Interactions between different media and follicle‐stimulating hormone supplementation on in vitro culture of preantral follicles enclosed in ovarian tissue derived from collared peccaries (Pecari tajacu Linneaus, 1758)

The aim was to verify the effect of follicle‐stimulating hormone (FSH) supplementation to α‐MEM+ or TCM199+ media on the in vitro development of ovarian preantral follicles (PFs) derived from collared peccaries. Ovaries (n = 5 pairs) were collected and divided into fragments destined to control group (non‐cultured) or treatments that were cultured for 7 days. The PFs morphology, growth and activation were evaluated by classical histology. The immunohistochemistry markers Ag‐NOR and PCNA were used for nuclear proliferation analysis, and the picrosirius red labelling was used for ovarian extracellular matrix (ECM) evaluation. After 7‐day culture, only the TCM199+ treatment maintained the proportion of intact PFs similar to day 1 (63.2%), but no differences were found among treatments (p > .05). In addition, a significant increase in the growing follicles proportion was verified for all the treatments, indicating follicular activation (p > .05). By the Ag‐NOR analysis, only the TCM199+/FSH maintained the nuclear proliferation similar to the first day (p > .05). The picrosirius red staining revealed that the ECM remained intact in all the treatments (p > .05). We suggest the use of TCM199+ medium supplemented of FSH for the in vitro development of peccaries PFs under 7‐day culturing conditions.     

ACE inhibition in goats under fixed‐time artificial insemination protocol increases the pregnancy rate and twin births

To assess the effect of the angiotensin‐converting enzyme (ACE) inhibition on the efficiency of the fixed‐time artificial insemination (TAI), 69 goats were divided randomly into two groups: enalapril (n = 35) and control (n = 34). In the experiment, all animals underwent the protocol of fixed‐time artificial insemination for 12 days. Enalapril group received enalapril maleate dissolved in saline (Enalapril, Lab Teuto Ltda) subcutaneously at the following doses: 0.2 mg/kg/day in D0‐D2; 0.3 mg/kg/day in D3‐D6 and 0.4 mg/kg/day in D7‐D11. The control group received the corresponding volume of 0.9% saline solution. We performed a single insemination 36 hr after sponge removal using frozen semen from two adult male goats with recognized fertility. The ultrasound pregnancy diagnosis was 30 days after the artificial insemination (AI). There was significant increase in pregnancy rates and twinning as well as a decrease in foetal loss in animals receiving enalapril (p < .01). The use of ACE inhibitors during the TAI protocol was shown to be a promising alternative to increase the efficiency of such reproductive biotechnology.     

Feeding behavior of feedlot lambs fed diets containing levels of cassava wastewater

In this study, we evaluated the effects of including cassava wastewater in the diet on the feeding behavior of feedlot lambs in 35 male uncastrated Santa Inês × Dorper crossbred lambs at an approximate age of 3 months, with an average live weight of 20.0?±?3.4 kg. Diets were formulated with hay of cassava shoots (roughage) and a concentrate based on corn and soybean, with a roughage:concentrate ratio of 50:50, plus inclusion of cassava wastewater at the levels of 0, 12, 24, 36, or 48 g/kg of the total diet. Feeding behavior was evaluated between the 46th and 52nd days of the experiment. Increasing cassava wastewater levels in the diet reduced (P?<?0.05) the intakes (kg/day) of dry matter and neutral detergent fiber as well as the efficiency of rumination (g/cud and g/h) of dry matter and neutral detergent fiber. The other behavioral parameters were not affected by wastewater inclusion in the diet. Therefore, the inclusion of up to 48 g/kg of cassava wastewater on fresh matter of diets is not recommended for feedlot lambs.     

Pathogenesis of reproductive failure induced by Trypanosoma vivax in experimentally infected pregnant ewes

The present study was aimed at investigating the effect of experimental infection by Trypanosoma vivax in different stages of pregnancy, determining the pathogenesis of reproductive failure, and confirming transplacental transmission. We used 12 pregnant ewes distributed into four experimental groups: G1, was formed by three ewes infected with T. vivax in the first third of pregnancy (30 days); G2 comprised three infected ewes in the final third of pregnancy (100 days); G3 and G4 were composed of three non-infected ewes with the same gestational period, respectively. Each ewe of G1 and G2 was inoculated with 1.25 × 10⁵ tripomastigotes. Clinical examination, determination of parasitemia, serum biochemistry (albumin, total protein, glucose, cholesterol, and urea), packed cell volume (PCV), serum progesterone, and pathological examination were performed. Placenta, amniotic fluid, blood and tissues from the fetuses and stillbirths were submitted to PCR. Two ewes of G1 (Ewe 1 and 3) presented severe infection and died in the 34^th and 35^th days post-infection (dpi), respectively; but both fetuses were recovered during necropsy. In G2, Ewe 5 aborted two fetuses on the 130^th day (30 dpi) of pregnancy; and Ewe 6 aborted one fetus in the 140^th day (40 dpi) of gestation. Ewes 2 and 4 delivered two weak lambs that died five days after birth. Factors possibly involved with the reproductive failure included high parasitemia, fever, low PCV, body score, serum glucose, total protein, cholesterol, and progesterone. Hepatitis, pericarditis, and encephalitis were observed in the aborted fetuses. The presence of T. vivax DNA in the placenta, amniotic fluid, blood, and tissues from the fetuses confirms the transplacental transmission of the parasite. Histological lesion in the fetuses and placenta also suggest the involvement of the parasite in the etiopathogenesis of reproductive failure in ewes.     

The Influence of Oxygen Tension on Cumulus Cell Viability of Canine COCs Matured in High-Glucose Medium

High in vitro oxygen (O₂) tensions are associated with enhanced levels of reactive oxygen species and cumulus oocyte complex (COC) apoptosis. The objective of this study was to determine the influence of O₂ tension on cumulus cell (CC) viability from canine oocytes. Cumulus oocyte complexes were distributed into three groups (CG, T20 and T5) and two O₂ tension levels (20% and 5%). The control group (CG) was matured in vitro in a humidified atmosphere with 5% CO₂ in air in TCM199 with 26.19 m m sodium bicarbonate, 10% (v/v) foetal calf serum (FCS), 0.10 m m gentamicin, 0.20 m m pyruvic acid, 20 μg/ml oestradiol, 0.5 μg/ml follicle-stimulating hormone, 0.03 IU/ml human chorionic gonadotropin, and 1.0 μg/ml human somatotropin. Groups T20 and T5 were matured under 20% or 5% O₂ tensions respectively in a high-glucose medium, without FCS. T20 and T5 were as CG, and supplemented with 0.1% Polyvinyl Alcohol, and 5.5 m m glucose. After 48 h of IVM, CCs from COCs were stained with propidium iodide (1.50 m m ). The results showed that viability of CCs (cytoplasmic features and nuclear morphological integrity) was different for the three groups. Rates of apoptosis were at 57.9% (521/900) for CG, 54.4% (490/900) for T20 and 38.9% (350/900) for T5 (p < 0.001). Predominant features in apoptotic cells (n = 1361) were DNA nuclear fragments (94.0%). It was concluded that CCs of canine COCs cultured in high-glucose medium showed significantly less apoptosis than those cultured in medium with FCS. Low O₂ tension was efficient in reducing apoptosis in canine CCs.     

Distribution of latent bovine herpesvirus 2 DNA in tissues of experimentally infected sheep

The biology of latent infection by bovine herpesvirus 2 (BoHV-2), the agent of mammillitis in cows, remains largely unknown. We herein report attempts to reactivate the latent infection and investigated the sites of BoHV-2 latency in experimentally infected sheep. Ewes inoculated with BoHV-2 in the udder’s skin shed virus for up to five days, developed mammillitis and seroconverted. However, attempts to reactivate latent infection by dexamethasone administration at day 40 pi failed. Nevertheless, viral DNA - and not infectious virus - was detected by PCR in several nerve ganglia and/or regional lymph nodes (LNs) of all animals at day 40 post-reactivation. Likewise, lambs previously inoculated with BoHV-2 in the nose harbored latent viral DNA in trigeminal ganglia, tonsils and regional LNs. These results demonstrate that BoHV-2 establishes latent infection in nerve ganglia and in regional lymphoid tissues, yet virus reactivation is not easily achieved by standard protocols used.     

Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Molecular, physiological and pathological characterization of Corynespora leaf spot fungi from rubber plantations in Sri Lanka

The plant pathogenic fungus Corynespora cassiicola causes a severe leaf spot disease on more than 70 host plant species including Hevea brasiliensis . Genetic variability in 32 isolates of C. cassiicola collected from diverse hosts and locations in Sri Lanka and Australia was assessed using restriction fragment length polymorphism (RFLP) analysis of the internal transcribed spacer (ITS) region of ribosomal DNA and random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis of total fungal DNA. Amplified ITS fragments from all 32 C. cassiicola isolates exhibited an identical size, and restriction analysis with seven different restriction endonucleases revealed identity in all of the detected DNA fragments. This finding of high genetic relatedness was further supported by the cloning and DNA sequencing of the ITS2 region from one Sri Lankan and one Australian isolate. However, RAPD-PCR profiles generated by 15 oligonucleotide decamer primers revealed significant polymorphism between groups of organisms. Genetic relationships among the isolates were determined by cluster analysis of the RAPD-PCR data and seven different RAPD groups were identified. Isolates showed strong correlations between the assigned RAPD group and the location and host plant genotype from which the isolate was collected. Correlations were also observed between the RAPD group, growth of the isolate and pathogenicity on different plant hosts.