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.     

Effect of dietary selenium and vitamin E on spermatogenic development in boars

An experiment involving a total of 61 crossbred boars evaluated the effects of dietary Se and vitamin E on spermatogenic development at various stages of sexual development and the prostaglandin F2alpha (PGF2alpha) content in the seminal vesicle and prostate glands at 18 mo of age. The experiment from 5.4 to 9 mo of age was conducted as a 2 x 2 factorial in a randomized complete block design. Dietary Se at 0 or .5 ppm was the first factor and vitamin E at 0 or 220 IU/kg diet was the second. From 9 to 18 mo of age, a group of sexually active and inactive boars was a third factor. Treatment diets were fed from weaning (28 d of age) to the end of the experiment. Three boars per treatment group at 5.4 (105 kg BW), 6.2 (130 kg BW), and 9.0 (150 kg BW) mo of age were killed and the testes collected. From 9 to 18 mo of age, three boars from each dietary treatment group were used for semen collection, and another set of three to four boars from each treatment group remained sexually inactive. At 18 mo, both sets of boars were killed and their testes, prostates, and seminal vesicles were collected. The testis at each age was evaluated for sperm reserve numbers and germ and Sertoli cell populations. At 5.4 or 6.2 mo of age, testicular sperm reserves were not affected by dietary Se (P > .15), at 9.0 mo of age there was a trend for a higher (P < .10) number of sperm reserves, and by 18 mo of age the Se-fed boars had higher (P < .01) numbers of sperm reserves. Vitamin E had no effect (P > .15) on testicular sperm reserves at any age period. Boars fed dietary Se had a greater number of Sertoli cells (P < .01) and round spermatids (P < .01) at 6.2 mo of age, but by 18 mo of age the boars fed Se had more Sertoli cells (P < .05), more secondary spermatocytes (P < .01), and more round spermatids (P < .05). Vitamin E did not affect Sertoli or germ cell populations at the various ages. Boars at 18 mo of age had lower PGF2alpha concentrations in the prostate (P < .05) and seminal vesicles (P < .01) when vitamin E was fed, whereas Se had no effect. Sexually active boars had lower PGF2alpha concentrations in the seminal vesicles (P < .01) than sexually inactive boars, but there was no effect (P > .15) of sexual activity on the number of Sertoli cells, primary or secondary spermatocytes, or round spermatids. Our results indicate that Se has a role in establishing the number of boar spermatozoal reserves and Sertoli cells, whereas supplemental vitamin E did not affect these criteria.     

Environmental and physiological controls over oxygen and carbon isotope composition of Tasmanian blue gum, Eucalyptus globulus

We measured oxygen isotope ratios (delta18O) of xylem sap, phloem sap, leaves, wood and bark of Eucalyptus globulus Labill. growing in southwestern Australia. Carbon isotope ratios (delta13C) were measured in the dry matter of phloem sap, leaves and wood. Results were used to test several aspects of a mechanistic model of 18O enrichment and provided insights into post-photosynthetic variations in dry matter delta13C. Xylem water delta18O varied little within the tree crown, whereas variation at the landscape-level was more pronounced, with plantations near the coast being enriched by up to 3 per thousand compared with plantations less than 100 km inland. Phloem water was significantly enriched in 18O compared with xylem water in two of three sampling campaigns; mean enrichments were 0.5 and 0.8 per thousand. Phloem sap sugars exported from E. globulus leaves closely reflected observed leaf water enrichment when diurnal variation in photosynthesis was taken into account. Photosynthetic rates were higher in the morning than in the afternoon, whereas leaf water 18O enrichment increased to maximum values in the afternoon. A non-steady-state model of leaf water 18O enrichment accurately predicted observed values through a full diel cycle. Mean estimates of the proportion of organic oxygen effectively exchanging with xylem water during cellulose synthesis were close to 0.40 for both leaves and wood. Carbon isotope ratios of nascent xylem tissues did not differ from those of phloem sap sugars collected concurrently, whereas nascent leaf tissues were depleted in 13C by 2 per thousand compared with phloem sap sugars, suggesting that, in E. globulus, 13C enrichment of sink tissues compared with source leaves does not result from an enriching process within the sink tissue.     

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.     

TRANSTHORACIC LUNG ULTRASOUND IN NORMAL DOGS AND DOGS WITH CARDIOGENIC PULMONARY EDEMA: A PILOT STUDY

Pulmonary edema is the most common complication of left‐sided heart failure in dogs and early detection is important for effective clinical management. In people, pulmonary edema is commonly diagnosed based on transthoracic ultrasonography and detection of B line artifacts (vertical, narrow‐based, well‐defined hyperechoic rays arising from the pleural surface). The purpose of this study was to determine whether B line artifacts could also be useful diagnostic predictors for cardiogenic pulmonary edema in dogs. Thirty‐one normal dogs and nine dogs with cardiogenic pulmonary edema were prospectively recruited. For each dog, presence or absence of cardiogenic pulmonary edema was based on physical examination, heartworm testing, thoracic radiographs, and echocardiography. A single observer performed transthoracic ultrasonography in all dogs and recorded video clips and still images for each of four quadrants in each hemithorax. Distribution, sonographic characteristics, and number of B lines per thoracic quadrant were determined and compared between groups. B lines were detected in 31% of normal dogs (mean 0.9 ± 0.3 SD per dog) and 100% of dogs with cardiogenic pulmonary edema (mean 6.2 ± 3.8 SD per dog). Artifacts were more numerous and widely distributed in dogs with congestive heart failure (P < 0.0001). In severe cases, B lines increased in number and became confluent. The locations of B line artifacts appeared consistent with locations of edema on radiographs. Findings from the current study supported the use of thoracic ultrasonography and detection of B lines as techniques for diagnosing cardiogenic pulmonary edema in dogs.