Decrease of CDDM and CDCE rates caused by adding 10% LLH in the diet may be attributed to an increase in excretion of dry matter by laying hens fed on diets with a higher proportion of fibers.
Variables (1) Treatments (2) Mean CV (%) 0% LLH 5% LLH 10% LLH CDDM (%) 72.96 a 71.60 ab 70.07 b 71.55 2.28 CDN (%) 51.88 a 47.27 a 45.38 a 48.18 10.52 CDCE (%) 76.32 a 74.86 ab 73.28 b 74.82 1.90 AME (kcal 3.341 a 3.285 a 3.255 a 3.293 1.90 [kg.sup.-1] DM) AMEn (kcal 3.204 a 3.166 a 3.116 a 3.162 1.72 [kg.sup.-1] DM) (1) CDDM = Coefficient of digestibility of dry matter; CDN = Coefficient of digestibility of nitrogen; CDCE = Coefficient of digestibility of Crude Energy; AME = Apparent metabolizable energy; AMEn = Apparent metabolizable energy corrected for Nitrogen.
In this report, we apply CDCE to screen for those variations in pooled DNA samples and show that families can be categorized by their CDCE profiles.
One of the primers had a GC-clamp added for optimal resolution of mutant fragments and/or a fluorescein group to permit detection by CDCE. PCR was carried out using Taq polymerase (Amersham) in 20-[micro]L reaction volumes containing 50 ng of genomic DNA, 10 pmol of each primer, and 400 pmol of deoxynucleotide triphosphates in the buffer provided.
The CDCE apparatus was as described by Khrapko et al.
DNA samples from different individuals of a family were pooled, amplified, and analyzed by CDCE, or the DNA from each individual was amplified and analyzed separately on the CDCE.
The optimal temperature for the resolution of fragment 13b was obtained by performing CDCE runs at different temperatures.
The detection limit of CDCE under the conditions used for the resolution of fragment 13b was next determined using DNA samples amplified from a homozygous (1987G) individual and a 1987A [right arrow] G heterozygote.
To evaluate the reproducibility of CDCE, we analyzed a sample containing a mixture of three mutants (50% 1853G [right arrow] T heterozygotes,16.7% 1987A [right arrow] G heterozygotes, and 33.3% 1853G [right arrow] T:1987A [right arrow] G compound heterozygotes) several times.
In the first screen for [alpha]-ENaC variants, exon 13 of the Scnn1a gene was amplified from pooled genomic DNA from all available members (n = 2-12) of any one of the 31 families and analyzed by CDCE. Analysis of the CDCE profiles of fragment 13a in the 31 families showed that the profiles for fragment 13a were similar to the standard Scnn1a sequence.
Regarding fragmented 13b, CDCE analysis of the 31 families showed that they could be categorized according to the observed peaks (CDCE profiles) into four groups.
The other 13 families clearly differed in their CDCE profiles from the standard sequence, indicating the presence of Scnn1a exon 13 variants.