The objective of this study was to compare the feeding effects of imported timothy hay, domestic Italian ryegrass silage (IRGS) and IRG dried by hot-air dry system (IRGHDS) under basic total mixed ration (bTMR) on rumination activity, milk production, and milk composition in lactating dairy cows. Eighteen Holstein dairy cows were divided into three groups: control (bTMR + imported timothy hay), treatment 1 (bTMR + IRGS) and treatment 2 (TMR + IRGHDS) groups. The study was conducted over a total period of 24 days, including a 10 days adaptation period and a 14 days main experimental period. The results indicate body weight was not significantly different between the three groups (p>0.05), and rumination time was significantly higher in the treatment 1 group compared with the control group (p<0.001). Milk fat, protein, and lactose were not significantly different between the three groups (p>0.05), however, somatic cell counts were significantly lower in the treatment 1 group compared with the control group (p<0.001). As a result of the economic feasibility analysis, it is expected that feeding TMR using IRGS and IRGHDS will increase profit by 402.8 won/day and 331.4 won/day per cow compared to imported timothy hay. Therefore, IRGS and IRGHDS can be used as substitutes for imported timothy hay in feeding lactating dairy cows.

The objective of this study was to investigate the comparison of feeding a total mixed ration (TMR) containing imported alfalfa hay and TMR containing hot-air dried domestic alfalfa on rumination activity, milk production, and composition in lactating dairy cows. Ten Holstein dairy cows were divided into two groups: control (imported alfalfa hay + TMR) and treatment (hot-air dried domestic alfalfa + TMR) groups. The study was conducted over a total period of 18 days, including a 13-days adaptation period and a 5-days main experimental period. The results indicated no differences in total digestible nutrient and net energy intake between the imported and hot-air dried alfalfa. Body weight was not significantly different between the two groups (p>0.05), however, total feed intake and rumination time were significantly and tendentially higher in the treatment group compared with control group, respectively (p<0.001; p=0.075). Milk yield was not significantly different between the two groups (p>0.05), however, milk fat (kg) and lactose (%) concentration were significantly higher in the treatment group compared with control group (p=0.016; p=0.02). This study confirms that feeding TMR with hot-air dried domestic alfalfa results in no differences of feed intake, rumination activity, and milk productivity. Therefore, it is considered that hot-air dried domestic alfalfa can be used as a substitute for imported alfalfa on lactating dairy cows.

Background: Sperm quality and the number of sperm introduced into the uterus during artificial insemination (AI) are pivotal factors influencing pregnancy outcomes. However, there have been no reports on the relationship between sperm concentration at AI and sperm quality in Hanwoo cattle. In this study, we examined sperm quality and pregnancy rates after AI using sperm inseminated at different concentrations. Methods: We evaluated the motility, viability, and acrosomal membrane integrity of sperm at different concentrations (10, 15, 18, and 20 million sperm/straw) in 0.5-mL straws. Subsequently, we compared the pregnancy rates after AI with different sperm concentrations. Results: After freeze-thawing, sperm at the assessed concentrations showed similar viability and acrosomal membrane integrity. After AI, cattle in the 10 million group had significantly lower pregnancy rates compared to those in the 18 and 20 million groups. Conversely, there were no statistically significant variances observed between cattle in the 10 and 15 million groups. Conclusions: Sperm at concentrations of 10, 15, 18 and 20 million per straw exhibited comparable motility, viability, and acrosomal membrane integrity. However, a concentration of at least 18 million sperm per straw is required to achieve a consistent rate of pregnancy rate in Hanwoo cattle after AI.

This study divided the area capable of producing domestic forage into grazing pasture, hay production area, and silage crop area, calculated the required area according to the forage production volume, and examined whether self-sufficiency in forage leads to cost savings. When the self-sufficiency rate of forage for dairy cows and Hanwoo is 80%, the improvement in profitability per heaf ranges from 3% to 9%, typically around 5%, which is considered a significant benefit for both corporate and individual businesses. The average profit per ranch is expected to increase about KRW 50 million per year, and the country as a whole is expected to reduce forage costs by KRW 0.9 trillion per year. Recently, efforts are being made by the government and local authorities to cultivate summer forage at the rice fields for improving self-sufficiency in forage feed to stabilize rice supply and demand. Furthermore, it is also necessary to conduct research on reducing the cost of concentrated feed and TMR (Total mixed ration).

Background: Estrus in cows can be detected through vaginal electrical resistance or conductivity. However, there are no studies measuring vaginal electrical resistance in Hanwoo cows. This study aims to measure the vaginal electrical resistance value in Hanwoo cows and compare it with estrus and ovulation. Methods: Vaginal electrical resistance values of 73 Hanwoo cows were measured before and after estrus at the Gyeongsangbuk-do Livestock Research Institute. Measurements were taken on days -6, -3, -2, -1, 0, 1, 2, 3, and 6 of artificial insemination. Large follicles and ovulation were confirmed using transvaginal ultrasonography. Results: The vaginal electrical resistance averaged 225.6 ± 6.3 Ω days before the artificial insemination date, decreasing until the day of artificial insemination. The average vaginal electrical resistance was 163.7 ± 4.6 Ω on the date of artificial insemination, and 188.8 ± 4.3 Ω one day after artificial insemination, when large follicles were observed. In addition, on the 6th day after artificial insemination, the vaginal electrical resistance averaged 231.4 ± 5.5, which was similar to the 6th day before artificial insemination (222.5 ± 6.3). Transvaginal ultrasonography showed that most of the cows ovulated one day after artificial insemination. Conclusions: The accuracy of estrus is high if the vaginal electrical resistance is measured for cows with confirmed estrus, making is a potentially useful for determining the timing of artificial insemination.

Background: Reproductive management practices play crucial roles to maximize the reproductive performance of cows, and thus contribute to farm profitability. We aimed to assess the reproductive management of cows currently practiced in the dairy farms in an urban farming system. Methods: A total of 62 dairy farms were randomly selected considering all size of farms such as small (1-5 cattle), medium (6-20 cattle) and large farms (> 20 cattle) from selected areas of Dhaka city in Bangladesh. The reproductive managementrelated parameters viz. estrus detection, breeding method, pregnancy diagnosis, dry cow and parturition management, vaccination and treatment of reproductive problems etc. were obtained in a pre-defined questionnaire during the farm visit. Results: The visual observation method was only used (100.0%; 62/62) for estrus detection irrespective of size of the farms; while farmers observed cows for estrus 4-5 times a day, but only for 20-60 seconds each time. Regardless of farm size, 89.0% (55/62) farms used artificial insemination (AI) for breeding the cows. Intriguingly, all farms (100.0%) routinely checked the cows for pregnancy at 35-40 days post-breeding using rectal palpation technique by registered veterinarian. However, only 6.5% (4/62) farms practiced dry cow management. Notably, all farms (100.0%) provided nutritional supplements (Vit D, Ca and P) during late gestation. However, proper hygiene and cleanliness during parturition was not practiced in 77.4% (48/62) farms; even though 96.7% (60/62) farms treated cows by registered veterinarian for parturition-related problems. Conclusions: While farmers used AI service for breeding and timely check their cows for pregnancy; however, they need to increase observation time (30 minutes/ observation, twice in a day: early morning and early night) for estrus detection, consider dry cow management and ensure hygienic parturition for maximizing production.

This study was performed to investigate immune changes by comparing the proportion and function of immune cells in the blood under high-temperature period and convalescence temperature period in Holstein dairy cows. The experiment was conducted using Holstein dairy cows of five animals per group (60 ± 20 months old, 175 ± 78 non-day) from the National Institute of Animal Science at high-temperature period (THI: 76 ± 1.2) and convalescence temperature period (THI: 66 ± 1.3). Complete blood count results showed no change in the number of immune cells between groups. In the analysis using Flow Cytometry of PBMCs, no significant differences were observed among B cells, Helper T cells, cytotoxic T cells, and γδ T cells between groups. However, there was an increase in Th17 cells producing IL-17a, while Th1 cells decreased during the convalescence temperature period. The results of gene expression analysis using qRT-PCR in PBMCs revealed an increase in IL-10 during the convalescence temperature period, while a decrease in HSP70 and HSP90 was observed. In conclusion, the increased expression of IL-10 and the decrease in HSP expression suggest the possibility of a weak recovery from heat stress. However, the lack of observed changes in B cells, T cells, and other immune cells indicates incomplete recovery from heat stress during the convalescence temperature period.

The impact of heat stress (HS) on reproductive performance and its problems in cows remains to be investigated in Bangladesh. The study was, therefore, aimed to evaluate the effect of HS on fertility and reproductive health problems of dairy cows in a selected area of Bangladesh. A total of 1,095 cows from 500 farms were included in this study. The climate-related data were recorded daily basis for every month in a year and temperature-humidity index (THI) values were calculated to determine the level of HS. Concurrently, data on fertility parameters [number of services per conception (NSC), conception rate (CR) and calving to the first service interval (CFSI)], and reproductive problems were collected through a pre-defined questionnaire. The results show that crossbred [Native x Holstein-Friesian, (HF)] cows were more vulnerable (p < 0.05) to a magnitude of HS effects considering physiological parameters of cows [age, body condition score (BCS), parity and milk yield]. Regarding fertility, HS had no effect on NSC, CR and CFSI in both native and crossbred cows (p > 0.05). The findings on the reproductive problems indicate that HS had significant influence on the prevalence of anestrus (χ2 = 21.814, p < 0.05) and retained placenta (χ2 = 24.632, p < 0.05) in cows. Of note, the prevalence of repeat breeding syndrome was 2.5 folds higher in stress condition than in no stress condition. Abortion and dystocia were not influenced by HS. In conclusion, HS does not influence the fertility parameters of cows studied; however, anestrus and retained placenta are likely to occur under HS conditions in cows.

This study investigated changes of milk production in dairy cows intramuscularly injected with drugs containing dexamethasone (DXM). Three types of dexamethasone formulations (Bueunde® (DXM 0.5 mg/mL), Dexason INJ.® (DXM 1 mg/mL) and Dexolone-20 inj.® (DXM 1 mg/mL)) were intramuscularly injected into sixteen healthy dairy cows each. Bueunde® was intramuscularly injected into 8 dairy cows with 5 mg (BED-1) and 10 mg (BED-2) of DXM once a day for 3 consecutive days, respectively. Dexason INJ.® was intramuscularly administered once into dairy cows with 20 mg (DXS-1, n=8) and 40 mg (DXS-2, n=8) of DXM, respectively. Dexolone-20 inj.® was intramuscularly injected once into dairy cows with 20 mg (DXS-1, n=8) and 40 mg (DXS-2, n=8) of DXM, respectively. Milk production (MP) of BED-1 and BED-2 significantly decreased during the drug administration and up to 48-hour post-drug treatment. Compared with the MP before drug administration, the MP of DXS-1 and DXL-1 was meaningfully decreased by 36 and 24-hour post-drug administration, respectively, and that in both DXS-2 and DXL-2 significantly decreased until 48-hour post-drug treatment. In conclusion, it was confirmed that the MP temporarily decreased by 48 hours after administration of DXM to dairy cows.

This study investigated chlorpheniramine maleate (CPM) residues in milk of intramuscularly dosed dairy cows and established the withdrawal time (WT) of CPM in milk. Sixteen healthy Holstein cows were injected with 10 (CPM-1) and 20 mL (CPM-2) of the drug containing 4 mg/mL of CPM, respectively. After administration of CPM, milk samples were collected from all cows at 12 hour intervals for 5 days. CPM residue concentrations in milk were determined using LC-MS/MS. The correlation coefficient of the calibration curve was 0.9956, and the limits of detection and quantification (LOQ) were 0.6 and 1.0 μg/kg, respectively. Recoveries ranged between 98.5-115.0%, and coefficient of variations were less than 10.96%. After treatment, CPM in CPM-1 and CPM-2 was detected below the LOQ in all milk samples at 12 hours. According to the European Medicines Agency’s guideline on determination of withdrawal periods for milk, withdrawal periods of both CPM-1 and CPM-2 in milk were established to 12 hours. In conclusion, the developed analytical method is sensitive and reliable for detection of CPM in milk, and the estimated WT of CPM in bovine milk will contribute to ensuring the safety of milk.

본 연구는 우리나라 Holstein의 번식형질에 대한 유전모수를 추정하기 위하여, 2009년부터 2013년까지 분만한 농협 젖소개량사업소의 검정자료 경산우 144,793두의 211,899개의 번식기록을 이용하였다. 7가지 형질의 유전 및 오차 (공)분산을 WOMBAT 패키지를 이용하여 분석하였다. CTFS, GL, NRR, NS, FSTC, DO 및 CI의 유전력은 각각 0.064, 0.060, 0.005, 0.014, 0.012, 0.021 및 0.021로 추정되었다. 형질들 간의 유전상관은 –0.910에서 0.995로 나타났다. NRR과 NS, NRR과 FSTC 간에는 부의 유전상관이 나타났으나, DC와 CI는 강한 양의 유전상관을 나타냈다. CTFS와 FSTC는 DO 및 CI와 유의하게 양의 유전상관관계를 나타냈다(0.479~0.862). CTFS와 FSTC 사이의 낮은 유전상관은 두 형질이 서로 다른 유전적 측면에 있다는 것을 의미한다. 본 연구는 번식능력 개량을 위한 기초 자료를 제공할 수 있을 것이라 판단한다.

This study was conducted to investigate the effect of dietary total mixed ration (TMR) supplementation and dry-aging period on the physico-chemical and sensory quality of striploin (longissimus lumborum) from multiparous Hanwoo cows. The experimental cows were divided into two treatments (n=3/treatment) according to the type of feed: in one treatment (slaughter age: 87±6 months old, parity: 5±1) the cows were fed on TMR for 7 months before slaughter, while in the other treatment (slaughter age: 83±9 months old, parity: 5±2) the cows were fed on concentrate and rice straw (CRS) for the whole rearing period. After slaughtering, the striploins were dry-aged for 20 or 40 days at 2±1℃, 85% relative humidity and 2 m/sec air flow velocity and then utilized for quality measurements. The pH value, cooking loss, and hypoxanthine content were higher (p<0.05) for the striploins dry-aged for 40 days than for those dry-aged for 20 days. Notably, the inosine 5’-monophosphate content was decreased (p<0.05) by increasing the dry-aging time. With regard to microbiological quality, the striploins dry-aged for 40 days exhibited lower (p<0.05) lactic acid bacteria counts and higher (p<0.05) coliform counts than those dry-aged for 20 days. Moreover, the tenderness and overall liking scores based on sensory evaluation were higher (p<0.05) for the dry-aged striploins of the TMR treatment and those dry-aged for 40 days than for the CRS treatment and those dry-aged 20 days, respectively. These findings suggest that dietary TMR supplementation leads to an increase in the sensory preference of dry-aged striploins from multiparous Hanwoo cows, and alteration of the CRS into the TMR may improve the quality of dry-aged Hanwoo beef.

The objective of the present study was to determine the best model to describe and quantify the changes in live body weight, height at withers, height at rump, body length and chest girth of Holstein cows raised under Korean feeding conditions for 50 months. The five standard growth models namely polynomial linear regression models, regression of growth variables on the first and second-order of ages in days (model 1) and regression of growth variables on age covariates from first to the third-order (model 2) as well as non-linear models were fitted and evaluated for representing growth pattern of Holstein cows raised in Korean feeding circumstances. Nonlinear models fitted were three exponential growth curve models; Brody, Gompertz, and von Bertalanffy functional models. For this purpose, a total of 22 Holstein cows raised in Korea used in the period from April 2016 to May 2020. Each model fitted to monthly growth curve records of dairy cows by using PROC NLIN procedure in SAS program. On the basis of the results, nonlinear models showed the lower root mean square of error (RMSE) for live body weight, height at withers, height at rump, body length and chest girth (12.22, 1.95, 1.55, 4.04, 2.06) with higher correlation coefficiency (R2) values for live body weight, height at withers, height at rump, body length and chest girth (0.99, 0.99, 0.99, 1.00, 1.00). Overall, the evaluation of the different growth models indicated that the Gompertz model used in the study seemed to be the most appropriate one for standard growth of Holstein cows raised under Korean feeding system.

The research work was undertaken to determine an effective fertilization medium, sperm separation method and sperm capacitating agent for optimum in vitro fertilization (IVF) rates of indigenous zebu cow oocytes. In experiment 1, tissue culture medium (TCM 199), Tyrode’s albumin lactate pyruvate (TALP) and Brackett and Oliphant (BO) medium were used as basic medium for IVF of oocytes of indigenous zebu cows. In experiment 2, three sperm separation methods namely centrifugation, swim up and percoll gradient methods were used for separation of motile and viable spermatozoa for IVF. In experiment 3, for capacitation of spermatozoa, IVF medium supplemented with the heparin, mixture of penicillamine, hypotaurine and epinephrine (PHE) or the combination of heparin with PHE were used for fertilization. In vitro culture (IVC) of presumptive zygotes was done in modified synthetic oviduct fluid (mSOF) medium using standard procedure 24 h after sperm-oocytes co-culture. The cleavage rate was determined to evaluate the efficacy of fertilization medium, sperm separation method and sperm capacitating agent 24 h after IVC. The cleavage rate was higher in oocytes fertilized in TALP (63.3%) than in TCM 199 (47.5%) (p < 0.05). The cleavage rate was higher in oocytes fertilized by spermatozoa separated by percoll gradient method (62.3%) than by centrifugation (51.6%) (p < 0.05). The cleavage rate of oocytes was higher when insemination was done with spermatozoa capacitated in TALP supplemented with heparin and PHE (61.3%) compared to control (40.9%) (p < 0.05). In conclusions, TALP based medium and percoll gradient sperm separation followed by capacitation with combination of heparin and PHE are suitable for IVF of indigenous zebu cow oocytes in Bangladesh.

The objective of this research work was to know ovarian dynamics and pregnancy rate of cyclic Murrah buffalo cows with induced estrus by administration of prostaglandin F2α (PGF2α) and timed artificial insemination (TAI) with frozen thawed semen. A total of 31 female buffaloes were selected for the study. The buffalos having matured CL observed by ultrasonography were given one intra muscular injection of cloprostenol 500 μg and TAI was performed using frozen thawed semen of Indian Murrah buffalo bull. Results showed that 90.32% (significantly, at p < 0.05) cows explore the sign of heat after injection of PG and 67.85% (significantly, at p < 0.05) cows were become pregnant out of 28 inseminated (TAI) cows. In the 28 inseminated (TAI) cows, average number of smaller and larger size of follicles were non-significantly (p > 0.05) higher at day 3 post PG injection, but the medium size of follicles was non-significantly (p > 0.05) higher at PG injection. At day 3 post PG injection the diameter of follicles was significantly (p < 0.05) higher, but the diameter of CL was significantly (p < 0.01) lower compared at PG injection. At PG injection the diameter of largest follicle was non-significantly differences (p > 0.05) in between pregnant and non-pregnant cows. But at day 3 post PG injection it was significantly (p < 0.01) higher in pregnant cows compared to non-pregnant cows. The number of small, medium, and large follicles at PG injection and at day 3 post PG injection were non-significantly (p > 0.05) difference in between pregnant and non-pregnant buffalo cows. Finally, it is concluded that the CL was effectively regresses and induced the sign of heat in buffalo cows and after AI the cows were become pregnant with significant rate. The study will help to the veterinarian and researcher to know the efficacy of PG injection and AI for reproductive efficiency in buffalo cows.

The present study was designed to determine the effect of barn or cycle of grazing on changes of biochemical metabolites in prepartum and changes of milk composition in postpartum of dairy cows. For this purpose, a total of sixteen 25 months old Holstein primiparous dairy cows were allocated in two groups (n=8) with an average body weight of 571.61 ± 35.30 kg (Barn) and 578.10 ± 39.20 kg (Grazing). The study was conducted from June 2018 to October 2018. Results revealed that barn raised dairy cows had a higher increase in their serum albumin and calcium level on day 14 prepartum. However, the level of palmitic acid, saturated fatty acid increased significantly, and the level of fat, oleic acid, γ-linoleic acid, arachidonic acid and unsaturated fatty acids decreased significantly in barn raised dairy cow’s milk on day 14 postpartum. There were no significant differences observed with respect to all other biochemical metabolites, fatty acids and minerals between barn raised and cycle grazing dairy cows during prepartum and postpartum. Our study results could serve to a better understanding of barn raised cow with respect to changes of biochemical metabolites in prepartum and changes of milk composition, fatty acids and minerals content in grazing dairy cows in postpartum for estimating their physiological status.

This study was performed to investigate the effect of heat-stressed environment on rumen microbial diversity in Holstein cows. Rectal temperature and respiration rate were measured and rumen fluid was collected under normal environment (NE; Temperature humidity index (THI)=64.6) and heat-stressed environment (HE; THI=87.2) from 10 Holstein cows (60±17.7 months, 717±64.4 kg) fed on the basis of dairy feeding management in National Institute of Animal Science. The rumen bacteria diversity was analyzed by using the Illumina HiSeq™ 4000 platform. The rectal temperature and respiratory rate were increased by 1.5ﾟC and 53 breaths/min in HE compared to that in NE, respectively. In this study, HE exposure induced significant changes of ruminal microbe. At phylum level, Fibrobacteres were increased in HE. At genus level, Ruminococcaceae bacterium P7 and YAD3003, Butyrivibrio sp. AE2032, Erysipelotrichaceae bacterium NK3D112, Bifidobacterium pseudolongum, Lachnospiraceae bacterium FE2018, XBB2008, and AC2029, Eubacterium celulosolvens, Clostridium hathewayi, and Butyrivibrio hungatei were decreased in HE, while Choristoneura murinana nucleopolyhedrovirus, Calothrix parasitica, Nostoc sp. KVJ20, Anabaena sp. ATCC 33047, Fibrobacter sp. UWB13 and sp. UWB5, Lachnospiraceae bacterium G41, and Xanthomonas arboricola were increased in HE. In conclusion, HE might have an effect to change the rumen microbial community in Holstein cows.

This study was conducted to analyze the specific genes associated with sex-determination in Korean native cow. The highly organized spermatogenesis requires accurate spatial and temporal regulation of gene expression, which is governed by transcriptional, post-transcriptional, and epigenetic processes. Recently, farmers have been interested in determining the sexual identity of the calves in their farm. We analyzed the sperm of Korean native and Holstein cows, which were supplied from Hanwoo Improvement Center. We evaluated sperm motility and expression of sperm-specific genes after treating semen with both male- and female reagents. Sperm motility in Korean native cows decreased by approximately 10% in the first 30 minutes after treatment with sex-determination reagent. However, sperm motility of Holstein cows decreased to 60-70% after 15 minutes and to 20-30% after 30 minutes. We selected six specific genes expressing in the spermatozoa to analysis the gene expression level. The Real-time PCR results suggest that the selected genes (Gimap4, Tmeff1, Rac2, Abi2, Rac1, and Clu) were highly expressed in the group treated with the male reagent compared to the group treated the female reagent and to the untreated-group (control). In the present study, we suggest that the selected genes play a pivotal role in sex-determination.

These studies were conducted to evaluate developmental competence of follicular oocyte collected from the ovaries of Hanwoo cows with the high offspring meat quality (1++ and 1+ grade). Cumulus oocyte complexes from individual cows were matured, fertilized and cultured using protocols of in-vitro maturation (IVM), in-vitro fertilization (IVF) and in-vitro culture (IVC). The rates of blastocyst development from Hanwoo cows with the offspring meat quality grades of 1++ and 1+ were 18.6 and 21.2%, respectively. The rates of blastocyst development were 26.3, 20.7, 20.7, 17.2 and 31.2% from Hanwoo cows with the meat quality grades of 1++, 1+, 1, 2 and 3, respectively. Fiftyseven transferable embryos were recovered from 11 Hanwoo donor cows (5.2/head) with the high offspring meat quality grades of 1++ and 1+ in vivo, and the pregnancy rate after embryo transfer was 61.1%. In conclusion, these results suggest that in vitro embryo production from the ovaries of cows with the high meat quality grades using individual culture system can be used an efficient method for livestock improvement. In addition, for the successful industrialization of embryo transfer, conception rate should be improved.

In this study, the effect of daily short-distance walking exercise on milk production and metabolic status of the lactating dairy cows was investigated. The experiment was conducted with 10 lactating dairy cows comprising 2 groups (5 dairy cows/group), which were walking activity and non-walking activity. The walking activity (WA) group lactating dairy cows walked outdoors for 1 km/d, and the non-walking activity (non-WA) lactating dairy cows were maintained within a cowshed from 18thApril to30th May. The activity volume of the neck was not significantly different between the groups, but the activity volume of the leg was higher in WA group compared with non-WA (p<0.05). The rectal temperature was higher in the WA group (38.6 ℃) compared with non-WA (38.2 ℃) (p<0.05). No significant differences in dietary NE intake, milk production and milk composition were found between the groups. The plasma cortisol concentration was higher in WA group (2.14 μg/dl) compared to non-WA (0.95 μg/dl) (p<0.05). However, plasma cortisol level of WA dairy cows was significantly decreased as compared with non-WA cows. On the contrary, the level of plasma melatonin was increased in WA cows than that of non-WA dairy cows. It may be interpreted as a change in physical fitness. From this study, we suggest that walking activity can be improved energy balance. Further investigation is needed to determine whether different combinations of distance, rate or doing in morning or afternoon will stimulate energy balance.