Bovine ovarian stem cells differentiate into germ cells and oocyte‐like structures after culture in vitro

Stem cells have been isolated from ovaries, and their ability to differentiate into oocytes in vitro has been demonstrated for mice and human, but not for bovine species. The aims of this study were to isolate germline stem cells from bovine ovaries and to evaluate the effects of bone morphogenetic proteins (BMPs) 2 and 4, and follicular fluid on the differentiation of these stem cells into oocyte‐like structures. The ovarian stem cells were isolated and cultured in α‐MEM⁺ supplemented with BMP2, BMP4 or follicular fluid. On days 0 and 14, cells were evaluated for their morphological appearance, viability, expression of alkaline phosphatase and for markers of germ cell formation (VASA and DAZL) and oocyte development (GDF9, ZPA and SCP3) by qPCR. Levels of mRNA were analysed using ANOVA and Bonferroni test (p < .05). The results showed that at day 0, ovarian stem cells expressed specific markers of pluripotency (OCT4, SOX). In addition, these cells were positive for alkaline phosphatase, which is a marker commonly used to identify primordial germ cells (PGCs). After the period of differentiation, cells had morphological features that resemble PGCs and oocyte‐like cells (OLCs). An increase, ranging from five to 14 times, in the expression of VASA was observed in cells cultured in medium supplemented with BMPs and follicular fluid, while the increase in DAZL expression ranged from four to six times. In addition, OLCs had an increase in expression of mRNAs for GDF9, ZPA and SCP3 that ranged from two to eight times. In conclusion, OLCs can be differentiated in vitro from ovarian stem cells and BMPs and follicular fluid are effective in stimulating the expression of mRNAs for germ cell and oocyte markers.     

Effect of polyunsaturated fatty acids on the fecundity of the Amazon river prawn Macrobrachium amazonicum (Heller, 1862)

Effects of the ratio of dietary fatty acids, namely n‐3 (mainly long chain polyunsaturated – LC‐PUFA) to n‐6 PUFA on the fecundity of Macrobrachium amazonicum were evaluated. In T1, the diet had equal and low levels of dietary n‐3 and n‐6 fatty acids (6 mg g^?1). In T2, the concentration of n‐3 (6 mg g^?1) was a half of the concentration of the n‐6 (12 mg g^?1), and in T3, the diet had equal and high concentrations of n‐3 and n‐6 (12 mg g^?1). Females with ovaries in stages I, III and V were collected. Higher gonadosomatic index (GSI) (6.89%) was observed in females in ovarian stage V than at other ovarian stages; however, the hepatosomatic index (HIS) showed high values in all females with ovaries in the stage III. A ratio of 1:2 n‐3:n‐6 fatty acids increased the GSI of mature females and the number of eggs spawned. Raising the level of both n‐3 and n‐6 fatty acids from ~0.6% to ~1.2% of the diet did not produce any effect on the GSI or on fecundity, suggesting that the ratio is more important than the absolute value of these two families of fatty acids.     

Efficiency of nitrogen-15-labelled fertilizers for rice and rye-grass cultivated in an Ultisol of Brazilian Amazonia

 The objective of this study was to determine the efficiency of two N fertilizers, (NH₄)₂SO₄ and urea, for rice (Oryza sativa L.) and rye-grass (Lolium multiflorum L.) cultivated in an Ultisol of central Amazonia using ¹⁵N as a tracer. Rice was cultivated in the field, while rye-grass was grown in a phytotron. Fertilization with (NH₄)₂SO₄ caused a 16% increase in the yield of rice grains and urea a 36% increase. In both crops total N uptake and N use efficiency of the fertilizers were higher for urea than for (NH₄)₂SO₄. The low values for N derived from fertilizer showed that the fertilizers contributed little to the total N absorbed by the plants. The "priming effect" or positive added N interaction (ANI) between the fertilizer N and soil organic N was observed, especially with urea. Immobilization by soil microorganisms was greater in the presence of urea, while losses were always higher with the (NH₄)₂SO₄ treatments. These losses were significant, and their reduction should allow more efficient use of this N fertilizer. It is possible that the N use efficiency was higher for urea due to a pH increase, caused by urea hydrolysis, which in turn may have favoured the activity of nitrifying bacteria in this extremely acid soil. Received: 6 April 1999     

Brucella-associated cervical bursitis in cattle

Bovine brucellosis poses a risk to human health and causes serious economic losses for the animal industry. This report describes the use of different diagnostic methods for the diagnosis of brucellosis in cattle affected by cervical bursitis from a slaughterhouse located in São Luís, Maranhão, Brazil. Serum samples from a total of 47 cattle with bursitis were collected and submitted to the Rose Bengal Test (RBT), and RBT-positive samples were further confirmed by the 2-mercaptoethanol (2-ME) assay. RBT indicated 85.1% (40/47) of positive samples, from which 78.7% (37/47) were confirmed by 2-ME. Immunohistochemistry detected Brucella spp. in 34.0% (16/47) of tissues with bursitis. PCR and/or bacterial isolation demonstrated that 63.8% (30/47) of samples were positive and morphologically compatible with Brucella sp. All colonies suggestive of Brucella sp. were confirmed by PCR. Isolates were further characterized by PCR Multiplex AMOS-ENHANCED, which indicated that the isolates corresponded to biovar 1, 2, 4 (43.33%). This study evidences an association between cervical bursitis and Brucella spp. infection in cattle, and that different biovars of Brucella circulate in bovine herds in Maranhão.

     

Efficiency of entomopathogenic fungi in the control of eggs and larvae of the horn fly Haematobia irritans (Diptera: Muscidae)

The present study assessed the pathogenic effect of isolates E9, IBCB425 and IBCB159 of the Metarhizium anisopliae fungus, JAB06, JAB07 and AM09 of Beauveria bassiana, IBCB133 and CB75 of Isaria fumosorosea (=Paecilomyces fumosoroseus) and CG189 and CG195 of Isaria farinosa (=Paecilomyces farinosus) against eggs and larvae of the horn fly Haematobia irritans. Eggs were inoculated with suspensions containing 10⁶, 10⁷ and 10⁸ conidia ml⁻¹ of the fungal isolates and observed after 48 h to determine viability. In the larvae study, eggs were allowed to hatch into fresh bovine feces that had been treated with 10⁸, 10⁷ or 10⁶ conidia mg feces⁻¹. In both studies, 5 days after initial procedures, all formed pupae were transferred to an incubator at 27 ± 0.5 °C until the emergence of the adult flies was complete. The M. anisopliae isolates did not cause the death of H. irritans eggs, but they did promote the death of larvae that hatched from treated eggs, and therefore increased the total mortality. Isolate E9 promoted 100% mortality of treated larvae at a concentration of 10⁸ conidia ml⁻¹. For the B. bassiana isolates, no activity was observed against insect eggs or larvae. Both I. fumosorosea isolates promoted significant mortality (p < 0.05) of eggs at every concentration of conidia. Isolate CG195 of I. farinosa increased the mortality of larvae and pupae that hatched from treated eggs and promoted significant total mortality (p < 0.05) of the insect at every concentration of conidia.