Ration formulation is a mixture of individual feed ingredients, and the metabolizable energy (ME) and net energy (NE) of the ration are assumed to be the sum of the individual ingredients. This assumes that the ME and NE values of the individual ingredients do not change when mixed with other feed products. However, some studies have documented associative effects among feed ingredients [1
], defined as interactions between the ration components that alter the nutritional value of the individual ingredients [4
]. Associative effects may be positive, negative, or absent [5
], and though widely discussed in theory, they are seldom considered in feed formulation.
Ruminant animals are fed diets of highly fibrous roughage, grains, brans, and hulls. Associative effects may be an important factor in ruminant rations, as interactions among these ingredients could modify the microbial fermentation processes in the rumen [6
]. Several studies have investigated the associative effects of barley straw and alfalfa (Medicago sativa
], grasses and legumes [9
], red clover (Trifolium pratense
) and kikuyu grass silage [10
], and corn stalks and alfalfa (Medicago sativa
Rice is a major crop in Asia and, consequently, rice straw is a common roughage for ruminant animals in most Asian countries. In 2014, approximately 538.88 million tons of rice straw, accounting for more than 90% of total world production, was produced in Asia [12
]. Prior studies have investigated the associative effects of rice straw with alfalfa hay and corn silage [13
], and those of chemically treated rice straw with grass hay or mulberry leaves [6
]. To date, however, no studies have tested the associative effects of rice straw with timothy hay and corn grain, which are commonly co-fed ruminants such as beef cattle. In particular, timothy hay is well known for its good quality and contributes a major proportion of imported forage in Korea [14
]. Compared to rice straw, timothy hay contains easily digestible carbohydrates, so that it has higher digestibility of fiber and dry matter [15
]. Timothy hay, however, is two to three times more expensive than rice straw, and thus it is mainly used as a forage source for calves and lactating dairy cows and as a supplement for rice straw for growing cattle in Korea [16
In vitro fermentation methods are widely used to study associative effects, as the digestibility and rumen fermentation of feed can be measured in a relatively simple manner, and numerous samples can be evaluated at one time [17
]. Among different in vitro methods, the in vitro gas production technique is commonly used to assess the differences between single and mixed feed substrates for different variables [6
]. An automated in vitro gas production system (AGPS) was first developed by Pell and Schofield [20
]—the AGPS continuously measures the gas produced during in vitro ruminal fermentation with electronic pressure transducers, which are sensitive enough to detect small changes due to associative effects. The AGPS developed by Pell and Schofield is inexpensive, easily adopted, and simple to maintain.
The objective of this study was to evaluate the associative effects of rice straw, timothy hay, and corn grain on the ruminal fermentation characteristics using an automated in vitro gas production system.
Rice straw is a commonly used forage in East Asia, therefore, we were interested in the associative effects of other feeds with rice straw. To date, no studies have investigated the associative effects of rice straw, timothy hay, and corn grain. Corn is an important grain source for cattle in Korea, so there is great interest in the associative effects of corn grain and rice straw. Furthermore, the associative effects of timothy hay, a high-quality forage source, may compensate for the lack of nutrients in rice straw. The objective of this study was to determine the associative effects of rice straw with timothy hay and corn grain using an automated gas production system in an in vitro fermentation experiment.
In this study, the pH was significantly lower for the three mixed feed treatments (RT, RC, and RTC) than for the rice straw (p
< 0.05), corresponding to higher total VFA concentrations in the mixed treatments (which contained more fermentable carbohydrates than the rice straw). The non-fiber carbohydrate (NFC) content of rice straw, timothy hay, and corn grain were 86, 204, and 792 g/kg DM, respectively (Table 1
). NFCs provide ruminants with fermentable carbohydrates such as starch, sugar, and pectin, and promote the growth of rumen microorganisms. It has been shown that high dietary NFC increases the total VFA production, decreases ruminal pH, and modifies the molar proportions of VFAs by decreasing the ratio of acetic acid/propionate acid and increasing the proportion of butyrate [25
]. Our results are consistent with Haddad [8
], who supplemented barley straw diets with 300 and 450 g alfalfa hay (i.e., more NFC) and observed higher total VFA and lower pH values than in non-supplemented barley straw.
-N concentration, TDMD, and V1max
(except for RT) were significantly higher in the mixed feed treatments (RT, RC, and RTC) than in the rice straw treatment (p
< 0.05). This can be explained by the higher NFC and lower NDF contents in the mixed feed treatments, which provided more fermentable carbohydrates to the rumen microorganisms [26
]. Previous studies have also reported a positive correlation between gas and VFA production, and total gas production at 48 h was primarily determined by the NFC content [27
]. The ash content of rice straw in this study was 12.2%, higher than both timothy hay (7.3%) and corn (1.2%). Normally, 80% of the ash contained in rice straw is silica [29
], and silica limits the digestion of fiber because it prevents bacterial colonization [30
]. Thus, the replacement of rice straw with timothy hay or corn gain significantly increased the growth and fermentation of rumen microbes.
When considering the associative effects, the observed NH3
-N concentrations were significantly higher than the predicted concentrations in the RT, RC, and RTC treatments. An increase in the NH3
-N concentration reflects greater catabolism of proteins and non-proteins [31
], and may also indicate improvements in the rumen environment [33
]. The growth of rumen microbes is stimulated by the digestibility of substrates that increases the NH3
-N concentration [35
]. Niderkorn et al. [36
] tested the associative effects of legumes and grass and found that the observed NH3
-N content was significantly higher than the predicted value in the legume-grass mixture. The mixture provided the rumen microorganisms with more nitrogen and other chemicals, and the interactions of these chemicals enhanced microbial growth and fermentation [36
The observed TDMD values were significantly higher than the predicted values (p
< 0.05; except RT), demonstrating a positive associative effect on dry matter digestibility; this result is consistent with other reports. Cho et al. [37
] reported positive associative effects on the dry matter digestibility, organic matter digestibility, and organic matter effective degradability at an appropriate feeding ratio of corn grain and pasture forage. The authors attributed these results to improved ruminal digestion and the asynchrony of nutrient release by the rumen microorganisms, e.g., the release of soluble carbohydrate from grain seemed to improve the digestibility of the cell wall [37
]. A corn grain-supplemented fescue hay diet also resulted in improved nutrient digestibility and rumen microorganism growth [38
In the RTC treatment, the observed value of total VFA was significantly higher than the predicted value. This might be due to a higher NFC content from the corn grain and easily fermentable cellulose and hemicellulose from the timothy hay. Other work has shown that easily fermentable cellulose and hemicellulose increased the number of cellulolytic bacteria, stimulating the digestibility of other less degradable fiber sources in the diet [39
]. Sun et al. [13
] reported positive associative effects for the in vitro gas, total VFA, and microbial protein production when alfalfa hay was fermented with corn silage. Together, these results suggest that the combination of rice straw, timothy hay, and corn increases the NFCs from corn grain and the easily fermentable cellulose and hemicellulose from timothy hay, which influences the rumen microorganisms and fermentation environment and produces more fermentable substances to increase the total VFA.
There was a trend for the observed values to be higher than predicted values for Vmax
, and k2
in the RTC treatment. Within the same treatment, the observed V1max
was also significantly higher than the predicted value, and the discrete lag time observed was significantly lower than the predicted value. A higher V1max
and lower lag time indicate that the soluble fraction constitutes a substrate of rapid fermentation that facilitated the adhesion and colonization of microorganisms, thereby increasing fermentation and reducing the lag period [40
]. Other studies have observed similar results, documenting a reduced lag time in the initiation of the in vitro digestion of fibers due to the positive associative effects of tropical grasses and legumes [41
]. Measuring the in vitro gas production is a popular technique for determining the forage digestion characteristics and kinetics of fermentation. Rumen microorganisms ferment substrates into CO2
, and VFA [6
], and the increase in gas production may be related to increased VFA, caused by the interaction of fiber and non-fiber decomposing microorganisms or ruminal bacteria and protozoa [6
]. When the rice straw was fermented with timothy hay and corn grain, interactions among the rumen microorganisms increased substrate fermentation and produced more gas and VFA, which resulted in the higher V1max
and a shorter lag time.
Nutrient deficiencies in the roughage may be detrimental to the growth of rumen microorganisms (e.g., nitrogen or sulfur) or the ruminant itself (e.g., phosphorus), but positive associative effects were commonly observed when other feed materials contained the needed nutrients. Some studies have suggested that the supplementation of forage with rapidly fermentable carbohydrate sources (i.e., corn grain) would improve microbial growth and feed degradability [43
], even in suboptimal nitrogen conditions, because of adaptations in the microbial populations [44
]. Niderkorn et al. [46
] studied the associative effects of temperate climate grass and legumes and found an influence on rumen protein degradation between the legume tannins and grass protein. Improvements to the rumen microflora were also demonstrated by the associative effects of cocksfoot (Dactylis glomerata
) and sainfoin (Onobrychis viciifolia
]. Thus, when rice straw was fermented with timothy hay and corn, non-fiber carbohydrate from the corn grain, and easily fermented cellulose and hemicellulose from the timothy hay may have contributed to the positive associative effects.