Chromic oxide

A standard digestibility study, in which the digestibility of a component in test feedstuff is determined, requires measuring the ingested amount of that component and the voided amount of given component of test feedstuff. The total collection and index methods have been widely used to determine the digestibility of components in swine and poultry diets. The total collection method requires an accurate measure of feed intake and fecal output for determining the amount of the component ingested and voided via feces, respectively. With these measurements, the digestibility of the component can be calculated as follows:
where C_input and C_output are the amount of component ingested and voided via the feces, respectively.
In a total collection study for swine, pigs are individually housed in crates and thereafter they are adapted to their crates and feed being given before fecal sample collection. The adaptation period usually lasts for 3 to 7 days before a collection period of 4 to 6 days (Adeola, 2001 ). During the adaptation period, a feeding level is adjusted to avoid feed refusal which results in additional work such as orts collection during the collection period as well as drying and analyzing the orts after the collection period. A level of feeding at 3 times maintenance (197 kcal/kg BW^0.60; NRC, 2012 ) or approximately 3% to 4% of body weight (BW) per d is suggested as the sufficient level of feeding for the digestibility study with total collection method. During the collection period, colored markers such as ferric oxide, chromic oxide, and indigo carmine are commonly used for the identification of fecal output from a given ingested feed (Adeola, 2001 ; Kim et al., 2006 (link); Son et al., 2013 ). Once pigs are adjusted to the crates and feed, the collection period begins and ends with feeding the first and the last marked feed, respectively. In this period, the feces that voided between the first and second appearances of the marker are collected as the representative output that is associated with the fed quantities given in the collection period.
In a balance study, it is difficult to identify urine that belongs to specific feed because marker does not appear in the urine, thus the urine collection is generally conducted based on time. The quantitative urine collection starts from the day of the first marked feed offered and ends at the day of the last marked feed. With measurement of components in the urine, the metabolizability of the component can be calculated as follows:
where C_urine is the amount of component voided via the urine.
In a digestibility study for poultry, the total collection method is not common for determining the fecal digestibility, because feces and urine are voided together in the form of excreta and it is difficult to separate the feces from the excreta and measure digestibility. There was an attempt to avoid this confounding effect of urine on the fecal digestibility using surgical technique such as colostomy (Okumura, 1976 (link)), however there are problems with the artificial anus including skin regrowth, intestinal stasis and hardening of fecal material (Paulson, 1969 ). Thus, the total collection method in poultry usually involves collecting excreta (feces+urine). Sibbald (1976) (link) developed the precision-fed rooster assay and McNab and Blair (1988) (link) later suggested some modification. In this assay, adult cockerels or roosters were fasted for 48 h prior to being fed test ingredients. During the fasting period, all birds are tube-fed 2 doses of 25 to 30 g of glucose (as an aqueous solution) at 8 and 32 h post-feed withdrawal, which partly alleviates the effects of starvation. At 48 h post-feed withdrawal, all birds are tube-fed 25 to 30 g of their assigned test ingredients that are in distilled water and ground through a 0.5 mm screen prior to feeding. Birds for determining endogenous losses are fed 50 g of glucose. The total collection of excreta is conducted for 48 h after feeding of test ingredients or glucose for endogenous losses determination. During 48-h collection period, all birds are given 50 ml of water by tube about 32 h after feeding to overcome any effects induced by low water intake.

The total collection method involves laborious quantitative records of feed intake and output whereas the index method can avoid these laborious procedures, but greatly relies on accurate chemical analysis of index compound in the feed and fecal output. In the use of an index, there are inherent fundamental assumptions which include that index compound should be i) completely inert in the gastrointestinal tract, ii) completely and regularly excreted, and iii) uniformly mixed with the digesta or fecal material. Thus, the amount of index compound in the feed and the amount voided in the output should be uniform over equal periods of time (Adeola, 2001 ). Several index compounds including chromic oxide, titanium dioxide and insoluble ash are commonly used for the determination of digestibility (Jagger et al., 1992 (link); Betancourt et al., 2012 ; Kim et al., 2012 (link); Olukosi et al., 2012 (link)) and are added to the diet at 0.1% to 0.5%. With the index method, digestibility is calculated as follows:
where CI_input and CI_output are the concentration of index compound in feed and feces, respectively; CC_input and CC_output are the concentration of component in feed and feces, respectively.

Individual pigs and amount of feed additions and refusals in each pen were
weighed and recorded to measure average daily gain (ADG), average daily feed
intake (ADFI), and ratio between ADG and ADFI (G:F) of pigs. Diarrhea of each
pig was checked and its visual score was recorded by 3 independent evaluators
with a score from 1 to 5 (1 = normal hard feces; 2 = slightly soft feces; 3 =
soft, partially formed feces; 4 = loose, semi-liquid feces; and 5 = watery,
mucous-like feces) each day for the first 2 weeks of this experiment [16 (link)]. Frequency of diarrhea was calculated
by counting pen days with average diarrhea score from individual pigs in each
pen of 4 or greater [17 (link),18 (link)]. Whole blood samples were collected
from the jugular vein of randomly selected 2 pigs in each pen using EDTA tubes
(Becton Dickinson Vacutainer Systems, Franklin Lakes, NJ, USA) containing
anticoagulant on day 1, 3, 7, and 14 after weaning. For the last week of the
experiment period, pigs were fed respective dietary treatments containing 0.2%
chromic oxide as an indigestible marker. Fecal samples were collected from
randomly selected 2 pigs in each pen by rectal palpation daily for the last 3
days after the 4-d adjustment period. The collected fecal samples were pooled
and stored at −20°C until analysis. Diet samples were also
collected and stored at −20°C until analysis. Randomly selected 2
pigs in each pen were anesthetized by an intra-muscular injection of a 2-mL
suxamethonium chloride (Succicholine^®, Ilsung Pharm. Co. Ltd.,
Seoul, Korea) at the end of this experiment. After anesthesia, pigs were
euthanized by CO₂ gas [19 (link)].
Ileal digesta samples were collected from distal ileum before the ileocecal
junction [11 ]. The collected ileal
digesta samples were stored at −20°C until analysis. Sections of
ileum of 3 cm length were collected, washed gently with distilled water, and
fixed in 10% neutral buffered formalin for histological analysis [20 (link),21 (link)].