Study on correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation

This investigation was carried out to study the correlation of sperm quality parameters with antioxidant and oxidant status of buffalo bull semen during various stages of cryopreservation. Semen samples were evaluated for sperm parameters (mass motility [MM], concentration [CON], progressive motility [PM], viability [VIB], acrosomal integrity [AI] and hypo‐osmotic swelling [HOS] response), antioxidants (superoxide dismutase [SOD], catalase [CAT], glutathione peroxidase [GPx] and total antioxidant capacity [TAC]) and oxidants (Lipid peroxidation [LPO] and reactive oxygen species [ROS]) at fresh, pre‐freeze and post‐thaw stages. Sperm parameters (PM, VIB, AI and HOS response) and antioxidants (SOD, CAT and TAC) were significantly (p < .05) reduced at fresh stage, and oxidants (LPO and ROS) were significantly (p < .05) increased at pre‐freeze and post‐thaw stages. At fresh stage, MM was negatively correlated with LPO (p < .05), and CON was positively correlated with SOD, TAC and CAT, negatively correlated with LPO and CAT was positively (p < .01) correlated with VIB and HOS response. At pre‐freeze stage, CAT was positively correlated with PM and AI (p < .05), and AI was negatively (p < .05) correlated with ROS. At post‐thaw stage, CAT was positively correlated with PM, VIB, HOS response and AI,, and LPO was negatively correlated with HOS, AI and VIB. The study of correlations of these parameters at different preservation stages with bull fertility may play an important role in developing models for predicting future fertility of bulls in the absence of conception rate data.     

A novel combination of silane‐coated silica colloid with hybrid RNA extraction protocol and RNA enrichment for downstream applications of spermatozoal RNA

Spermatozoa are specialised cells with low RNA content as compared to somatic cells. The suitable sperm RNA extraction and enrichment protocols for downstream applications are available for human, cattle, stallion and mouse but not for buffalo spermatozoa. Therefore, the present work was conducted to find out suitable colloidal solution for sperm purification and appropriate protocol for sperm RNA extraction and enrichment/amplification of RNA. For purification, we used PVP‐coated silica colloidal solution (PVP‐Si), silane‐coated silica colloidal solution (Silane‐Si) and iodixanol. Sperm recovery rate, total sperm motility and progressive sperm motility were significantly improved after separation by Silane‐Si and iodixanol compared to PVA‐Si method. The combined guanidinium thiocyanate–phenol–chloroform (GTPC) with silica matrix (SM)‐based RNA extraction yielded more quantity of RNA in compared to individual method. The hybrid of SM and GTPC into a single protocol yielded 360–450 ng RNA from 30 million buffalo spermatozoa. For the first time, we adopted new way to enrich sperm RNA that increased the RNA concentration 4–5 times that was sufficient for downstream applications. The linear amplification of sperm RNA increased RNA concentration around 27–45 times. In summary, Silane‐Si colloid for sperm separation, hybrid SM and GTPC protocol for sperm RNA extraction followed by enrichment or amplification of RNA was found suitable for high‐throughput analyses of buffalo sperm RNA.     

The Socioeconomic Impacts of Clinically Diagnosed Haemorrhagic Septicaemia on Smallholder Large Ruminant Farmers in Cambodia

Haemorrhagic septicaemia (HS) is an acute fatal infectious disease of mainly cattle and buffalo and outbreaks occur commonly in Cambodia. Disease outbreak reports were examined to select five villages from three provinces for a retrospective investigation of HS epidemiology and socioeconomic impact on smallholders, with an aim of identifying potential benefits from improving disease prevention through biosecurity and vaccination. The Village Animal Health Worker (VAHW) or Chief in each village and 66 affected smallholders were surveyed. At the village level, 24% of all households were affected with an estimated mean village herd morbidity of 10.1% and mortality of 28.8%. Affected farmers reported HS disease morbidity and mortality at 42.7% and 63.6% respectively. Buffalo had a higher morbidity (OR = 2.3; P = 0.003) and mortality (OR = 6.9; P < 0.001) compared with cattle, and unvaccinated large ruminants a higher morbidity (OR = 2.9; P = 0.001). The financial impact varied depending on whether the animal survived, provision of treatment, draught replacement and lost secondary income. The mean cost per affected household was USD 952.50 based on ownership of five large ruminants. The impact per affected animal was USD 375.00, reducing the pre‐disease value by 66.1%. A partial budget revealed an overwhelming incentive for farmers to practice biannual vaccination, with a net benefit of USD 951.58 per household based on an annual disease incidence rate of 1. Sensitivity analysis showed that a net benefit of USD 32.42 remained based on an outbreak every 20 years. This study indicates HS can cause a catastrophic financial shock to smallholders and remains a critical constraint to improving large ruminant productivity and profitability. Addressing HS disease control requires a focus on improving smallholder farmer knowledge of biosecurity and vaccination and should be priority to stakeholders interested in addressing regional food insecurity and poverty reduction.     

Detection of Bovine Papilloma Viruses in Wart‐Like Lesions of Upper Gastrointestinal Tract of Cattle and Buffaloes

In present investigation, etiopathological characterization of upper gastrointestinal tract (GIT) tumours of cattle and buffaloes was undertaken. A total of 27 GIT wart‐like lesions in rumen, reticulum, mouth and oesophagus of cattle and buffaloes revealed the presence of small nodular to larger spherical or slender growths with thin base present on mucosa and ruminal pillar. Histopathologically, these cases were diagnosed as fibropapilloma/papilloma. This is the first world record on ruminal papillomatosis in buffaloes. Ruminal warts of cattle and buffaloes revealed the presence of BPV‐5, ‐1 & ‐2, which is the first report of presence of these BPVs in the ruminal warts from India. Quantitative real‐time PCR revealed that DNA samples of different GIT wart‐like lesions contained varying amount of BPV DNA copy numbers. Immunohistochemistry revealed that the PCNA and Ki67 immunopositivity was present in the basal and spinosum layer of the fibropapilloma/papilloma, indicating these as the cellular proliferation site. In conclusion, the present investigation revealed that BPV‐5, ‐1 & ‐2 are associated with certain ruminal wart‐like lesions/growths in cattle and buffaloes, and the basal and spinosum layer of the ruminal fibropapilloma/papilloma were cellular proliferation sites.     

Molecular cloning and expression of the dihydrofolate reductase (DHFR) gene from adult buffalo fly (Haematobia irritans exigua): effects of antifolates

The folate analogues methotrexate, aminopterin and pyrimethamine were toxic when fed in a blood meal to adult buffalo flies (Haematobia irritans exigua), but aminopterin caused greater mortality than methotrexate, while trimethoprim was not toxic to adult flies. This is the first recorded instance of mortality in adult insects caused by ingestion of folate analogues. In order to investigate the mechanism of this toxicity, the dihydrofolate reductase (DHFR) gene was cloned from adult buffalo fly cDNA using a PCR-based approach. The full-length DHFR coding sequence (BF-DHFR) was 887 bp and contained an open reading frame encoding a protein of 188 amino acids. The deduced protein sequence identities between BF-DHFR and the other known insect DHFR sequences were: Drosophila melanogaster, 75%; Aedes albopictus, 54%; Heliothis virescens, 43%. The BF-DHFR gene has a single 52 bp intron, an organization more similar to Dipteran species (Drosophila and Aedes). The cDNA encoding BF-DHFR was inserted into an Escherichia coli expression vector and the recombinant protein was expressed to levels representing about 25% of total cell protein. The active enzyme was purified by affinity chromatography on methotrexate-agarose and displayed a relatively low affinity (IC50 = 30 nm) for methotrexate.     

自拟当归芦荟水牛角汤治疗过敏性紫癜疗效观察

目的探讨运用自拟当归芦荟水牛角汤口服治疗过敏性紫癜反复发作的效果。方法对20例门诊过敏性紫癜患者单方运用"自拟当归芦荟水牛角汤"口服治疗,治疗1-2个疗程,观察治疗效果。结论过敏性紫癜采用"当归芦荟水牛角汤"治疗见效快,疗效稳定持久,服用方便且经济,值得临床推广应用。     

重组日本血吸虫26 kDa GST抗原免疫水牛后抗体动态及免疫保护性效果

目的　观察重组日本血吸虫 2 6kDaGST抗原 (reSjc2 6GST)免疫役用放养水牛 (简称水牛 )后抗体动态及免疫保护性的效果。　方法　试验组 96头水牛 ,用reSjc2 6GST免疫 3次 ,每次间隔 2wk ,3次剂量分别为 0 2、 0 2和 0 1mg。对照组 90头水牛不作免疫 ;观察 2组水牛免疫前及免疫后 2、 5、 9、 12、 15和 2 0个月的抗体水平及血吸虫感染率的变化。　结果　试验组机体产生特异性抗reSjc2 6GST抗体 ,其抗体水平呈明显的梯形升高趋势。试验组免疫后 2 0个月血吸虫感染率比免疫前下降了 62 2 % ,比同期对照组低 67 7%。　结论　用reSjc2 6GST免疫水牛能产生特异性抗体 ,在免疫后 2 0个月内维持较高水平 ,有一定的抗血吸虫自然感染的保护力。     

Sequence analysis and genetic variability of stearoyl CoA desaturase (SCD) gene in the Italian Mediterranean river buffalo

Stearoyl-CoA desaturase (SCD) plays a key metabolic role by changing the saturated FA content of ruminant milk and meat. In this study we characterized for the first time the stearoyl-CoA desaturase (SCD) gene in river buffalo (Bubalus bubalis) and investigated its genetic variability. On a total of 78 buffaloes, 15 SNPs were detected and 6 of them were preliminarily genotyped. In particular, the g.133A>C SNP was found to create a new consensus site for the SP1 binding site, thus generating a new tandem repeat in the promoter region. A preliminary association study with the milk fatty acid content showed that the C allele significantly affects the total desaturation index (P < 0.01). Linkage disequilibrium analysis allowed identification of 7 haplotypes and 4 tag SNPs. Such polymorphisms could represent useful genetic markers for association studies with fatty acid composition, but further studies are needed to evaluate their potential use to improve the nutritional quality of the dairy products.     

Addition of pomegranate juice (Punica granatum) in tris‐based extender improves post‐thaw quality,motion dynamics and in vivo fertility of Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa

The aim of the present study was to determine the protective effects of pomegranate juice in tris‐based extender on semen parameters, computer‐assisted sperm analysis (CASA) motion characteristics and field fertility of post‐thawed Nili Ravi buffalo (Bubalus bubalis) bull spermatozoa. Two consecutive ejaculates/collection from each of the five adult Nili Ravi buffalo bulls were collected with artificial vagina at 42°C for a period of 7 weeks, diluted in extender containing different concentrations of pomegranate juice (0.0%, 2.5%, 5%, 7.5% and 10%). Diluted samples were packed and frozen in 0.54 ml French straws. The addition of 10% pomegranate juice in extender significantly improved post‐thaw sperm morphology (%), motilities (CASA total motility, progressive motility (%) as well as VAP, VSL, VCL, STR, DAP, DSL) compared to the control group (p < 0.05). Plasma membrane, acrosome membrane and DNA integrity were significantly higher in extender with 10% pomegranate juice than the control group (p < 0.05). Field fertility rate (60.39% vs. 46.53%) was higher (p < 0.05) in extender with 10% pomegranate juice as compared to the control. It is therefore concluded that the addition of 10% pomegranate juice in tris‐based extender improves post‐thaw semen parameters, CASA motion dynamics and field fertility in Nili Ravi buffaloes.     

水牛化疗控制鄱阳湖地区血吸虫病传播的现场实验研究

目的探索水牛治疗是否在湖沼地区血吸虫病传播中的作用,为制定防治策略提供依据。方法将江西省鄱阳湖区2个血吸虫病重度流行村,分为实验(干预)组及对照组。实验组用吡喹酮治疗所有耕牛,以消灭可能血吸虫传染源;对照组,不治疗耕牛。两村居民按常规方法进行血吸虫病防治。结果连续5年观察结果表明,实验组居民感染率比对照组下降70%;3岁以下水牛感染率为2%~18%,3岁以上水牛感染率为0~2.9%。结论通过干预和对照组的研究表明,在血吸虫重度感染区,单纯对居民进行化疗是难以有效地控制血吸虫病的传播的,而通过化疗水牛在一定程度上能降低居民的再感染。本研究通过5年的观察,证实了对水牛进行治疗对控制血吸虫病的传播的效果可达70%,同时也证实了在感染的水牛中的大部分(89%)是小于3岁的水牛,这对我们今后制定有效的控制湖沼地区的血吸虫病对策提供了依据。