• Asian-Australasian Journal of Animal Sciences
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• 제26권6호
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• pp.845-855
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• 2013

Requirements of dietary chloride (dCl) and chloride salts were determined by using $4{\times}2$ factorial arrangement under four phase feeding program. Four levels (0.31, 0.45, 0.59 and 0.73%) and two sources ($NH_4Cl$ and $CaCl_2$) of the dCl were allocated to 1,472 chicks in eight dietary treatments in which each treatment was replicated four times with 46 birds per replicate. The four phase feeding program was comprised of four dietary phases: Prestarter (d 1 to 10), Starter (d 11 to 20), Grower (d 21 to 33) and Finisher (d 34 to 42); and diets were separately prepared for each phase. The cations, anions, pH, dissolved oxygen (DO), temperature, electrical conductivity (EC), total dissolved solids (TDS) and salinity were analyzed in drinking water and were not affected by dietary treatments. BW gain (BWG; $p{\leq}0.009$) and feed:gain (FG; $p{\leq}0.03$) were improved in $CaCl_2$ supplemented diets during d 1 to 10. The maximum response of BWG and FG was observed at 0.38% and 0.42% dCl, respectively, for d 34 to 42. However, the level of dCl for BWG during d 21 to 33 ($p{\leq}0.04$) and d 34 to 42 ($p{\leq}0.009$) was optimized at 0.60% and 0.42%, respectively. The level of dCl for optimized feed intake (FI; $p{\leq}0.006$), FG ($p{\leq}0.007$) and litter moisture (LM; $p{\leq}0.001$) was observed at 0.60%, 0.38% and 0.73%, respectively, for d 1 to 42. Water intake (DWI) was not affected by increasing dCl supplementation (p>0.05); however, the ratio between DWI and FI (DWI:FI) was found highest at 0.73% dCl during d 1 to 10 ($p{\leq}0.05$) and d 21 to 33 ($p{\leq}0.009$). Except for d 34 to 42 ($p{\leq}0.006$), the increasing level of dCl did not result in a significant difference in mortality during any phase. Blood pH and glucose, and breast and thigh weights (percentage of dressed weight) were improved while dressing percentage (DP) and gastrointestinal health were exacerbated with $NH_4Cl$ as compared to $CaCl_2$ supplemented diets ($p{\leq}0.001$). Higher plasma $Na^+$ and $HCO_3{^-}$ and lower $Cl^-$ and $Ca^{{+}{+}}$ were observed in $NH_4Cl$ supplemented diets ($p{\leq}0.001$). Increasing supplementation of dCl increased plasma $Cl^-$ ($p{\leq}0.04$; quadratically) and linearly reduced plasma $K^+$ ($p{\leq}0.001$), $Ca^{{+}{+}}$ ($p{\leq}0.003$), $HCO_3{^-}$ ($p{\leq}0.001$), and $Na^+$ ($p{\leq}0.001$; quadratically). Consequently, higher requirements of dietary chloride are suggested for feed intake; nevertheless, lower levels of dietary chloride are sufficient to support optimal BWG and FG with increasing age. The $NH_4Cl$ supplemented diets ameliorate breast and thigh meat yield along with overall energy balance (glucose).

• Asian-Australasian Journal of Animal Sciences
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• 제29권11호
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• pp.1653-1663
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• 2016

Meat quality is a complex trait influenced by many factors, including genetics, nutrition, feeding environment, animal handling, and their interactions. To elucidate relevant factors affecting pork quality associated with oxidative stress and muscle development, we analyzed protein expression in high quality longissimus dorsi muscles (HQLD) and low quality longissimus dorsi muscles (LQLD) from Duroc pigs by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomic analysis. Between HQLD (n = 20) and LQLD (n = 20) Duroc pigs, 24 differentially expressed proteins were identified by LC-MS/MS. A total of 10 and 14 proteins were highly expressed in HQLD and LQLD, respectively. The 24 proteins have putative functions in the following seven categories: catalytic activity (31%), ATPase activity (19%), oxidoreductase activity (13%), cytoskeletal protein binding (13%), actin binding (12%), calcium ion binding (6%), and structural constituent of muscle (6%). Silver-stained image analysis revealed significant differential expression of lactate dehydrogenase A (LDHA) between HQLD and LQLD Duroc pigs. LDHA was subjected to in vitro study of myogenesis under oxidative stress conditions and LDH activity assay to verification its role in oxidative stress. No significant difference of mRNA expression level of LDHA was found between normal and oxidative stress condition. However, LDH activity was significantly higher under oxidative stress condition than at normal condition using in vitro model of myogenesis. The highly expressed LDHA was positively correlated with LQLD. Moreover, LDHA activity increased by oxidative stress was reduced by antioxidant resveratrol. This paper emphasizes the importance of differential expression patterns of proteins and their interaction for the development of meat quality traits. Our proteome data provides valuable information on important factors which might aid in the regulation of muscle development and the improvement of meat quality in longissimus dorsi muscles of Duroc pigs under oxidative stress conditions.

• Asian-Australasian Journal of Animal Sciences
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• 제33권10호
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• pp.1544-1557
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• 2020

Objective: Genomic selection (GS) is becoming popular in animals' genetic development. We, therefore, investigated the single-step genomic best linear unbiased prediction (ssGBLUP) as tool for GS, and compared its efficacy with the traditional pedigree BLUP (pedBLUP) method. Methods: A total of 9,952 males born between 1997 and 2018 under Hanwoo proven-bull selection program was studied. We analyzed body weight at 12 months and carcass weight (kg), backfat thickness, eye muscle area, and marbling score traits. About 7,387 bulls were genotyped using Illumina 50K BeadChip Arrays. Multiple-trait animal model analyses were performed using BLUPF90 software programs. Breeding value accuracy was calculated using two methods: i) Pearson's correlation of genomic estimated breeding value (GEBV) with EBV of all animals (r_M1) and ii) correlation using inverse of coefficient matrix from the mixed-model equations (r_M2). Then, we compared these accuracies by overall population, info-type (PHEN, phenotyped-only; GEN, genotyped-only; and PH+GEN, phenotyped and genotyped), and bull-types (YBULL, young male calves; CBULL, young candidate bulls; and PBULL, proven bulls). Results: The r_M1 estimates in the study were between 0.90 and 0.96 among five traits. The r_M1 estimates varied slightly by population and info-type, but noticeably by bull-type for traits. Generally average r_M2 estimates were much smaller than r_M1 (pedBLUP, 0.40 to0.44; ssGBLUP, 0.41 to 0.45) at population level. However, r_M2 from both BLUP models varied noticeably across info-types and bull-types. The ssGBLUP estimates of r_M2 in PHEN, GEN, and PH+ GEN ranged between 0.51 and 0.63, 0.66 and 0.70, and 0.68 and 0.73, respectively. In YBULL, CBULL, and PBULL, the r_M2 estimates ranged between 0.54 and 0.57, 0.55 and 0.62, and 0.70 and 0.74, respectively. The pedBLUP based r_M2 estimates were also relatively lower than ssGBLUP estimates. At the population level, we found an increase in accuracy by 2.0% to 4.5% among traits. Traits in PHEN were least influenced by ssGBLUP (0% to 2.0%), whereas the highest positive changes were in GEN (8.1% to 10.7%). PH+GEN also showed 6.5% to 8.5% increase in accuracy by ssGBLUP. However, the highest improvements were found in bull-types (YBULL, 21% to 35.7%; CBULL, 3.3% to 9.3%; PBULL, 2.8% to 6.1%). Conclusion: A noticeable improvement by ssGBLUP was observed in this study. Findings of differential responses to ssGBLUP by various bulls could assist in better selection decision making as well. We, therefore, suggest that ssGBLUP could be used for GS in Hanwoo proven-bull evaluation program.

• 복식
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• 제15권
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• pp.177-194
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• 1990

Koh(袴) was a type of dress worn on the lower part of the body which was commonly used in the Northeast Asia. It was originally used by the Northern race for the need of nomadism or hunting. The origin of the Koh which appeared in the area would be found from the trousers of the Huns who influenced in the Northeast Asia, and became in the part of the Scythian culture. The Scythians are the nomadizing race inhabited in the Northern Caucasas on the wast of the Black Sea and influenced on the inland Eurasian steppe as the first typical horse-riding race. The objectives of Koh which had been worn in the Scythian, Mongolia, Korea as well as Japan as a part of Dongho dress and ornaments and to contemplate the transmission process by cultural exchange among different races for the period from 4th century to 7th century. 1. The Origination of the Koh The Koh was originated by the environmental factor to protect the cold in the North but also from the heat in the South, and was changed and developed as gradually satisfying to the needs of the times. In the Northeast Asia the Koh was in the class of the Northern Chinese garment, and was used widely by the horse riding Scythians who moved widely from the Eurasian inland to Japan. The oldest original which could reflect the type of the Northern clothes was a pair of trousers discovered in the Huns remains of Noin Ula. This showed the exact form of hunting clothes and had a similar form with the Korean female tro-users. Since the same form of trousers drawn on the wall painting of which was excavated 4-5th century ancient Koguryo(高句麗) tomb was the same form the trousers of Noin Ula seemed to be the original form of Koh in the Northeast Asia. 2. The Chinese Trousers It was the time of the King Mooryung(武靈王) in the Cho(趙) Dynasty B.C. 3th century that the trousers used regularly in China. However, the Koh had been used as undergarment which functioned for the protection of the cold not the horseriding garment. The trousers seemed to be not very obviously shown off since the Poh (袍) was long, but mainly used by the people from lower class. As people learned the adapted the trousers. It was essential for the times of war and quarrel. The king himself started wearing the Koh. The Chinese trousers were influenced by the Huns, the Northern clothes of the Scythian culture, and similar to the Korean clothes. 3. The Korean Trousers Korean was a race bared from the Eastern foreign group. It was obvious that the clothes was Baji-Jeogori(바지 저고리), the garment of the Northern people. This had the same form of the Scythian dress and ornaments which was excavated from the Mongolian Noin Ula. The Scythian dress and ornaments were influenced from the Ancient West Asia Empire and transmitted to the Northeast Koguryu by the horseriding Scythian. The trousers were kept in the traditional style by the common people in Korea were transmitted to Japan which were for behind in cultural aspect, as well as got used to the Chinese as the efficient clothes though active cultural exchange. 4. The Japanese Trousers The ancient Japanese clothes were influenced by the Southern factor but not the form of the Koh. As the Korean people group was moving towards Japan and conquer the Japanese in the 4-5th century, however, North Altaic culture was formed and at the same time the clothes were also developed. The most influenced clothes at this time were those of Baekge(百濟) and the trousers form called Euigon became the main form. Because of the climatic regional factor, it was tied not at the ankle but under the knee. From the view the ancient Japanese clothes disappeard about that time, it could be due to the conquest of the culturally superior race but not the transmission of the culture. In the latest 7th century both the Chinese and Japanese dress forms were present, but the Dongho(東胡) dress and its ornament from Korea was still the basic of the Japanese dress form.

• Asian-Australasian Journal of Animal Sciences
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• 제14권9호
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• pp.1260-1266
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• 2001

The effect of protein supplementation, $O_2$ concentration and co-culture on the development of embryos produced by nuclear transfer using cultured cumulus cell was investigated. Recipient oocytes and cumulus cells were obtained from the ovaries of the slaughtered Hanwoo cows. Donor cumulus cells were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum at 5% $CO_2$ in air at $38.5^{\circ}C$. The 1 to 6 passages of cumulus cells were isolated and used as donor cells. The in vitro matured oocytes were enucleated and then the isolated donor cells were introduced. One $15{\mu}s$ pulse of 180 volts was applied to induce the fusion between karyoplast and cytoplast. The fused embryos were activated with $10{\mu}M$ calcium ionophore for 5 min and 2 mM 6-dimethylaminopurine for 3 h. To examine the effect of protein supplementation, nuclear transfer (NT) embryos were cultured in one of the following 4 treatments : 1) CR1aa + 3 mg/ml BSA for 7 days ; 2) CR1aa + 10% FBS for 7 days ; 3) CR1aa + 1.5 mg/ml BSA + 5% FBS for 7 days ; and 4) CR1aa + 3 mg/ml BSA for first 3 days and then CR1aa + 1.5 mg/ml BSA + 5% FBS for 4 days. Culture took place at 5% $CO_2$, 5% $O_2$ and 90% $N_2$ at $38.5^{\circ}C$. Although there were no significant differences in cleavage rate among different protein supplements, the rates of blastocyst formation were significantly different. When NT embryos were cultured in the medium supplemented with only BSA, they could develop to only morula not to blastocyst. However, when FBS was supplemented, NT embryos developed to blastocyst stage. In order to investigate the effect of $O_2$ concentration and co-culture, NT embryos were cultured in CR1aa + 1.5 mg/ml BSA + 5% FBS with or without cumulus cell co-culture at an atmosphere of 5% $CO_2$ in air (20% $O_2$) or 5% $CO_2$, 5% $O_2$, 90% $N_2$ (5% $O_2$) at $38.5^{\circ}C$ for 7 days. The percentage of blastocyst development was significantly higher when the NT embryos were cultured at an atmosphere of 5% $O_2$ than that of 20% $O_2$ (p<0.05). However, there was no significant difference between with and without cumulus cell co-culture at an atmosphere of 5% $O_2$ or 20% $O_2$. Fifty embryos were transferred to 25 recipients and 5 recipients were pregnant at 100 days. From 5 pregnant cows, only one cow was delivered of female twin. In conclusion, the embryos reconstructed by enucleation of metaphase II oocytes and introduction of the cycling and quiescent cumulus donor cells in Hanwoo had developmental potential to term after embryo transfer to recipient cows.

• Asian-Australasian Journal of Animal Sciences
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• 제14권10호
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• pp.1450-1459
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• 2001

These experiments were conducted to investigate the effects of different feeding and processing methods of diets on performance, morphological changes in the small intestine and nutrient digestibility of growing-finishing pigs. One-hundred fifty growing pigs ($Yorkshire{\times}Landrace{\times}Duroc$; initial body weight of $23.33{\pm}0.75kg$) and one-hundred twenty finishing pigs ($Yorkshire{\times}Landrace{\times}Duroc$; initial body weight of $59.22{\pm}0.56kg$) were used in Exp. 1 and Exp. 2, respectively. Pigs were grouped on the basis of body weight and gender, and randomly allotted into 6 different treatments with 5 replications in each treatment in a $2{\times}3$ factorial arrangement. Treatments were 1) dry feeding with a mash diet (DM), 2) dry feeding with a pelleted diet (DP), 3) dry feeding with an expanded crumble diet (DEC), 4) dry/wet feeding with a mash diet (WM), 5) dry/wet feeding with a pelleted diet (WP), and 6) dry/wet feeding with an expanded crumble diet (WEC). In Exp. 1 (growing phase), there was no significant difference in average daily gain (ADG) and average daily feed intake (ADFI) among treatments during the entire experimental period, but feed conversion ratio (FCR) was significantly (p<0.05) improved in pigs fed pelleted diets regardless of feeding method. FCR was best in pigs fed a DP diet and worst in pigs fed a WM diet. Pigs fed a pelleted diet showed a 6.2% or 4.0% improvement in FCR compared with those fed a mash diet or an expanded crumble diet. Water disappearance was not significantly affected by dry/wet feeding or feed processing. Significant differences in villus height were not found among treatments, but villus height tended to be improved by dry/wet feeding. Dry/wet feeding or feed processing did not affect crypt depth. Digestibilities of calcium and phosphorus were significantly (p<0.05) improved in pigs fed an expanded crumble diet compared with pigs fed mash diets. Especially, pigs fed a WEC diet digested 8.1% more P than those fed a DM diet. Feed cost per kg weight gain (FCG) tended to be increased by dry/wet feeding rather than dry feeding. In Exp. 2 (finishing phase), ADG and ADFI were not significantly different among treatments, but a significant difference in FCR was found among feed processing forms. The best FCR was obtained in pigs fed a pelleted diet. Pigs fed a DP diet showed a 11.3% improvement compared with those fed a DEC diet. Water disappearance was significantly (p=0.0408) decreased by feeding the mash diet. However, water disappearance was not affected by dry/wet feeding during the finishing period. The villus height and crypt depth were not significantly different among treatments. However, crypt depth tended to be decreased by dry/wet feeding at the mid part of the small intestine. Fat digestibility was improved by dry feeding rather than dry/wet feeding, and was improved by 4.8% by feeding pellet diets compared with expanded crumble diets. Except for carcass grade, carcass characteristics were not significantly (p<0.05) different among treatments. Carcass grade was the best in pigs fed a WP diet. Feed cost per kg weight gain (FCG) was significantly decreased in pigs fed a pelleted diet compared with those fed an expanded crumble diet, and tended to be decreased by dry/wet feeding. In conclusion, these studies suggest that feeding the pelleted diet to growing-finishing pigs can be beneficial in terms of FCR and production cost. Dry/wet feeding can be helpful for the maintenance of villus height, but may not be reflected in improved growth performance or reduction of production costs.

• Asian-Australasian Journal of Animal Sciences
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• 제21권1호
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• pp.107-119
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• 2008

To determine the true digestible phosphorus (TDP) requirement of growing pigs, two experiments were designed with the experimental diets containing five true digestible P levels (0.16%, 0.20%, 0.23%, 0.26% and 0.39%) and the ratio of total calcium to true digestible P (TDP) kept at 2:1. In Experiment 1, five barrows (Duroc${\times}$Landrace${\times}$Yorkshire) with an average initial body weight of 27.9 kg were used in a $5{\times}5$ Latin-square design to evaluate the effect of different dietary P levels on the digestibility and output of P and nitrogen. In Experiment 2, sixty healthy growing pigs (Duroc${\times}$Landrace${\times}$Yorkshire) with an average body weight (BW) of 21.4 kg were assigned randomly to one of the five dietary treatments (12 pigs/diet), and were used to determine the true digestible phosphorus (TDP) requirement of growing pigs on the basis of growth performance and serum biochemical indices. The results indicated that the true digestibility of P increased (p<0.05) linearly with increasing dietary TDP level below 0.26%. The true P digestibility was highest (56.6%) when dietary TDP was 0.34%. Expressed as g/kg dry matter intake (DMI), fecal P output increased (p<0.05) linearly with increasing P input. On the basis of g/kg fecal dry matter (DM), fecal P output was lowest for Diet 4 and highest (p<0.05) for Diet 5. The apparent digestibility of crude protein (CP) did not differ (p>0.05) among the five diets, with the average nitrogen output of 12.14 g/d and nitrogen retention of 66% to 74% (p>0.05), which suggested that there was no interaction between dietary P and CP protein levels. During the 28-d experimental period of Experiment 2, the average daily gain (ADG) of pigs was affected by dietary TDP levels as described by Eq. (1): $y=-809,532x^4+788,079x^3-276,250x^2+42,114x-1,759$; ($R^2=0.99$; p<0.01; y = ADG, g/d; x = dietary TDP, %), F/G for pigs by Eq. (2): $y=3,651.1x^4-3,480.4x^3+1,183.8x^2-172.5x+10.9$ ($R^2=0.99$; p<0.01; y = F/G; x = dietary TDP, %), and Total P concentrations in serum by Eq. (3): $y=-3,311.7x^4+3,342.7x^3-1,224.6x^2+195.6x-8.7$ (R2 = 0.99; p<0.01; y = total serum P concentration and x = dietary TDP, %). The highest ADG (782 g/d), the lowest F/G (1.07) and the highest total serum P concentration (3.1 mmol/L) were obtained when dietary TDP level was 0.34%. Collectively, these results indicate that the optimal TDP requirement of growing pigs is 0.34% of the diet at a total Ca to TDP ratio of 2:1.

• Asian-Australasian Journal of Animal Sciences
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• 제28권5호
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• pp.605-611
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• 2015

A synthetic strain of ducks (Anas platyrhynchos) was developed by introducing genes for long duration of fertility to be used as mother of mule ducklings and a seven-generation selection experiment was conducted to increase the number of fertile eggs after a single artificial insemination (AI) with pooled Muscovy semen. Reciprocal crossbreeding between Brown Tsaiya LRI-2 (with long duration of fertility) and Pekin L-201 (with white plumage mule ducklings) ducks produced the G0. Then G1 were intercrossed to produce G2 and so on for the following generations. Each female duck was inseminated 3 times, at 26, 29, and 32 weeks of age. The eggs were collected for 14 days from day 2 after AI. Individual data regarding the number of incubated eggs (Ie), the number of fertile eggs at candling at day 7 of incubation (F), the total number of dead embryos (M), the maximum duration of fertility (Dm) and the number of hatched mule ducklings (H) with plumage colour were recorded. The selection criterion was the breeding values of the best linear unbiased prediction animal model for F. The results show high percentage of exhibited heterosis in G2 for traits to improve (19.1% for F and 12.9% for H); F with a value of 5.92 (vs 3.74 in the Pekin L-201) was improved in the G2. Heritabilities were found to be low for Ie ($h^2=0.07{\pm}0.03$) and M ($h^2=0.07{\pm}0.01$), moderately low for Dm ($h^2=0.13{\pm}0.02$), of medium values for H ($h^2=0.20{\pm}0.03$) and F ($h^2=0.23{\pm}0.03$). High and favourable genetic correlations existed between F and Dm ($r_g=0.93$), between F and H ($r_g=0.97$) and between Dm and H ($r_g=0.90$). The selection experiment showed a positive trend for phenotypic values of F (6.38 fertile eggs in G10 of synthetic strain vs 5.59 eggs in G4, and 3.74 eggs in Pekin L-201), with correlated response for increasing H (5.73 ducklings in G10 vs 4.86 in G4, and 3.09 ducklings in Pekin L-201) and maximum duration of the fertile period without increasing the embryo mortality rate. The average predicted genetic response for F was 40% of genetic standard deviation per generation of selection. The mule ducklings' feather colour also was improved. It was concluded that this study provided results for a better understanding of the genetics of the duration of fertility traits in the common female duck bred for mule and that the selection of a synthetic strain was effective method of improvement.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.500-508
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• 2016

This study was conducted to investigate the effects of Momordica charantia saponin (MCS) on ruminal fermentation of maize stover and abundance of selected microbial populations in vitro. Five levels of MCS supplements (0, 0.01, 0.06, 0.30, 0.60 mg/mL) were tested. The pH, $NH_3-N$, and volatile fatty acid were measured at 6, 24, 48 h of in vitro mixed incubation fluids, whilst the selected microbial populations were determined at 6 and 24 h. The high dose of MCS increased the initial fractional rate of degradation at t-value = 0 ($FRD_0$) and the fractional rate of gas production (k), but decreased the theoretical maximum of gas production ($V_F$) and the half-life ($t_{0.5}$) compared with the control. The $NH_3-N$ concentration reached the lowest concentration with 0.01 mg MCS/mL at 6 h. The MSC inclusion increased (p<0.001) the molar proportion of butyrate, isovalerate at 24 h and 48 h, and the molar proportion of acetate at 24 h, but then decreased (p<0.05) them at 48 h. The molar proportion of valerate was increased (p<0.05) at 24 h. The acetate to propionate ratio (A/P; linear, p<0.01) was increased at 24 h, but reached the least value at the level of 0.30 mg/mL MCS. The MCS inclusion decreased (p<0.05) the molar proportion of propionate at 24 h and then increased it at 48 h. The concentration of total volatile fatty acid was decreased (p<0.001) at 24 h, but reached the greatest concentration at the level of 0.01 mg/mL and the least concentration at the level of 0.60 mg/mL. The relative abundance of Ruminococcus albus was increased at 6 h and 24 h, and the relative abundance of Fibrobacter succinogenes was the lowest (p<0.05) at 0.60 mg/mL at 6 h and 24 h. The relative abundance of Butyrivibrio fibrisolvens and fungus reached the greatest value (p<0.05) at low doses of MCS inclusion and the least value (p<0.05) at 0.60 mg/mL at 24 h. The present results demonstrates that a high level of MCS quickly inhibits in vitro fermentation of maize stover, while MCS at low doses has the ability to modulate the ruminal fermentation pattern by regulating the number of functional rumen microbes including cellulolytic bacteria and fungi populations, and may have potential as a feed additive applied in the diets of ruminants.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.487-499
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• 2016

The aim of the present study was to examine the effects of testosterone (T) and estradiol-$17{\beta}$ ($E_2$) on the production of progesterone ($P_4$) by granulosa cells, and of the $E_2$ on the production of $P_4$ and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest ($F_1$) and third largest ($F_3$) preovulatory follicle were incubated for 4 h in short-term culture system, $P_4$ production by granulosa cells of both $F_1$ and $F_3$ was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or $E_2$. In the second experiment, $F_1$ and $F_3$ granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and $E_2$. Basal $P_4$ production for 48 h during 48 to 96 h of the cultured was about nine fold greater by $F_1$ granulosa cells than by $F_3$ granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not $E_2$, stimulated in a dose-dependent manner $P_4$ production in both $F_1$ and $F_3$ granulosa cells. In addition, when the time course of $P_4$ production by $F_1$ granulosa cells in response to oLH, cVIP, T and $E_2$ was examined for 48 h during 48 to 96 h of culture, although $E_2$ had no effect on $P_4$ production by granulosa cells of $F_1$ during the period from 48 to 96 h of culture, $P_4$ production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, $P_4$ production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when $F_1$ granulosa cells were precultured with $E_2$ for various times before 4 h culture with oLH at 96 h of culture, the increase in $P_4$ production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of $F_1$, $F_2$ and the largest third to fifth preovulatory follicles ($F_{3-5}$) were incubated for 4 h in short-term culture system with increasing doses of $E_2$. The production of $P_4$ and T by theca internal cells were increased with the addition of $E_2$ of $10^{-6}M$. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on $P_4$ production, and on $E_2$ in long-term action which may enhance the sensitivity to LH for $P_4$ production, and thus, in theca internal cells, $E_2$ in short term action may stimulate the production of $P_4$ and T.