Nowadays, most available information on the degradative behaviour of feeds in ruminants is based on in situ incubation in the rumen, and it is adopted by many feed evaluation systems currently in use for ruminants.  However, the outcome of this technique might be affected by many factors such as sequence of nylon bags incubation in the rumen.  The objective of current study was to investigate effects of sequence of nylon bag incubation on degradative behavior of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF) and acid detergent fiber (ADF) in some feed ingredients commonly used in dairy rations, including alfalfa haylage, corn silage, corn grain and soybean meal.  Four multiparous Holstein lactating cows fitted with permanent ruminal cannulas were used.  The nylon bags containing feed samples either were placed in the rumen at once and removed at designated time intervals (all in-gradually out method; AG) or were placed in the rumen at designated time points and retrieved at once (gradually in-all out method; GA).  Fractional rate of degradation of potentially degradable fraction, lag time and effective rumen degradability (ED) of DM and CP were significantly higher in the AG compared to the GA method (P<0.05).  Fractional rates of DM and CP degradation was higher in alfalfa haylage samples incubated in the rumen using the AG method compared to that using the GA method (0.138 h^–1 vs. 0.073 h^–1 and 0.002 h^–1 vs. 0.1125 h^–1, for DM and CP, respectively; P<0.05).  Due to a higher fractional rate of degradation (K_d) of DM and CP, the ED of DM and CP at different fractional passage rates were higher in the AG than those in the GA method (P<0.05).  Potentially degradable fraction and lag time of NDF were higher in the AG method compared to the GA method (P<0.05).  Placing all bags in the rumen at once and removing them at designated time intervals compared with introduction of bags in reverse sequence and removing them all at once led to a lower undegradable fraction (U) of NDF in alfalfa (1.8% vs. 4.0%, respectively; P<0.05) and corn silage (3.3% vs. 6.7%, respectively; P<0.05) samples.  Potentially degradable fraction of ADF was significantly higher in the AG method compared with the GA method (P<0.05).  Bag incubation sequence had profound effects on kinetics of degradation of DM, CP and NDF in situ in the feed samples studied.  The effects were more evident in the forages (especially alfalfa haylage) than in the concentrate ingredients

Odorant binding proteins (OBPs) in insects are postulated to solubilize and transport the hydrophobic odorants across the hydrophilic antennal lymph to the olfactory receptors (ORs) located on the dendrite membrane of the sensory neurons. OBPs in adult insects have been intensively reported, but those in larvae are rarely addressed. In our study, a full-length OBP cDNA, namely SexiOBP13, was cloned by RT-PCR and RACE strategy from the heads of Spodoptera exigua larvae. The quantitative real-time PCR (qPCR) measurement indicated that SexiOBP13 was highly expressed in larval head, but very low in other parts of larva and was not detected in any tissues of adult. The binding affinities of SexiOBP13 to plant volatiles and female sex pheromone components were measured by competitive binding assays. Interestingly, SexiOBP13 displayed a high binding affinity (Ki=3.82 μmol L–1) to Z9,E12–14:Ac, the major sex pheromone component of S. exigua, while low affinities to the tested host plant volatiles (Ki>27 μmol L–1). The behavioral tests further confirmed that Z9,E12–14:Ac was indeed active to elicit the behavioral activity of the third instar larvae of S. exigua. Taken together, our results suggest that SexiOBP13 may play a role in reception of female sex pheromone in S. exigua larvae. The ecological significance of the larvae preference to the adult female sex pheromone was discussed.

The normalization of quantitative real-time PCR (qPCR) is important to obtain accurate gene expression data, and the most common method for qPCR normalization is to use reference genes. However, reference genes can be regulated under different conditions. qPCR has recently been used for gene expression study in Laodelphax striatellus, but there is no study on validation of the reference genes. In this study, five new housekeeping genes (LstrTUB1, LstrTUB2, LstrTUB3, LstrARF and LstrRPL9) in L. striatellus were cloned and deposited in the GenBank with accession numbers of JF728809, JF728810, JF728811, JF728807 and JF728806, respectively. Furthermore, mRNA expressions of the five genes and β-actin were measured by qPCR with insect samples of different instar at nymph stage, and the expression stabilities were determined by the software geNorm and NormFinder. As a result, ARF and RPL9 were consistently more stable than β-actin, while three TUB genes were less stable than β-actin. To determine the optimal number of reference genes used in qPCR, a pairwise variations analysis by geNorm indicated that two references ARF and RPL9 were required to obtain the accurate quantification. These results were further confirmed by the validation qPCR experiment with chitinase gene as the target gene, in which the standard error of the mRNA quantification by using binary reference ARF-RPL9 was much lower than those by ARF, RPL9 or β-actin alone. Taken together, our study suggested that the combination of ARF-RPL9 could replace β-actin as the reference genes for qPCR in L. striatellus.