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.     

半干旱黄土丘陵区不同植被类型的土壤水分特征及其稳定性

[目的]研究5种不同植被类型土壤水分动态特征,为区域内优化配植模式提供理论依据。[方法]以甘肃省定西市半干旱黄土丘陵区5种典型的植被类型为研究对象,使用土壤水分速测仪对2017,2018年0—200 cm土层土壤含水量进行观测,并对不同植被类型土壤水分特征及其稳定性进行分析。[结果]①不同植被类型土壤水分呈现出:山杏×侧柏混交林地(11.24%)退耕苜蓿草地(10.97%)自然荒地(10.66%)退耕林地(9.99%)柠条林地(9.55%)。②5种植被类型在0—200 cm土层依据有序聚类法可分为动态变化层(0—40 cm),利用层(40—100 cm)和弱利用层(100—200 cm)3个层次。在动态变化层(0—40 cm),土壤水分呈现出:自然荒地(14.23%)最高,柠条林地(11.04%)最低;在利用层(40—100 cm):退耕苜蓿草地(12.16%)最高,柠条林地(9.15%)最低;在弱利用层(100—200 cm):自然荒地(10.81%)最高,退耕林地(8.61%)最低。③不同植被类型土壤水分土层稳定性(变异系数)呈现出:退耕林地(19.9%)山杏×侧柏混交林地(21.3%)自然荒地(21.9%)退耕苜蓿草地(22.6%)柠条林地(23.6%)。④不同植被类型土壤水分时间稳定性(变异系数)呈现出:自然荒地(25%)退耕林地(25.2%)山杏×侧柏混交林地(26.5%)柠条林地(27.1%)退耕苜蓿草地(31.9%)。[结论]柠条林地土壤水分含量最低,在土层和时间上不稳定,山杏×侧柏混交林地较其他植被类型土壤水分含量更高,更具有稳定性。