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.     

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.     

Genetic variation in seed production and its components in four cultivars of the pasture grass Setaria sphacelata

Variation in seed production and its components was investigated between and within four cultivars of the pasture grass Setaria sphacelata, in two experiments over two years, as a basis for future cultivar improvement. The study sought to determine the basis for the considerable differences in seed production of the four cultivars Nandi, Narok, Solander and Kazungula, to determine the extent of genetic variation in the four cultivars in attributes which contribute to seed yield, and to investigate genotypic consistency in seed production over years and seasons. Each experiment comprised 50 genotypes of each cultivar. In the first experiment, plants were harvested a set number of days after median flowering date whereas in the second experiment, which was unreplicated, each genotype was harvested a set number of days after it had flowered. In the first experiment, seed yields were generally highest for Kazungula, lowest for Narok and intermediate for Nandi and Solander. All measured attributes contributing to seed yield exhibited a high order of variation between and within cultivars, but the basis for the large difference in seed yield per plant between cultivars was tiller fertility rather that total tiller number. Averaged over the four harvests, there was a six fold to > 100–fold intra-cultivar genetic range in seed production, associated with differences in tiller fertility, which were associated with differences in date of first flowering. Broad sense heritability for seed yield averaged 0.68 for the four cultivars and showed little change over the four harvests. Genotypes which produced high seed yields in summer were also more productive of seed in autumn and the 0ore productive genotypes in the first year were also more productive in the second year. Cultivars differed in the relative importance of factors which contributed to the high seed yield of high-yielding genotypes. In the second experiment, genotypes with a high seed yield also generally had the highest tiller fertility, even though all genotypes were harvested the same number of days after first flowering. Within-cultivar correlations in seed yield between the two experiments were generally significant and the elite 20% of genotypes from this experiment had 1.2–2.9 times the seed yield of the same genotypes with a very different harvesting regime in the first experiment. It is concluded that opportunities exist in all four cultivars for improvement in seed production and that the selection criterion offering the best opportunity for advance would be fertile tiller number. In Narok, Solander and Nandi, this would result in increased tiller fertility, whereas in Kazungula, it would result in an increase in total tiller number. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of topical phenylephrine on the equine pupil

The mydriatic effect of 10% phenylephrine was evaluated in 9 horses. Base-line pupillary size in mesopic conditions and during light stimulation was ascertained before application of pharmacologic agents. In study 1, 10% phenylephrine was applied to each eye (n = 5 horses). After 15 minutes, the pupillary size was determined in both lighting conditions. Phenylephrine was again applied to each eye, and after an additional 15 minutes, the pupillary size was determined. In study 2, 1% tropicamide was applied to each eye (n = 4 horses), and after 30 minutes, the pupillary size was determined in both lighting conditions. Tropicamide and phenylephrine were applied to each eye immediately after determination of pupillary size, and after an additional 30 minutes, the pupillary size was determined. There was no significant change (P greater than 0.9) in pupillary size when phenylephrine was used alone or when phenylephrine was used in an eye that had been dilated with tropicamide.     

Ocular angiosarcoma in four horses

In 4 horses with ocular angiosarcomas, the common characteristics were that all horses were aged, and the neoplasms developed initially on conjunctival surfaces, grew slowly, and metastasized despite excision and radiation treatment. Histopathologically, the neoplasms were of 2 types: a capillary pattern of scant stroma and endothelial-lined vascular channels, and a solid pattern composed of sheets of pleomorphic cells with hyperchromatic nuclei and poorly defined cytoplasmic borders. Definitive diagnosis was made by use of factor VIII:RAg, which stained the tissues intensely.