- Editorial - Guidelines for experimental design and statistical analyses in animal studies submitted for publication in the Asian-Australasian Journal of Animal Sciences.

Animal experiments are essential to the study of animal nutrition. Because of the large variations among individual animals and ethical and economic constraints, experimental designs and statistical analyses are particularly important in animal experiments. To increase the scientific validity of the results and maximize the knowledge gained from animal experiments, each experiment should be appropriately designed, and the observations need to be correctly analyzed and transparently reported. There are many experimental designs and statistical methods. This editorial does not aim to review and present particular experimental designs and statistical methods. Instead, we discuss some essential elements when designing an animal experiment and conducting statistical analyses in animal nutritional studies and provide guidelines for submitting a manuscript to the Asian-Australasian Journal of Animal Sciences for consideration for publication.

Molecular cloning, purification, expression, and characterization of ß-1, 4-endoglucanase gene (Cel5A) from Eubacterium cellulosolvens sp. isolated from Holstein steers' rumen.

OBJECTIVE: This study was conducted to isolate the cellulolytic microorganism from the rumen of Holstein steers and characterize endoglucanase gene (Cel5A) from the isolated microorganism. METHODS: To isolate anaerobic microbes having endoglucanase, rumen fluid was obtained from Holstein steers fed roughage diet. The isolated anaerobic bacteria had 98% similarity with Eubacterium cellulosolvens (E. cellulosolvens) Ce2 (Accession number: AB163733). The Cel5A from isolated E. cellulolsovens sp. was cloned using the published genome sequence and expressed through the Escherichia coli BL21. RESULTS: The maximum activity of recombinant Cel5A (rCel5A) was observed at 50°C and pH 4.0. The enzyme was constant at the temperature range of 20°C to 40°C but also, at the pH range of 3 to 9. The metal ions including Ca2+, K+, Ni2+, Mg2+, and Fe2+ increased the endoglucanase activity but the addition of Mn2+, Cu2+, and Zn2+ decreased. The Km and Vmax value of rCel5A were 14.05 mg/mL and 45.66 µmol/min/mg. Turnover number, Kcat and catalytic efficiency, Kcat/Km values of rCel5A was 96.69 (s-1) and 6.88 (mL/mg/s), respectively. CONCLUSION: Our results indicated that rCel5A of E. cellulosolvens isolated from Holstein steers had a broad pH range with high stability under various conditions, which might be one of the beneficial characteristics of this enzyme for possible industrial application.

Production of Endoglucanase, Beta-glucosidase and Xylanase by Bacillus licheniformis Grown on Minimal Nutrient Medium Containing Agriculture Residues.

Bacillus licheniformis was grown in minimal nutrient medium containing 1% (w/v) of distillers dried grain with soluble (DDGS), palm kernel meal (PKM), wheat bran (WB) or copra meal (CM), and the enzyme activity of endoglucanase, ß-glucosidase, xylanase and reducing sugars was measured to investigate a possibility of using cost-effective agricultural residues in producing cellulolytic and hemicellulolytic enzymes. The CM gave the highest endoglucanase activity of 0.68 units/mL among added substrates at 48 h. CM yielded the highest titres of 0.58 units/ml of ß-glucosidase, compared to 0.33, 0.23, and 0.16 units/mL by PKM, WB, and DDGS, respectively, at 72 h. Xylanase production was the highest (0.34 units/mL) when CM was added. The supernatant from fermentation of CM had the highest reducing sugars than other additional substrates at all intervals (0.10, 0.12, 0.10, and 0.11 mg/mL respectively). It is concluded that Bacillus licheniformis is capable of producing multiple cellulo- and hemicellololytic enzymes for bioethanol production using cost-effective agricultural residues, especially CM, as a sole nutrient source.

Effects of Methylcellulose on Fibrolytic Bacterial Detachment and In vitro Degradation of Rice Straw.

Two in vitro experiments were conducted to evaluate the effect of methylcellulose (MC) on i) bacterial detachment from rice straw as well as ii) inhibition of bacterial attachment and fiber digestibility. To evaluate the effect of MC on fibrolytic bacterial detachment (Exp 1), in vitro bacterial cultures with 0.1% (w/v) MC solution were compared with cultures without MC after 8 h incubation. The effect of MC on inhibition of bacterial attachment was determined by comparing with real-time PCR the populations of F. succinogenes, R. flavefaciens and R. albus established on rice straw pre-treated with 0.1% MC with those on untreated straw after incubation for 0, 6 and 12 h (Exp 2). The major fibrolytic bacterial attachment on rice straw showed significantly lower populations with either the addition of MC to the culture or pre-treated rice straw compared to controls (p<0.05). Also, the digestibility of rice straw with MC was significantly lower compared with control (p<0.05). The F. succinogenes population did not show detachment from rice straw, but showed an inhibition of attachment and proliferation on rice straw in accordance with a decrease of fiber digestion. The detachments of Ruminococcus species co-existed preventing the proliferations with subsequent reduction of fiber degradation by MC during the incubation. Their detachments were induced from stable colonization as well as the initial adhesion on rice straw by MC in in vitro ruminal fermentation. Furthermore, the detachment of R. albus was more sensitive to MC than was R. flavefaciens. These results showed the certain evidence that attachment of major fibrolytic bacteria had an effect on fiber digestion in the rumen, and each of fibrolytic bacteria, F. succinogenes, R. flavefaciens and R. albus had a specific mechanism of attachment and detachment to fiber.

Effects of Methylcellulose on Cellulolytic Bacteria Attachment and Rice Straw Degradation in the In vitro Rumen Fermentation.

An in vitro experiment was conducted to evaluate the effect of methylcellulose on the attachment of major cellulolytic bacteria on rice straw and its digestibility. Rice straw was incubated with ruminal mixture with or without 0.1% methylcellulose (MC). The attachment of F. succinogenes, R. flavefaciens and R. albus populations on rice straw was measured using real-time PCR with specific primer sets. Methylcellulose at the level of 0.1% decreased the attachment of all three major cellulolytic bacteria. In particular, MC treatment reduced (p<0.05) attachment of F. succinogenes on rice straw after 10 min of incubation while a significant reduction (p<0.05) in attachment was not observed until 4 h incubation in the case of R. flavefaciens and R. albus. This result indicated F. succinogenes responded to MC more sensitively and earlier than R. flavefaciens and R. albus. Dry matter digestibility of rice straw was subsequently inhibited by 0.1% MC, and there was a significant difference between control and MC treatment (p<0.05). Incubated cultures containing MC had higher pH and lower gas production than controls. Current data clearly indicated that the attachment of F. succinogenes, R. flavefaciens and R. albus on rice straw was inhibited by MC, which apparently reduced rice straw digestion.

Characterization of Cellulolytic and Xylanolytic Enzymes of Bacillus licheniformis JK7 Isolated from the Rumen of a Native Korean Goat.

A facultative bacterium producing cellulolytic and hemicellulolytic enzymes was isolated from the rumen of a native Korean goat. The bacterium was identified as a Bacillus licheniformis on the basis of biochemical and morphological characteristics and 16S rDNA sequences, and has been designated Bacillus licheniformis JK7. Endoglucanase activities were higher than those of ß-glucosidase and xylanase at all temperatures. Xylanase had the lowest activity among the three enzymes examined. The optimum temperature for the enzymes of Bacillus licheniformis JK7 was 70°C for endoglucanase (0.75 U/ml) and 50°C for ß-glucosidase and xylanase (0.63 U/ml, 0.44 U/ml, respectively). All three enzymes were stable at a temperature range of 20 to 50°C. At 50°C, endoglucanse, ß-glucosidase, and xylanase had 90.29, 94.80, and 88.69% residual activity, respectively. The optimal pH for the three enzymes was 5.0, at which their activity was 1.46, 1.10, and 1.08 U/ml, respectively. The activity of all three enzymes was stable in the pH range of 3.0 to 6.0. Endoglucanase activity was increased 113% by K(+), while K(+), Zn(+), and tween 20 enhanced ß-glucosidase activity. Xylanase showed considerable activity even in presence of selected chemical additives, with the exception of Mn(2+) and Cu(2+). The broad range of optimum temperatures (20 to 40°C) and the stability under acidic pH (4 to 6) suggest that the cellulolytic enzymes of Bacillus licheniformis JK7 may be good candidates for use in the biofuel industry.

Effects of synchronicity of carbohydrate and protein degradation on rumen fermentation characteristics and microbial protein synthesis.

A series of in vitro studies were carried out to determine i) the effects of enzyme and formaldehyde treatment on the degradation characteristics of carbohydrate and protein sources and on the synchronicity of these processes, and ii) the effects of synchronizing carbohydrate and protein supply on rumen fermentation and microbial protein synthesis (MPS) in in vitro experiments. Untreated corn (C) and enzyme-treated corn (EC) were combined with soy bean meal with (ES) and without (S) enzyme treatment or formaldehyde treatment (FS). Six experimental feeds (CS, CES, CFS, ECS, ECES and ECFS) with different synchrony indices were prepared. Highly synchronous diets had the greatest dry matter (DM) digestibility when untreated corn was used. However, the degree of synchronicity did not influence DM digestibility when EC was mixed with various soybean meals. At time points of 12 h and 24 h of incubation, EC-containing diets showed lower ammonia-N concentrations than those of C-containing diets, irrespective of the degree of synchronicity, indicating that more efficient utilization of ammonia-N for MPS was achieved by ruminal microorganisms when EC was offered as a carbohydrate source. Within C-containing treatments, the purine base concentration increased as the diets were more synchronized. This effect was not observed when EC was offered. There were significant effects on VFA concentration of both C and S treatments and their interactions. Similar to purine concentrations, total VFA production and individual VFA concentration in the groups containing EC as an energy source was higher than those of other groups (CS, CES and CFS). The results of the present study suggested that the availability of energy or the protein source are the most limiting factors for rumen fermentation and MPS, rather than the degree of synchronicity.

Effect of γ-Aminobutyric Acid (GABA) Producing Bacteria on In vitro Rumen Fermentation, Biogenic Amine Production and Anti-oxidation Using Corn Meal as Substrate.

The effects and significance of γ-amino butyric acid (GABA) producing bacteria (GPB) on in vitro rumen fermentation and reduction of biogenic amines (histamine, methylamine, ethylamine, and tyramine) using corn meal as a substrate were determined. Ruminal samples collected from ruminally fistulated Holstein cows served as inoculum and corn was used as substrate at 2% dry matter (DM). Different inclusion rates of GPB and GABA were evaluated. After incubation, addition of GPB had no significant effect on in vitro fermentation pH and total gas production, but significantly increased the ammonia nitrogen (NH3-N) concentration and reduced the total biogenic amines production (p<0.05). Furthermore, antioxidation activity was improved as indicated by the significantly higher concentration of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) among treated samples when compared to the control (p<0.05). Additionally, 0.2% GPB was established as the optimum inclusion level. Taken together, these results suggest the potential of utilizing GPB as feed additives to improve growth performance in ruminants by reducing biogenic amines and increasing anti-oxidation.

Leadership of AAAP scientists and journals in animal science: achievements, limitations, and challenges.

The Asian-Australasian Association of Animal Production Societies (AAAP), the only international scientific organization in animal science representing Asia and Pacific region, showed a remarkable development since its inception in 1980. The number of member countries increased from 8 to 19 while 19 congresses were held in 11 different member countries. The AAAP also helped creating the official journal, Asian-Australasian Journal of Animal Sciences in 1988 with the title being changed to Animal Bioscience in 2021. It is an open access journal indexed by most global databases and has become one of the most respected global journals in animal science. Through scientific meetings and journal publication, the AAAP has made a tremendous contribution to the advancement of animal science and industry throughout its member countries over the last 40 years. This paper summarizes the advances in animal science in the Asia-Pacific region, together with the roles of the AAAP scientists and journals in developing animal science.

Isolation, screening and identification of Swine gut microbiota with ochratoxin a biodegradation ability.

The potential for ochratoxin A (OTA) degradation by swine intestinal microbiota was assessed in the current study. Intestinal content that was collected aseptically from swine was spiked with 100 ppb OTA and incubated for 6 and 12 h at 39°C. An OTA assay was conducted using the incubated samples, and it was found that 20% of the OTA toxin was detoxified, indicating the presence of microbes capable of OTA degradation. Twenty-eight bacterial species were isolated anaerobically in M 98-5 media and 45 bacterial species were isolated using nutrient broth aerobically. Screening results showed that one anaerobic bacterial isolate, named MM11, detoxified more than 75% of OTA in liquid media. Furthermore, 1.0 ppm OTA was degraded completely after 24 h incubation on a solid 'corn' substrate. The bacterium was identified by 16S rDNA sequencing as having 97% sequence similarity with Eubacterium biforme. The isolation of an OTA-degrading bacterium from the swine natural flora is of great importance for OTA biodegradation and may be a valuable potential source for OTA-degradation enzymes in industrial applications.

Effects of synchronization of carbohydrate and protein supply in total mixed ration with korean rice wine residue on ruminal fermentation, nitrogen metabolism and microbial protein synthesis in holstein steers.

Three Holstein steers in the growing phase, each with a ruminal cannula, were used to test the hypothesis that the synchronization of the hourly rate of carbohydrate and nitrogen (N) released in the rumen would increase the amount of retained nitrogen for growth and thus improve the efficiency of microbial protein synthesis (EMPS). In Experiment 1, in situ degradability coefficients of carbohydrate and N in feeds including Korean rice wine residue (RWR) were determined. In Experiment 2, three total mixed ration (TMR) diets having different rates of carbohydrate and N release in the rumen were formulated using the in situ degradability of the feeds. All diets were made to contain similar contents of crude protein (CP) and neutral detergent fiber (NDF) but varied in their hourly pattern of nutrient release. The synchrony index of the three TMRs was 0.51 (LS), 0.77 (MS) and 0.95 (HS), respectively. The diets were fed at a restricted level (2% of the animal's body weight) in a 3×3 Latin-square design. Synchronizing the hourly supply of energy and N in the rumen did not significantly alter the digestibility of dry matter, organic matter, crude protein, NDF or acid detergent fiber (ADF) (p>0.05). The ruminal NH3-N content of the LS group at three hours after feeding was significantly higher (p<0.05) than that of the other groups; however, the mean values of ruminal NH3-N, pH and VFA concentration among the three groups were not significantly different (p>0.05). In addition, the purine derivative (PD) excretion in urine and microbial-N production (MN) among the three groups were not significantly different (p>0.05). In conclusion, synchronizing dietary energy and N supply to the rumen did not have a major effect on nutrient digestion or microbial protein synthesis (MPS) in Holstein steers.

Effects of dietary potential Acid production value on productivity in dairy cows.

This study was conducted to estimate the potential acid production value (PAPV) of major diets and to determine the relationship between dietary PAPV and dairy production traits. Estimation of PAPV of major cattle feeds was based on an in vitro technique, which determined the degree of Ca dissociation from CaCO3. Data on feeds and production traits were collected on 744 multiparous lactating Holstein dairy cows from five different farms. Grains had high PAPV with variable protein sources and by-products. High PAPV feedstuffs had a higher total gas production and lower pH compared to those with low PAPV. Dietary PAPV had a positive correlation with intake of dry matter, NDF, ADF, milk yield and milk solid production but a negative correlation with milk protein and milk fat concentration. Current results indicate that dietary PAPV can be utilized in predicting dairy production traits.

Effect of Total Mixed Ration with Fermented Feed on Ruminal In vitro Fermentation, Growth Performance and Blood Characteristics of Hanwoo Steers.

In this study, two experiments were conducted to evaluate the total mixed ration with fermented feed (TMRF) and total mixed ration (TMR) by rumen in vitro fermentation and their effects on the growth performance and blood characteristics of Hanwoo steers. In experiment 1, three Hanwoo steers (600±47 kg), each permanently fitted with a ruminal cannula were used. In this experiment, three diets designated as T1, TMRF (18.4% fermented feed, tall fescue, mammoth wild rye forage and whole crop barley); T2, TMRF (17.7% fermented feed, rice straw and whole crop barley); and T3, TMR (rice straw, whole crop barley and probiotics, but no fermented feed), which were subjected to rumen in vitro fermentation for 48 h. The results demonstrated that DM disappearance rate gradually increased with advancing fermentation time, but T1 and T2 were higher than the T3 (p<0.05) from 3 h to 12 h, but insignificant (p>0.05) at 24 and 48 h. None of the specific VFAs were affected except for acetic and non volatile lactic acids, which were produced more in T2 than in T1 and T3 at 24 h and 48 h of incubation. A/P was lower in T1 and T2 than inT3 at 24 h (p<0.05) and 48 h (p>0.05) of incubation. These results confirmed that TMRF-related treatment shows a superior performance to that of TMR during the ruminal fermentation period. In experiment 2, the three diets in experiment 1 plus 1 more control diet (concentrates, probiotics and 2% rice straw of body weight) were fed to the 48 Hanwoo steers (160±10 kg) for a period of 168 d. The results demonstrated that the daily and total live weight gain and feed efficiency were higher (p<0.05) in the TMRF and TMR groups than in the control group. SGOT, SGPT and BUN (p<0.05) were reduced in TMRF relative to the control and TMR groups by 168 d which confirmed that TMRF shows better blood profiles than the TMR and control groups. Overall, these results appear to show that TMRF has better in vitro ruminal characteristics than those of TMR; growth performance and blood profiles were also found to be superior in TMRF than in the TMR and control groups. Thus, our findings suggest that TMRF-based feed supplies are favorable for Hanwoo cattle.

Hemato-biochemical and Cortisol Profile of Holstein Growing-calves Supplemented with Vitamin C during Summer Season.

Effect of vitamin C (VC) on biochemical, hematological and cortisol profile of growing Holstein calves during summer was investigated. Eighteen calves between 14 and 16 weeks of age were divided equally into two groups. One group received a diet supplemented with VC (20 g/d) for 60 days, while other non-supplemented diet fed group served as a control (CON). The temperature humidity index (THI) was recorded and computed during the experiment. From days 0 to 60, the THI exceeded 70. Blood samples were collected from the jugular vein of each calf at days 0, 15, 30, 45 and 60. Serum albumin and total protein decreased (p<0.05) in CON and VC calves with age. Serum glutamic-oxaloacetic transaminase concentrations were not affected by treatments. Serum creatinine, albumin and glutamic-pyruvic transaminase concentrations were higher in calves in the VC group than the CON group. While red blood cells, hemoglobin and hematocrit were lower (p<0.05) in VC calves, mean corpuscular volume, mean corpuscular hemoglobin, red blood cell distribution width and mean platelet volume were higher (p<0.05) in these VC supplemented calves. Leukocyte parameters including white blood cells and full term for lymphocytes were not affected by the treatments. Also, serum cortisol was not affected by treatments. At day 15, 30 and 45, the total VC in plasma was higher (p<0.05) in calves fed with VC. In conclusion, serum cortisols were not affected by plasma VC concentration, while some blood parameters were positively influenced in calves fed with VC.

Comparative study on the aflatoxin B1 degradation ability of rumen fluid from Holstein steers and Korean native goats.

The aflatoxin B1 degrading abilities of two different ruminants were compared in this study. One set of experiments evaluated the aflatoxin B1 degradation ability of different rumen fluid donors (steers vs. goats) as well as the rumen fluid filtration method (cheese cloth filtered vs. 0.45 microm Millipore) in a 2 x 2 factorial arrangement. Additional studies examined aflatoxin B1 degradation by collecting rumen fluid at different times (0, 3, 6, 9 and 12 h) after feeding. Cannulated Holstein steers (740 +/- 10 kg bw) and Korean native goats (26 +/- 3 kg bw) were fed a 60% timothy and 40% commercial diet with free access to water. Rumen fluid from Korean native goats demonstrated higher (p < 0.01) aflatoxin B1 degradability than Holstein steers. However, filtration method had no significant influence on degradability. In addition, aflatoxin degradation did not depend upon rumen fluid collection time after feeding, as no significant differences were observed. Finally, a comparison of two types of diet high in roughage found aflatoxin degradability in goats was higher with timothy hay opposed to rice straw, although individual variation existed. Thus, our findings showed the aflatoxin degradability is comparatively higher in goats compared to steers.

Effects of road transportation on metabolic and immunological responses in Holstein heifers.

This study examined the effects of road transportation on metabolic and immunological responses in dairy heifers. Twenty Holstein heifers in early pregnancy were divided into non-transported (NT; n = 7) and transported (T; n = 13) groups. Blood was collected before transportation (BT), immediately after transportation for 100 km (T1) and 200 km (T2), and 24 h after transportation (AT). The T heifers had higher (P < 0.05) blood cortisol and non-esterified fatty acid concentrations after T1 and T2 than did NT heifers. By contrast, the T heifers had lower (P < 0.05) serum triglyceride concentrations after T1 and T2 than had the NT heifers. The serum cortisol and triglyceride concentrations returned (P > 0.05) to the BT concentrations at 24 h AT in the T heifers. The granulocyte-to-lymphocyte ratio and the percentage of monocytes were higher (P < 0.05) after T2 in the T heifers than in the NT heifers, suggesting that transportation stress increased the numbers of innate immune cells. T heifers had higher (P < 0.01) plasma haptoglobin concentrations than NT heifers 24 h AT. In conclusion, transportation increased cortisol secretion and was correlated with increased metabolic responses and up-regulation of peripheral innate immune cells in dairy heifers.

The stress of weaning influences serum levels of acute-phase proteins, iron-binding proteins, inflammatory cytokines, cortisol, and leukocyte subsets in Holstein calves.

The purpose of our study was to investigate changes in immunological parameters induced by weaning stress (including milk restriction) in calves. Fifteen Holstein calves were subjected to weaning at 6 weeks of age. Blood samples were collected at -14, -7, -2, 1, 3, and 5 days post-weaning (DPW; 0 DPW = 42 days). Weaning caused significant (p < 0.01) increases in the neutrophil (NE):lymphocyte (LY) ratio at 5 DPW with a significant (p < 0.05) reduction of LYs. The concentration of acute-phase proteins (haptoglobin and serum amyloid A) also increased significantly (p < 0.05) at 3 and 5 DPW compared to -2 DPW. Levels of the iron-binding protein lactoferrin decreased significantly (p < 0.05) after weaning. Serum tumor necrosis factor-α and cortisol levels were elevated (p < 0.05) at 3 DPW, while those of serum interferon-γ decreased (p < 0.05) at 1 and 3 DPW compared to levels observed before weaning. Weaning significantly (p < 0.05) decreased the percentage of CD25(+) T cells in the peripheral blood. In conclusion, weaning stress affected the NE:LY ratio along with the levels of acute phase proteins, lactoferrin, cortisol, and inflammatory cytokines in the peripheral blood of calves. Weaning stress may induce an acute phase response possibly through the elevation of cortisol production and modulation of inflammatory cytokines.