A universal test to determine the integrity of RNA, and its suitability for reverse transcription, in animal tissue laboratory specimens

Degradation of RNA in diagnostic specimens can cause false-negative test results and potential misdiagnosis when tests rely on the detection of specific RNA sequence. Current molecular methods of checking RNA integrity tend to be host species or group specific, necessitating libraries of primers and reaction conditions. The objective here was to develop a universal (multi-species) quality assurance tool for determining the integrity of RNA in animal tissues submitted to a laboratory for analyses. Ribosomal RNA (16S rRNA) transcribed from the mitochondrial 16S rDNA was used as template material for reverse transcription to cDNA and was amplified using polymerase chain reaction (PCR). As mitochondrial DNA has a high level of conservation, the primers used were shown to reverse transcribe and amplify RNA from every animal species tested. Deliberate degradation of rRNA template through temperature abuse of samples resulted in no reverse transcription and amplification. Samples spiked with viruses showed that single-stranded viral RNA and rRNA in the same sample degraded at similar rates, hence reverse transcription and PCR amplification of 16S rRNA could be used as a test of sample integrity and suitability for analysis that required the sample's RNA, including viral RNA. This test will be an invaluable quality assurance tool for determination of RNA integrity from tissue samples, thus avoiding erroneous test results that might occur if degraded target RNA is used unknowingly as template material for reverse transcription and subsequent PCR amplification.

A universal equation to predict methane production of forage-fed cattle in Australia

The methods for estimating methane emissions from cattle as used in the Australian national inventory are based on older data that have now been superseded by a large amount of more recent data. Recent data suggested that the current inventory emissions estimates can be improved. To address this issue, a total of 1034 individual animal records of daily methane production (MP) was used to reassess the relationship between MP and each of dry matter intake (DMI) and gross energy intake (GEI). Data were restricted to trials conducted in the past 10 years using open-circuit respiration chambers, with cattle fed forage-based diets (forage >70%). Results from diets considered to inhibit methanogenesis were omitted from the dataset. Records were obtained from dairy cattle fed temperate forages (220 records), beef cattle fed temperate forages (680 records) and beef cattle fed tropical forages (133 records). Relationships were very similar for all three production categories and single relationships for MP on a DMI or GEI basis were proposed for national inventory purposes. These relationships were MP (g/day) = 20.7 (±0.28) × DMI (kg/day) (R2 = 0.92, P < 0.001) and MP (MJ/day) = 0.063 (±0.008) × GEI (MJ/day) (R2 = 0.93, P < 0.001). If the revised MP (g/day) approach is used to calculate Australia’s national inventory, it will reduce estimates of emissions of forage-fed cattle by 24%. Assuming a global warming potential of 25 for methane, this represents a 12.6 Mt CO2-e reduction in calculated annual emissions from Australian cattle.

Hydration in non-suckling neonatal Brahman-cross calves

Objective To identify measures that most closely relate to hydration in healthy Brahman-cross neonatal calves that experience milk deprivation. Methods In a dry tropical environment, eight neonatal Brahman-cross calves were prevented from suckling for 2–3 days during which measurements were performed twice daily. Results Mean body water, as estimated by the mean urea space, was 74 ± 3% of body weight at full hydration. The mean decrease in hydration was 7.3 ± 1.1% per day. The rate of decrease was more than three-fold higher during the day than at night. At an ambient temperature of 39°C, the decrease in hydration averaged 1.1% hourly. Measures that were most useful in predicting the degree of hydration in both simple and multiple-regression prediction models were body weight, hindleg length, girth, ambient and oral temperatures, eyelid tenting, alertness score and plasma sodium. These parameters are different to those recommended for assessing calves with diarrhoea. Single-measure predictions had a standard error of at least 5%, which reduced to 3–4% if multiple measures were used. Conclusion We conclude that simple assessment of non-suckling Brahman-cross neonatal calves can estimate the severity of dehydration, but the estimates are imprecise. Dehydration in healthy neonatal calves that do not have access to milk can exceed 20% (>15% weight loss) in 1–3 days under tropical conditions and at this point some are unable to recover without clinical intervention.