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.     

Studies on production performance and toxin residues in tissues and eggs of layer chickens fed on diets with various concentrations of aflatoxin AFB1

1. An experiment on 1-week-old, White Leghorn female chicks was conducted to study the effect of aflatoxin AFB1 on weight gain, feed intake, feed gain ratio, age at sexual maturity, production and quality of eggs, retention of nutrients, pathoanatomical and histopathological parameters, and also on AFB1 residues in eggs and muscles of hens. The chicks were assigned to 4 dietary treatments: D1 (without AFB1), D2 (2.50 mg/kg AFB1), D3 (3.13 mg/kg AFB1), D4 (3.91 mg/kg AFB1) up to the age of 40 weeks. 2. At the end of the experiment, the mean body weight gain and feed intake were significantly lower in all aflatoxin-fed groups compared to control. The feed gain ratios were noted as 13.41, 13.59, 13.82 and 14.71, with the group fed the highest concentration of AFB1 showing a significantly poorer ratio than other groups. 3. Age at sexual maturity was also affected adversely by dietary AFB1: 193 d for D4 as compared to as early as 148 d for D1. Hen-d egg production was recorded as 96.92, 74.67, 65.98 and 50.75 in D1, D2, D3 and D4, respectively. 4. Average egg weights at the end of the experiment were 57.77, 57.49, 57.54 and 54.66 for D1, D2, D3 and D4, respectively. Shape index was significantly lower in D4 as compared to control. Contrary to this, albumen index was significantly higher in D4 as compared to D1. The values of yolk indices and eggshell thickness did not differ significantly among treatment groups. However, colour of yolk was reduced in all aflatoxin-fed groups compared to control. 5. Retentions of dry matter, crude protein, ether extract, calcium and metabolisable energy were adversely affected at various levels of AFB1 compared to control. 6. Pathoanatomical and histopathological studies showed various adverse changes in liver, kidney, heart, ovaries and bursa of Fabricius in AFB1-fed groups. 7. Different amounts of aflatoxin residues were detected in eggs and breast muscles of hen in all AFB1-fed groups.     

Evaluation of cell-mediated immunity during chronic organophosphate pesticide intoxication in mice and goats

The status of cell-mediated immunity (CMI) after pesticide exposure was assessed in mice with the help of skin sensitivity and graft versus host reaction tests. It was observed that at 24 hours post-challenge CMI values did not differ significantly from the control, indicating no effect of quinalphos treatment in mice. Goats receiving monocrotophos at a dose rate of 1.0 mg kg-1 body mass for 40 days gave a similar result when CMI was tested with the help of the chemical sensitizer dinitrofluorobenzene (DNFB). Thus the results clearly indicate that the tested organophosphates do not interfere with cellular immunity in the intoxicated animals.     

Comparative Expression Analysis of Gametogenesis‐Associated Genes in Foetal and Adult Bubaline (Bubalus bubalis) Ovaries and Testes

This study was conducted to identify and analyse the expression of gametogenesis‐associated genes and proteins in foetal and adult buffalo gonads of both the sexes. Relative quantification of the genes was determined by qPCR and Western blotting. Immunohistochemistry was also performed for various gametogenesis‐associated proteins in foetal and adult gonads of both the sexes. We observed significantly (p < 0.05) increased expression of primordial germ cell‐specific, meiotic as well as genes associated with oocyte maturation and development in foetal ovaries as compared to the adult ones. However, significantly (p < 0.05) increased expression of proteins associated with oocyte maturation like GDF9 and ZP4 was found in adult ovaries, indicating temporal regulation of mRNA translation during oogenesis. Meiotic genes showed significantly (p < 0.05) increased expression in adult testes as compared to foetal testes and ovaries, indicating onset of meiosis at a later stage in spermatogenesis. In general, the expression of primordial germ cell‐associated as well as meiotic genes was higher in adult testes, indicating the increased biological activity in the organ. Immunohistochemistry revealed localized expression of gametogenesis‐associated proteins in ovarian follicles and seminiferous tubules of testes, while the surrounding somatic tissues were devoid of these proteins. The study gives an understanding of the sequential and temporal events of gene expression as well as mRNA translation during male and female gametogenesis. It could also be concluded that follicles and seminiferous tubules are the functional units of the female and male gonads, respectively, and their function could be enhanced by appropriate chemical and genetic intervention of the somatic tissue immediately surrounding them. This assumes importance in the context that buffalo attains sexual maturity at an older age of 2–3 years and have smaller ovaries with lesser number of primordial follicles in comparison with cattle, which is suggested to be the main reason of their poor breeding performance.