Apoptosis in the Antral Follicles of Swamp Buffalo and Cattle Ovary: TUNEL and Caspase-3 Histochemistry

The present study was carried out to investigate the pattern of apoptosis in the healthy antral and atretic follicles of Philippine swamp buffaloes (BU) in comparison with Holstein-Friesian (HF) cows. Paraffin sections of healthy follicles and various stages of atretic follicles were stained using the terminal deoxynucleotidyl transferase (TdT)-mediated biotinylated deoxyuridine triphosphates (dUTP) nick end-labelling (TUNEL) method to detect DNA fragmentation and cleaved caspase-3 antibody to detect cells committed to undergo apoptosis. Five equidistant areas of a follicle were counted for the presence of TUNEL- and caspase-3-positive cells. Healthy follicles of BU and HF contained no TUNEL-positive cells in the granulosa and theca layer but showed some caspase-3 positivity. The granulosa layer of advanced atretic follicles showed a significantly higher frequency of caspase-3 positivity than the healthy and early atretic follicles in both breeds. The frequency of caspase-3-positive cells of BU was significantly higher than HF in the granulosa layer of healthy, early atretic and advanced atretic follicles. In the theca interna layer, BU and HF showed a significantly lower and higher frequency of TUNEL-positive cells in the late atretic follicles compared with advanced atretic follicle, respectively. However, the frequency of caspase-3-positive cells of both BU and HF in the late atretic follicles was significantly higher than the advanced atretic follicles in the theca interna layer. These results indicate that caspase-3 and DNA fragmentation is involved in the buffalo ovarian apoptotic process.     

Spatial patterns of shrub cover after different fire disturbances in the Pyrenees

? Woody encroachment into grasslands is a worldwide phenomenon. In the Pyrenees, fire has been used as a management tool to transform part of the encroached land to grassland.

? This study aims to compare the spatial patterns of shrub cover 4 y after 4 different fire disturbances (prescribed burning, repeated prescribed burning, wildfire in 20 year-old shrubs and wildfire in 5 year-old shrubs); and also to compare shrub cover after different fire disturbances, accounting for spatial autocorrelation. The study focuses on the shrub Cytisus balansae. Two-dimensional transects (20 × 0.5 m) were established to monitor shrub cover for 4 y after each disturbance type. Autoregressive models and Markov models were used with a Monte Carlo procedure to account for the presence of spatial autocorrelation.

? Shrub cover was greater after prescribed burning than after repeated prescribed burning, and it increased with shrub age before disturbance. Differences in spatial patterns were detected in shrub patch size, with repeated prescribed fires and wildfires reducing shrub patch size by half in comparison with prescribed burning.

? From the management point of view, the effects of repeated prescribed burning were similar to those of a wildfire on reducing shrub cover and shrub patch size.

     

Litter decomposition and faunal activity in Mediterranean forest soils: effects of N content and the moss layer

Accumulation of soil carbon is mainly controlled by the balance between litter production and litter decomposition. Usually In Mediterranean forests there are contrasting conditions in the distribution of faunal activity and the moss layer that may have different effects on litter decomposition. Decomposition and faunal activity were studied by exposing litter of contrasting quality (Pinus halepensis Mill. and Quercus ilex L.) for 3.5 yr in three Mediterranean pine forests of the eastern Iberian Peninsula. The effects of mosses on decomposition and on faunal activity were studied by exposing P. halepensis litter either on moss patches or directly on the forest floor. Faecal pellet production was used as an indication of faunal activity. Water availability or soil characteristics seem to limit faunal activities in the drier sites. Faecal pellets were not found during the first stages of decomposition and in all sites they appeared when about a 30% of the initial litter had decomposed. Under wet conditions faecal pellet production was very high and a mass balance suggested that soil faunal activity may result in a net flow of organic matter from the lower organic horizons to the surface Oi horizon. Mosses slightly increased mass loss of pine litter probably as a consequence of high potentially mineralizable nitrogen in the Oa horizon of moss patches and also, perhaps, as a consequence of the higher moisture content measured in the Oi horizon needles sampled among the mosses. In contrast, moss patches reduced faunal activity. The effect of litter quality on mass loss was not always significant, suggesting an interaction between litter quality and site conditions. During the first stages of decomposition there was N immobilisation in P. halepensis litter (poorer in N) and N release from Q. ilex litter (richer in N). In conclusion, in these forests soil microclimate and/or N availability appear to be more important controlling litter decomposition than the distribution of faunal activity.     

Plant diversity and soil properties in pristine and managed stands from Bosnian mixed forests

We compared pristine and managed beech–fir mixed forestsin Bosnia to assess the impact of management on plant speciesdiversity, vegetation structure and soil–vegetation interaction.Traditionally, management of Bosnian mixed forests has beenby the opening of small gaps, similar to those occurring naturally.Species composition revealed three successional vegetation groups,two mainly in managed forests (regenerating and mature) andone mainly in pristine old growth forests. Managed sites showeda tendency to be more diverse in plant species than pristinesites, and were also more heterogeneous. Diversity was clearlyexplained by vegetation groups; mature managed stands showingthe highest plant diversity and regenerating stands the lowest,both in terms of species richness and abundance distributions.Plant diversity decreased with canopy cover and increased withcarbon concentration in the A_h horizon, being accurately modelledby both factors. Although topographical conditions were homogeneousamong sites, vegetation was very sensitive to small changesin environmental variables at the local scale. Vegetation reflecteda combination of past situations, represented by C in the A_hhorizon, and current conditions, represented by canopy coverand the C : N ratio in the LF organic layers, suggesting bothsensitivity to present-day environmental conditions as wellas the imprint of past events.     

Cryopreservation of Piau‐Breed Wild Boar Sperm: Assessment of Cooling Curves and Centrifugation Regimes

This study aimed to assess the effects of different cooling curves and centrifugation regimes used in cryopreservation protocols on the post‐thaw viability of Piau‐breed wild boar (Sus scrofa) sperm using in vitro assessment tests. Two centrifugations (800  g for 10 min and 2400  g for 3 min) and two cooling curves (conventional cooling using nitrogen vapour – freezing 1 and automated cooling using a programmed freezing machine – freezing 2) were tested. Therefore, the treatments were divided into M3 – centrifugation at 2400  g for 3 min and freezing 2; M10 – centrifugation at 800  g for 10 min and freezing 2; R3 – centrifugation at 2400  g for 3 min and freezing 1; and R10 – centrifugation at 800  g for 10 min and freezing 1. No significant differences (p > 0.05) between treatments occurred post‐thawing regarding the total sperm motility means recorded. The mean values of the different treatments were not different from each other regarding the supravital staining (SV), hypo‐osmotic test (HO), sperm–egg binding assay or sperm morphology. This study showed that both the cooling curve and the centrifugation regime affected the quality of post‐thaw sperm, and centrifugation for shorter times and cooling curves using automated cooling are the most suitable for minimizing sperm injury.     

Effect of nitrogen source in high-concentrate, low-protein beef cattle diets on microbial fermentation studied in vivo and in vitro

In Exp. 1, four Holstein heifers (112+/-5.5 kg BW) fitted with ruminal cannulas were used in a 4 x 4 Latin square to evaluate the effects of N source on ruminal fermentation and urinary excretion of purine derivatives. A 2 x 2 factorial arrangement of treatments was used; the factors were the type of protein source (soybean meal, SBM, vs a 50:50 mixture of fish meal and corn gluten meal, FMCGM) and the partial substitution of protein source by urea (with vs without). Heifers were allowed to consume concentrate and barley straw on an ad libitum basis. Barley straw:concentrate ratio (12:88) and average ruminal pH (6.25) were not affected (P > 0.05) by treatment. Ruminal NH3 N concentration and urinary excretion of purine derivatives were not affected (P > 0.05) by supplemental N source. In situ CP degradability of supplemented SBM was very low (50%). In Exp. 2, eight dual-flow continuous-culture fermenters were used to study diet effects on microbial fermentation and nutrient flow, using forage:concentrate ratio, solid and liquid passage rates, and pH fluctuation to simulate in vivo conditions. The treatment containing SBM without urea reached the greatest total VFA concentration (P < 0.01), molar percentage of acetate (P < 0.05), and NH3 N concentration (P < 0.05), followed by treatments with partial substitution of protein source by urea, and finally by the treatment containing FMCGM. True OM digestion tended to increase (P = 0.13) in treatments containing SBM. These results suggest that amino N from SBM and NH3 N concentration stimulated nutrient digestion. Microbial protein synthesis was lowest in treatments with FMCGM and without urea, indicating that rapidly available N limited microbial growth. The low CP degradability of SBM observed may have contributed to the limitation in N supply for microbial growth. Efficiency of microbial protein synthesis increased in treatments containing urea (P < 0.05). Protein source affected total (P < 0.05) and essential AA (P < 0.10) flows, which were greater in treatments containing FMCGM. Partial replacement of protein supplements by urea did not affect total and essential AA flows. Because mean dietary protein contribution to total N effluent was 46%, the AA profile of supplemental protein sources had a great impact on total AA flow and its profile.