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Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet.

ABSTRACT: PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (epsilon) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sample collected; serial dilutions of DNA were used similarly to calculate PCR efficiency. Species of Entodinium, the most prevalent of the rumen protozoa, were enumerated in rumen samples collected from 100 1-year-old merino wethers by microscopy and real-time PCR. Both the counts developed by the real-time PCR method and microscopic counts were accurate and repeatable, with a strong correlation between them (R2= 0.8), particularly when the PCR efficiency was close to optimal (i.e., two copies per cycle). The advantages and disadvantages of each procedure are discussed. Entodinium represented on average 98% of the total protozoa, and populations within the same sheep were relatively stable, but greater variation occurred between different sheep (10(0) and 10(6) entodinia per gram of rumen contents). With this inherent variability, it was estimated that, to detect a statistically significant (P = 0.05) 20% change in Entodinium populations, 52 sheep per treatment group would be required.

SUBMITTER: Skillman LC

PROVIDER: S-EPMC1352179 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet.

Skillman Lucy C LC Toovey Andrew F AF Williams Andrew J AJ Wright André-Denis G AD

Applied and environmental microbiology 20060101 1

PCR and real-time PCR primers for the 18S rRNA gene of rumen protozoa (Entodinium and Dasytricha spp.) were designed, and their specificities were tested against a range of rumen microbes and protozoal groups. External standards were prepared from DNA extracts of a rumen matrix containing known numbers and species of protozoa. The efficiency of PCR (epsilon) was calculated following amplification of serial dilutions of each standard and was used to calculate the numbers of protozoa in each sampl ...[more]

PMID: 16391043

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Project description:Oats have the characteristics of drought tolerance, cold resistance, strong adaptability, high forage yield, and high nutritional value. However, there are few reports on the most appropriate amount of oat hay in ruminant diets, the digestion and metabolism of ruminants, and the rumen microflora. To study the effects of oat hay content in diets on nutrient digestion and metabolism and the rumen microflora in sheep, 9 German Merino and Mongolian crossbred rams of similar body condition and weight with permanent fistulas were selected. The 3 × 3 Latin square design was used to randomly divide the rams into 3 groups, with 3 animals in each group. The three groups were fed different kinds of roughage: whole-plant corn silage only (corn silage group, CSG), oat hay mixed with whole-plant corn silage (1:1) (mixed group, MG), and oat hay only (oat hay group, OHG). The nutrient digestion and metabolism of each group were measured, and the pH and rumen microflora were examined after feeding for different durations. Dynamic changes in microbial communities were detected. The nutrient digestion and metabolism results showed that, with an increase in the content of oat hay in the diet, the intake and apparent digestibility of dry matter (DM) and organic matter (OM) showed an increasing trend, and the intake, digestion, and stability of acid detergent fiber (ADF) and neutral detergent fiber (NDF) increased in the OHG. The apparent digestibility, dietary nitrogen, deposited nitrogen, and nitrogen retention rate in this group were significantly higher than those in the CSG (p < 0.05). The rumen pH and sequencing results showed that the rumen fluid pH of the CSG was significantly lower than that of the OHG at 1 and 5 h (p < 0.05). The main microbial in the rumen of the three groups of sheep were Bacteroides, Sclerotium, and Proteus. The dominant taxon in the CSG was Prevotella, followed by Vibrio syringae, and the dominant taxon in the MG and OHG was Prevotella, followed by Rikenellaceae. Redundancy analysis showed that ADF and NDF in the feed had an effect on the abundance of Fibrobacteres, Ruminococcaceae, and Prevotella. Our findings indicate that the use of oat hay roughage in the diet significantly improves the apparent digestibility of NDF and ADF and helps maintain the stable state of the sheep's rumen internal environment and the growth of rumen microorganisms.

Dataset Information

Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet.

Publications

Development and validation of a real-time PCR method to quantify rumen protozoa and examination of variability between entodinium populations in sheep offered a hay-based diet.

Similar Datasets

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