Effect of EGF on expression and localization of maturation‐promoting factor,mitogen‐activated protein kinase,p34cdc2 and cyclin B during different culture periods on in vitro maturation of canine oocytes

This study aimed to investigate the localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins, before and after treatment with EGF during different moments of oocyte maturation. The ovaries obtained from 350 domestic dogs were aseptically isolated, immersed in physiological solution and transported at 4°C. In the laboratory, the ovaries were sectioned for the release of cumulus–oocyte complexes. Cumulus–oocyte complexes were selected and divided into treatment groups with and without EGF and cultured for 24, 48 and 72 hr. Immunofluorescence was used for the detection and the localization of MAPK, MPF, p34^cdc2 and cyclin B1 proteins. We observed that the expression and localization of MPF, MAPK, p34^cdc2 and cyclin B1 proteins are associated with meiosis resumption and cell cycle progression, and that EGF influences cell signalling pathways by promoting alterations in the localization of these proteins, improving the acquisition of oocyte competence. This is the first report of the localization of crucial proteins for meiosis progression in domestic dogs and identification of the expression and localization of proteins for cell cycle progression performed in this study represented a step of great importance to elucidate the mechanisms involved in the meiosis block in domestic dogs, allowing the advance in this research area.     

Use of trichostatin A alters the expression of HDAC3 and KAT2 and improves in vitro development of bovine embryos cloned using less methylated mesenchymal stem cells

The aim of this work was to investigate the methylation and hydroxymethylation status of mesenchymal stem cells (MSC) from amniotic fluid (MSC‐AF), adipose tissue (MSC‐AT) and fibroblasts (FIB‐control) and to verify the effect of trichostatin A (TSA) on gene expression and development of cloned bovine embryos produced using these cells. Characterization of MSC from two animals (BOV1 and BOV2) was performed by flow cytometry, immunophenotyping and analysis of cellular differentiation genes expression. The cells were used in the nuclear transfer in the absence or presence of 50 nM TSA for 20 hr in embryo culture. Expression of HDAC1, HDAC3 and KAT2A genes was measured in embryos by qRT‐PCR. Methylation results showed difference between animals, with MSC from BOV2 demonstrating lower methylation rate than BOV1. Meanwhile, MSC‐AF were less hydroxymethylated for both animals. MSC‐AF from BOV2 produced 44.92 ± 8.88% of blastocysts when embryos were exposed to TSA and similar to embryo rate of MSC‐AT also treated with TSA (37.96 ± 15.80%). However, when methylation was lower in FIB compared to MSC, as found in BOV1, the use of TSA was not sufficient to increase embryo production. MSC‐AF embryos expressed less HDAC3 when treated with TSA, and expression of KAT2A was higher in embryos produced with all MSC and treated with TSA than embryos produced with FIB. The use of MSC less methylated and more hydroxymethylated in combination with embryo incubation with TSA can induce lower expression of HDAC3 and higher expression of KAT2A in the embryos and consequently improve bovine embryo production.     

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.     

Genotype–environment interaction for age at first calving in buffaloes,using the reaction norm model

The objective of this study was to evaluate the genotype–environment interaction effect on age at first calving in buffaloes. The records were analysed using two approaches: (a) standard animal model and (b) reaction norm model. For the reaction norm analysis, two environmental gradients were formed, using age of first calving or milk yield group contemporary average. The results showed differences in the heritability estimates when using the two approaches. The reaction norm model indicated high heritability in more favourable environments and low magnitude genetic correlations between extreme environments. Based on our findings, we verified the significance of the genotype–environment interaction effect on age at first calving in buffaloes.     

Growth differentiation factor‐9 improves development,mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

This study analysed the effect of growth differentiation factor‐9 (GDF‐9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α‐MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α‐MEM⁺—control medium) or α‐MEM⁺ supplemented with 1, 10, 50 or 100 ng/ml GDF‐9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF‐9 than other treatments, except for 10 ng/ml of GDF‐9 (p > 0.05). Treatment containing 100 ng/ml GDF‐9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF‐9 showed more oocytes in MI than α‐MEM⁺, 1 or 50 ng/ml GDF‐9 (p < 0.05). In conclusion, 100 ng/ml GDF‐9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.     

Proteomic analysis of amniotic and allantoic fluid from buffaloes during foetal development

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 μg) was used for in‐solution tryptic digestion, and mass spectrometry analysis (nano‐LC‐MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty‐one proteins were found in the amniotic fluid, and twenty‐one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha‐1‐antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha‐2‐macroglobulin and alpha‐2‐HS‐glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha‐2‐macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.     

Spray‐dried porcine plasma improves feed intake of weaned piglets subjected to heat stress

The objective of this study was to evaluate the effects of spray‐dried porcine plasma (SDPP) on performance, measurement of thyroid hormones triiodothyronine (T3) and thyroxine (T4), body temperature and relative organ weights of weaned piglets exposed to a heat stress or a thermoneutral environment. One hundred and forty‐four piglets with initial BW of 5.82 ± 0.33 kg (28 d old) were used. The experimental design was a 4 x 2 factorial arrangement, four plasma levels (0.0, 2.5, 5.0 and 7.5%) and two environments (34º [heat stress (HS)] and 24°C [thermoneutral (TN)]). SDPP was fed from weaning (28 d of age) to the end of the trial (piglets were weighed at the beginning, 7, 21 and 35 days of trial). Rectal, neck, shoulder and leg temperatures were measured every seven days. At 21 days of trial, one piglet per pen was slaughtered to determine the weight of the carcass and organs. Blood samples were collected at 18 and 35 days of trial to determine levels T3 and T4. The rectal, neck, shoulder and leg temperatures were increased (p < 0.05) 0.4, 1.5, 1.0 and 1.3ºC, respectively, and relative weight of the lungs (p < 0.05) and total T3 (p < 0.05) were decreased 0.001 kg/kg and 0.19 ng/dl, respectively, for pigs in the HS environment. The estimated inclusion of 5.1% of SDPP had the greatest (p < 0.05) weight gain during the first week, and throughout the nursery phase, the 5% of inclusion had a tendency of improvement (p = 0.075), regardless of ambient temperature. For the entire period, pigs reared in the HS environment had the greatest (p < 0.05) estimated feed intake by the quadratic regression with the inclusion of 4.3% of SDPP.     

Changes in testicular size,echotexture, and arterial blood flow associated with the attainment of puberty in Dorper rams raised in a subtropical climate

There is a paucity of information on the relationships of testicular morphology, echotextural attributes, and blood flow dynamics with pubertal development of rams raised in a subtropical climate. Forty‐five Dorper rams (24 rams aged 8–11 months and 21 rams aged 12–24 months) were examined using a portable ultrasound scanner connected to a 7.5‐MHz transducer. Computer‐assisted analyses of testicular ultrasonograms utilized commercially available Image ProPlus^® analytical software. Spectral Doppler scans of testicular arteries were performed immediately after scrotal (B‐mode) ultrasonography to determine peak systolic velocity (PSV), end‐diastolic velocity (EDV), resistive index (RI = [PSV?EDV]/PSV), and pulsatility index (PI = [SPV–EDV]/mean velocity) of the blood vessels. The length of the testes (9.7 ± 0.3 compared with 9.0 ± 0.2 cm) and scrotal circumference (33.3 ± 0.5 compared with 31.8 ± 0.4 cm) were greater (p < 0.05) but testicular depth (4.5 ± 0.1  compared with 4.9 ± 0.08 cm) was less (p < 0.05) in sexually mature compared with peripubertal rams. [Corrections added on 9 Jan 2019 after initial online publication: The testicular size values in the sentence were corrected.] There were no differences (p > 0.05) between the two age groups of Dorper rams in blood flow indices of testicular arteries. Mean numerical pixel values (100.5 ± 4.1 compared with 89.2 ± 4.8) and pixel heterogeneity (25.6 ± 0.6 compared with 23.6 ± 0.5) of testicular parenchyma were greater (p < 0.05) in peripubertal than in postpubertal rams. Semen volume was negatively correlated with PI of testicular arteries (r = ?0.57, p = 0.04). In summary, the attainment of sexual maturity in the rams of the present study was associated with significant changes in testicular length and depth, scrotal circumference, and parenchymal echogenicity/hetrogeneity but not in testicular volume and blood perfusion rates. Testicular artery PI can be used to predict the volume of ejaculate in rams.     

Twinning rate in buffaloes: A case report

The information of twin pregnancy frequencies in buffaloes is scarce in the literature. Evidence even indicates that twin pregnancies are not carried to term, but it does not seem to be the case. In this case report, the event of twin pregnancy in buffaloes is reported as well as its frequency. Twin birth is the presence of two or more embryos in a single gestation. Multiple births are uncommon in buffaloes since the species is uniparous. We studied a population of Murrah buffaloes in which slightly more than 13,000 births were observed. In this study, the frequency of occurrence of twin births in Murrah buffaloes was 0.14%. This low incidence impaired the estimation of genetic parameters. Twins can have advantages and disadvantages depending on the production system, which are discussed in this case report.     

Modelling biodiversity trends in the montado (wood pasture) landscapes of the Alentejo,Portugal

Context

Montados are dynamic agroforestry systems of southern Portugal, with high economic and ecological values. Changes in land use and cover have important implications for landscape-level biodiversity and its conservation.

Objectives

Our objectives were to evaluate the biodiversity values and trends in a montado system in the Alentejo, Portugal so as to inform landscape level conservation approaches. In doing so, we aimed to develop a replicable and robust approach drawing together field observation, expert opinion, and remote sensing to produce predictions relevant to land management planning.

Methods

Field sampling and subsequent analysis of data on the birds, butterflies and plants in eight distinct land covers allowed the identification of two principal habitat groupings of importance: ‘montado mosaic’ and ‘shrubland’. Morphological spatial pattern analysis was performed on Landsat-derived GIS habitat layers for 1984 and 2009, generating maps and statistics for change in the different landscape functional classes. In addition, we demonstrated how the modelling of ecotones between open and closed biomes can identify the preferred hunting grounds of the threatened Iberian lynx and black vulture, flagship species whose conservation provides benefits to the area’s wider biodiversity values.

Results

Total and core area of montado mosaics and shrubland increased over the 25 year period, whilst the amount of habitat connectivity declined in the case of shrubland. Considerable local variation in these trends highlighted targetable areas for conservation action (e.g. through agri-environment spending).

Conclusions

A rapid and robust approach was demonstrated, with potentially wider utility for biodiversity assessment and planning.