haplotypes. Patients infected with GT2/3 and carriers of the CTA haplotype had lower odds of achieving an SVR (aOR = 0.08; p = 0.004) and the CAG haplotype (favorable alleles) had higher odds of achieving an SVR than other haplotypes (aOR = 21.96; p < 0.001). IL7RA polymorphisms seem to play a significant role in the virological response to pegIFNα/ribavirin therapy in HIV/HCV-coinfected patients, in particular among patients infected with HCV GT2/3.

Relative Susceptibility of New Olive Cultivars to Spilocaea oleagina, Colletotrichum acutatum, and Pseudocercospora cladosporioides.

The evaluation of the relative susceptibility of new cultivars to the main diseases of a crop is a key point to consider prior to their release to the nursery industry. This study provides a rigorous characterization of the resistance of 15 new olive cultivars and their genitors ('Arbequina,' 'Frantoio,' and 'Picual') to the three main aerial diseases, peacock spot, anthracnose, and cercosporiosis caused by Spilocaea oleagina, Colletotrichum acutatum, and Pseudocercospora cladosporioides, respectively. To do so, developing leaves and detached green-yellowish fruit were inoculated in laboratory tests with S. oleagina and C. acutatum, respectively, using conidial suspensions of both pathogens. Additionally, a previously validated rating scale was used to assess the incidence of leaves with symptoms of S. oleagina or P. cladosporioides and the fruit rot incidence of C. acutatum in the trees for four years under field conditions. As a result, only two of the cultivars were susceptible to peacock spot, most likely because these new cultivars were previously screened for resistance to the disease on previous phases of the breeding program. Conversely, the 15 cultivars were susceptible or moderately susceptible to cercosporiosis. Five of the 15 new cultivars were classified as resistant to anthracnose, with four of them descendants of 'Frantoio' × 'Picual' crosses. In addition, the cultivars resistance to C. acutatum showed a negative linear correlation with the total phenols content of olive oil. This information regarding disease reaction of the new olive cultivars is essential for nursery industry and growers.

Are parent-reared red-legged partridges (Alectoris rufa) better candidates for re-establishment purposes?

Aiming to produce game birds suitable for re-establishment purposes, we studied the survival and behavior of 31 adult red-legged partridges reared by their natural parents in captivity (parent-reared) and 67 wild red-legs (35 adults and 32 subadults). Birds were radio-tracked and released in a game estate where management targeting small game species was conducted and shooting was not allowed. Survival of parent-reared partridges was shorter (mean 108 d) than wild adults (mean 160 d), though no significant differences were found. Parent-reared birds showed a longer escape reaction (mean 11.7 sec) than wild adults (mean 0.4 sec) and were mainly predated by terrestrial predators (52%), whereas the most important source of predation in wild red-legs was raptors (49%). The home range of parent-reared (mean 23 ha) was not significantly lower than wild adults (mean 27 ha). Nineteen percent of parent-reared and 31.4% of wild adults paired successfully, and we recorded 6 pairs in which one bird was wild and the other parent-reared and one pair in which both birds were parent-reared. A small proportion of red-legs started incubation (parent-reared 12.9%, wild 20%), and the proportion completing incubation was similar among wild and parent-reared birds. Overall, parent-reared red-legs showed similar survival and behavior compared to wild red-legs and better than intensively reared, so parent-reared red-legs should be chosen for the re-establishment of wild populations.

Hepatoma-derived growth factor: from the bovine uterus to the in vitro embryo culture.

Early in cow embryo development, hepatoma-derived growth factor (HDGF) is detectable in uterine fluid. The origin of HDGF in maternal tissues is unknown, as is the effect of the induction on developing embryos. Herein, we analyze HDGF expression in day 8 endometrium exposed to embryos, as well as the effects of recombinant HDGF (rHDGF) on embryo growth. Exposure to embryos did not alter endometrial levels of HDGF mRNA or protein. HDGF protein localized to cell nuclei in the luminal epithelium and superficial glands and to the apical cytoplasm in deep glands. After uterine passage, levels of embryonic HDGF mRNA decreased and HDGF protein was detected only in the trophectoderm. In fetal fibroblast cultures, addition of rHDGF promoted cell proliferation. In experiments with group cultures of morulae in protein-free medium containing polyvinyl alcohol, adding rHDGF inhibited blastocyst development and did not affect cell counts when the morulae were early (day 5), whereas it enhanced blastocyst development and increased cell counts when the morulae were compact (day 6). In cultures of individual day 6 morulae, adding rHDGF promoted blastocyst development and increased cell counts. Our experiments with rHDGF indicate that the growth factor stimulates embryonic development and cell proliferation. HDGF is synthesized similarly by the endometrium and embryo, and it may exert embryotropic effects by autocrine and/or paracrine mechanisms.

Elements of functional genital asymmetry in the cow.

Asymmetry in the cow affects ovarian function and pregnancy. In this work we studied ovarian and uterine asymmetry. Synchronised animals, in which in vitro-produced embryos (n=30-60) had been transferred on Day 5 to the uterine horn ipsilateral to the corpus luteum (CL), were flushed on Day 8. Ovulatory follicle diameter, oestrus response and total protein flushed did not differ between sides. However, a corpus luteum in the right ovary led to plasma progesterone concentrations that were higher than when it was present in the left ovary. Fewer embryos were recovered from the left than the right horn. Among 60 uterine proteins identified by difference gel electrophoresis, relative abundance of nine (acyl-CoA dehydrogenase, very long chain; twinfilin, actin-binding protein, homologue 1; enolase 1; pyruvate kinase isozymes M1/M2 (rabbit); complement factor B Bb fragment ; albumin; fibrinogen gamma-B chain; and ezrin differed (P<0.05) between horns. Glucose concentration was higher, and fructose concentration lower, in the left horn. In a subsequent field trial, pregnancy rates after embryo transfer did not differ between horns (51.0±3.6, right vs 53.2±4.7, left). However, Day 7 blood progesterone concentrations differed (P=0.018) between pregnant and open animals in the left (15.9±1.7 vs 8.3±1.2) but not in the right horn (12.4±1.3 vs 12.4±1.2). Progesterone effects were independent of CL quality (P=0.55). Bilateral genital tract asymmetry in the cow affects progesterone, proteins and hexoses without altering pregnancy rates.

Prediction of pregnancy viability in bovine in vitro-produced embryos and recipient plasma with Fourier transform infrared spectroscopy.

We analyzed embryo culture medium (CM) and recipient blood plasma using Fourier transform infrared (FTIR) metabolomics to predict pregnancy outcome. Individually cultured, in vitro-produced (IVP) blastocysts were transferred to recipients as fresh and vitrified-warmed. Spent CM and plasma samples were evaluated using FTIR. The discrimination capability of the classifiers was assessed for accuracy, sensitivity (pregnancy), specificity (nonpregnancy), and area under the receiver operator characteristic curve (AUC). Within all IVP fresh embryos (birth rate=52%), high AUC were obtained at birth, especially with expanded blastocysts (CM: 0.80±0.053; plasma: 0.89±0.034). The AUC of vitrified IVP embryos (birth rate = 31%) were 0.607±0.038 (CM, expanded blastocysts) and 0.672±0.023 (plasma, all stages). Recipient plasma generally predicted pregnancy outcome better than did embryo CM. Embryos and recipients with improved pregnancy viability were identified, which could increase the economic benefit to the breeding industry.

The dynamic changes in olive fruit phenolic metabolism and its contribution to the activation of quiescent Colletotrichum infection.

Anthracnose, the most critical disease affecting olive fruits, is caused by Colletotrichum species. While developing olive fruits are immune to the pathogen regardless of the cultivar, the resistance level varies once the fruit ripens. The defense mechanisms responsible for this difference in resistance are not well understood. To explore this, we analyzed the phenolic metabolic pathways occurring in olive fruits and their susceptibility to the pathogen during ripening in two resistant cultivars ('Empeltre' and 'Frantoio') and two susceptible cultivars ('Hojiblanca' and 'Picudo'). Overall, resistant cultivars induced the synthesis of aldehydic and demethylated forms of phenols, which highly inhibited fungal spore germination. In contrast, susceptible cultivars promoted the synthesis of hydroxytyrosol 4-O-glucoside during ripening, a compound with no antifungal effect. This study showed that the distinct phenolic profiles between resistant and susceptible cultivars play a key role in determining olive fruit resistance to Colletotrichum species.

Local and systemic exposure of cisplatin during hyperthermic intrathoracic chemotherapy perfusion after pleurectomy and decortication for treatment of pleural malignancies.

BACKGROUND: Assessing the pharmacokinetics of intrapleurally administered cisplatin during hyperthermic intrathoracic chemotherapy perfusion (HITHOC) following pleurectomy/decortication in patients with malignant pleural mesothelioma or advanced thymoma with pleural spread. METHODS: Pharmacokinetic analysis (ICP-MS) of intrapleural cisplatin with a dosage of 100 mg/m(2) (n = 5) or 150 mg/m(2) (n = 5) at 42°C perfusate temperature. Simultaneous pleural perfusion fluid and serum samples were collected at the beginning and every 15 min. Serum samples were collected at the end of the operation, 6, 12, and 24 hr postoperative. RESULTS: Mean cisplatin levels in the perfusate slightly decreased during the HITHOC. The mean area under the curve ratios (AUC perfusate :AUC serum ) of cisplatin were nearly similar. The mean AUCs of cisplatin in the perfusate were approximately 58 and 55 times greater than detected in the serum. The mean peak of cisplatin in the serum was reached after 1 hr of HITHOC. The AUC of cisplatin in the serum did not significantly differ (P = 0.18) between both groups up to 24 hr after perfusion. CONCLUSIONS: HITHOC with cisplatin provides a pharmacological advantage of high local intrapleural cisplatin concentrations. Elevation of the cisplatin dosage to 150 mg/m(2) did not lead to a significant increase of the systemic cisplatin concentration.

Cell counts and survival to vitrification of bovine in vitro produced blastocysts subjected to sublethal high hydrostatic pressure.

This work analyses the effects of a high hydrostatic pressure (HHP) treatment on in vitro survival of in vitro produced (IVP) bovine embryos vitrified with the Cryologic Vitrification Method (CVM). Consequences on embryo quality in terms of cell proliferation and differentiation, and levels of embryonic Heat Shock Protein 70 (Hsp-70) were also examined. Day 7 and 8 bovine in vitro-produced blastocysts were submitted to an HHP treatment (60 MPa, at 32 °C for 1 h) and allowed to recover for 1 or 2 h in culture medium. The HHP treatment did not improve blastocyst survival rates after vitrification/warming. Survival (24 h post-warming) and hatching (48 h post-warming) rates were 79.3 ± 4.9 and 51.8 ± 4.2 vs 73.9 ± 4.2 and 44.7 ± 4.1 for untreated controls and HHP-treated embryos, respectively. Total cell numbers measured in fresh embryos were reduced after 1 h at 32 °C, with or without HHP treatment, indicating that cell proliferation was stopped as a result of stress. Vitrified HHP-treated embryos that hatched at 48 h after warming showed increased cell numbers in their ICM compared with untreated controls (50.2 ± 3.1 vs 38.8 ± 2.7), indicating higher embryo quality. Treatment of blastocysts with HHP did not alter the level of the Hsp-70 protein. In our conditions, HHP treatment did not affect the cryoresistance of these embryos. However, combination of HHP treatment and vitrification in fibreplugs resulted in an increase in the ICM cell number of hatched embryos 48 h post-warming.

Assessment of meiotic spindle configuration and post-warming bovine oocyte viability using polarized light microscopy.

The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(-) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes.

Influence of genetic and interannual factors on the fatty acids profile of virgin olive oil.

Among olive oil nutritional benefits, it is worth mentioning its fatty acids composition with predominance of monounsaturated fatty acids (MUFAs). We have evaluated the influence of the cultivar and interannual factors on the fatty acids profile of virgin olive oil samples obtained from 45 and 71 cultivars along three and two consecutive crop seasons, respectively. The cultivars were classified in two groups according to the fatty acids composition: (1) high content in MUFAs and moderate content in saturated and polyunsaturated fatty acids (SFAs and PUFAs, respectively) and (2) moderate content in MUFAs and high content in SFAs/PUFAs. We also observed variations in the fatty acids content with the climate conditions, which can significantly alter the saturated and unsaturated profiles. Thus, a significant decrease in MUFAs and an increase in SFAs/PUFAs concentrations was found when the precipitation accumulated within the June-October period was reduced.

Demographic, clinical and microbiological characteristics of the first 30 human monkeypox confirmed cases attended in a tertiary hospital in Madrid (Spain), during the May-June 2022 international outbreak.

The present outbreak of Human Monkeypox (HMPX) that has begun in May 2022 and has spread across all continents in less than two months has qualitative and quantitative characteristics that make it different from the pattern of human disease previously caused by this virus. It has spread with enormous ease, affects almost exclusively adults, behaves as a sexually transmitted disease and focuses on very specific groups and transmission conditions. The high incidence in the city of Madrid in males that have sex with males (MSM) has allowed us to observe and report the experience with the first 30 cases diagnosed in our institution. Patients presented with febrile symptoms, genital and paragenital skin lesions reminiscent of smallpox, but less extensive and severe. The disease may also cause proctitis, pharyngitis and perioral lesions. The PCR test for diagnostic confirmation has been shown to be very sensitive and effective, not only in skin lesions but also in blood and other fluids such as pharyngeal, rectal exudates and blood. A very high proportion of patients with HMPX also have other sexually transmitted diseases that must be actively detected in this context. The spontaneous evolution of our patients has been good and hospitalization has been practically unnecessary. Transmission to non-sexual cohabitants and health personnel has been nonexistent and the lesions have disappeared in less than 30 days without leaving sequelae and no need for specific antiviral treatment.