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.     

Cell Proliferation in the Atretic Follicles of Buffalo and Cattle Ovary

The present study was carried out to describe the proliferative activity of granulosa and theca cells in healthy antral and atretic follicles of Philippine buffaloes (BU) and Holstein-Friesian (HF) cows. Paraffin sections of ovary were immunostained with mouse monoclonal antibody to proliferating cell nuclear antigen (PCNA). Then the follicles were classified into healthy and various stages of atretic follicles. The granulosa layer of healthy follicles had a significantly higher frequency of PCNA-positive cells than the early and advanced atretic follicles in both breeds. In the theca interna, significantly reduced populations of the PCNA-positive cells were found in both breeds as atresia progressed. Moreover, HF had significantly higher PCNA-positive cells in the theca interna of healthy, early atretic and advanced atretic follicles than BU. A reduction of PCNA-positive cells during atresia was also noted in the theca externa in both animals although differences were not significant. The results of the present work suggest that the proliferative activity of granulosa and theca cells decreases in association with follicular atresia in the BU similar to HF. Furthermore, a significantly deficient cell proliferative activity of theca interna was found in BU compared with HF.     

Ergovaline does not alter the severity of ryegrass staggers induced by lolitrem B

AIM: To investigate a possible interaction between lolitrem B and ergovaline by comparing the incidence and severity of ryegrass staggers in sheep grazing ryegrass (Lolium perenne) containing lolitrem B or ryegrass containing both lolitrem B and ergovaline.

METHODS: Ninety lambs, aged approximately 6 months, were grazed on plots of perennial ryegrass infected with either AR98 endophyte (containing lolitrem B), standard endophyte (containing lolitrem B and ergovaline) or no endophyte, for up to 42 days from 2 February 2010. Ten lambs were grazed on three replicate plots per cultivar. Herbage samples were collected for alkaloid analysis and lambs were scored for ryegrass staggers (scores from 0–5) weekly during the study. Any animal which was scored ≥4 was removed from the study.

RESULTS: Concentrations of lolitrem B did not differ between AR98 and standard endophyte-infected pastures during the study period (p=0.26), and ergovaline was present only in standard endophyte pastures. Ryegrass staggers was observed in sheep grazing both the AR98 and standard endophyte plots, with median scores increasing in the third week of the study. Prior to the end of the 42-day grazing period, 22 and 17 animals were removed from the standard endophyte and AR98 plots, respectively, because their staggers scores were ≥4. The cumulative probability of lambs having scores ≥4 did not differ between animals grazing the two pasture types (p=0.41).

CONCLUSIONS AND CLINICAL RELEVANCE: There was no evidence for ergovaline increasing the severity of ryegrass staggers induced by lolitrem B. In situations where the severity of ryegrass staggers appears to be greater than that predicted on the basis of concentrations of lolitrem B, the presence of other tremorgenic alkaloids should be investigated.     

Suppression of mitogenic response of bovine lymphocytes during experimental ketosis in calves

Experimental ketosis was induced by feeding calves a diet containing 1,3 butanediol for 9 or 10 days. Blood beta-hydroxybutyrate was significantly (P less than 0.05) elevated during clinical ketosis. The mitogenic response of the lymphocytes collected during ketosis was significantly (P less than 0.01) suppressed, and the suppression persisted for 2 weeks. During ketosis, all calves fed a 1,3 butanediol diet had clinical signs of an upper respiratory tract infection. Possible relationships between the suppressed function of lymphocytes, the increased concentration of 1,3 butanediol, and the increased susceptibility of the calves to infections during ketosis were considered.     

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 tissue culture explant source on sugarcane yield components

Clonal propagation of sugarcane(interspecific hybrids of Saccharum)is conducive to spread of systemicdiseases, such as ratoon stunting disease,caused by Leifsonia xyli subsp. xyli. This important disease iscontrolled by obtaining and plantinghealthy seed-cane. In Louisiana, commercialseed-cane initially produced through tissueculture is available to sugarcane farmersand is being widely planted. Long-termacceptability of this seed-cane productionmethod depends on the production of healthyplants that do not differ significantly inphenotypic and yield characteristics fromthe clones originally selected and releasedas commercial cultivars. To determinewhether tissue culture affects yield or itscomponents, three cultivars, CP 70-321, LCP85-384, and HoCP 85-845, were compared inthree successive crops initially plantedwith stalks from three sources: plantsderived from callus culture of the leafroll above the apical meristem, directregeneration from the apical meristem, andconventional bud propagation. Stalks ofplants derived from both explant sourceswere typical of seed-cane farmers wouldpurchase for planting that had beenpreviously rogued for phenotypic variantsand increased by bud propagation.Differences in yield components amongtissue culture explant sources and budpropagated cane only occurred in CP 70-321.Stalk diameter and stalk weight were lowerand stalk population was higher for plantsderived from leaf roll callus compared tobud propagated cane. Yield components weresimilar for plants derived from an apicalmeristem and bud propagation. Individualplant phenotypic variants resulting fromsomaclonal variation were not observed inany of the cultivars derived from eitherexplant source. In summary, genotype andexplant source affected persistent, uniformphenotypic variation resulting from tissueculture that changed some yield components. However, apical meristem culture wassuitable for production of seed-cane, assugarcane derived by meristem culture ofthree cultivars did not differsignificantly from the original germplasmfor any measured yield trait.     

Initiation of behavior by single neurons: the role of behavioral context

Flying crickets avoid sources of ultrasound, possibly echolocating bats, by making rapid steering movements that turn them away from the stimulus. Electrical stimulation of a single, identified sensory interneuron (Int-1) elicits avoidance steering; depressing its response to ultrasound abolishes avoidance steering. Int-1 is necessary and sufficient for this behavior but only while the cricket is in flight. Thus, the sufficiency of Int-1 for eliciting this behavior is contingent on behavioral context.     

Genetic control of song specificity in crickets

The calling song of male field crickets is composed of stereotyped rhythmic pulse intervals, which are predictable expressions of genotype. Females identify conspecific males by their song. Two species of crickets were found to exhibit species-specific song preference, and hybrids between them preferred hybrid calls over either parental call. These results imply genetic control of song reception as well as transmission.