MATERIALS AND METHODS

This experiment was conducted in Dairy Cow Units, Department of Agriculture, Daerah Istimewa Yogyakarta. Analysis of samples was conducted in Laboratory of Dairy Science and Milk Industry, Faculty of Animal Science and Laboratory of Pathology Clinic, Faculty of Veterinary Medicine, Universitas Gadjah Mada.

Animals and diets: This experiment used twelve early lactation holstein friesian cows were divided into two groups, permanent enclosure models stanchion barn with cement floors which feed and drink water. Six cows were used as a control group and other 6 cows as HURP group. Both control and HRUP group obtained basal diet with forage to concentrate ratio 60:40 (DM basis), but different concentrate composition for each groups (Table 1). Rumen undegraded protein for control group was 27.47% and HRUP group was 32.78%. Feeding twice daily, morning and afternoon. The cow feeds after parturition. Drink water was given by ad libitum. The equipment used in blood sampling and analysis was vacutainer needle merk BD Vacutainer, 3 mL venoject tube merk vaculab, holder vacutainer merk brand, cotton, centrifuge merk eppendorf centrifuge 5417C, 1.5 mL eppendrof tube merk brand, refrigerators merk sharp, freezers merk modena, microtiter plate reader, micropipette, absorbent paper, spectrophotometer merk microlab, timer and distilled water.

Data and sample collection
Data of reproduction performance: Data collection was performed by jotting reproduction performance on each friesian holstein dairy cows. Reproduction performance includes postpartum mating, service per conception and days open were collected. Postpartum mating refers to the time from when a cow calves until the first time she is bred. Service per conception refers to the average number of time the cow mates to that cow were pregnant. Days open refers to the time from when a cow calves until when she conceives.

Sampling and analysis of blood urea nitrogen: At 7 and 90 day of the experiment, blood samples from each individual animal were collected aseptically via caudalis artery (3-4 h after the morning feeding) into 3 mL vacutainer K3 Ethylene Diamine Tetraacetic Acid (EDTA) tubes. The tubes were rolled gently several times to ensure enough anticoagulant mixing with methods of Delany et al. (2010) and Zetina-Cordoba et al. (2012). Blood samples were centrifuge for 15 min at 3000 rpm and the plasma was taken using a micropipette and then transferred into 1 mL eppendorf tube. After that, the sample plasma is stored at a temperature of -20°C freezer until blood urea nitrogen analysis. After all collected, plasma concentration of urea nitrogen were determined using an auto-analyzer.

Statistical analysis: The data obtained were examined by t-test analysis using Statistical Program for Social Science or SPSS version 16.0 (Supardi, 2013).

RESULTS AND DISCUSSION

Chemical composition of feed ingredients concentrates: Chemical composition of feed ingredients concentrates consist of: G-Pro, soy bean meal-HCHO, kapok seed meal, copra meal, corn gluten feed, wheat pollard, palm kernel meal, coffee husk, corn tumpi, cassava waste, molasses and mineral mix. The control group getting concentrate containing Dry Matter (DM) 88.24%, Crude Protein (CP) 18.55%, Total Digestible Nutrient (TDN) 56.94% and Rumen Undegraded Protein (RUP) 27.47%, while the HRUP group getting concentrate containing Dry Matter (DM) 89.06%, Crude Protein (CP) 18.83%, Total Digestible Nutrient (TDN) 58.91% and Rumen Undegraded Protein (RUP) 32.78%. Content ingredients and nutrient compositions concentrates formulation of experimental diet was presented in Table 1.

Feed intake: Table 2 showed the average intake of dairy cows during the experiment. The results showed that intake of DM, OM, CP, CF and the TDN of cow given HRUP supplementation and those of control were not different, which were DM 11.64, 10.15, 1.50, 3.10, 6.63 vs. 11.54, 10.10, 1.50, 3.05, 6.67 kg DM/head/day, respectively. This was similar to the report of Widyobroto et al. (2001) and Widyobroto (2013) which found that the increase of RUP in the ration was not influenced by the intake of DM. The intake of OM was improved with decreasing RUP in the current study. The high protein intake was caused by concentrates in ration has high protein content.

Table 1:	Ingredients and nutrient compositions concentrate formulation of experimental diet
DM: Dry matter, OM: Organic matter, CP: Crude protein, CF: Crude fibre, EE: Ether extract, RUP: Rumen undegraded protein, RDP: Rumen degraded protein and TDN: Total digestible nutrient, a: Results of the analysis in sacco degradation and formulas Widyobroto (1997), b: Results of the formula Hartadi et al. (2005)

Table 2:	Feed and nutrient intake of dairy cows receiving control and HRUP
DM: Dry matter, OM: Organic matter, CP: Crude protein, TDN: Total digestible nutrient

Dry Matter Intake (DMI) has special importance to meet nutrient requirement of fresh cows to maintain their health and production. Le Floc'h et al. (2004) and Sejrsen et al. (2006) that reported low DMI and deficiency in nutrient supply, specially protein and asam amino acids has led to immunosuppression and incidence of metabolic disorders on body cows. Thus, diets that have higher levels of crude protein and RUP are effective in maintaining of production and Body Condition Score (BCS) (NRC., 2001).

Dietary fibre content (CF) had no influence on OM intake in this trial. Commonly, an increase in dietary CF can be achieved by increasing the level of concentrates. Therefore, the results of the current study are comparable with those obtained by Cantalapiedra-Hijar et al. (2009) and Ramos et al. (2009), who found no effect of dietary concentrate: forage ratios (30:70 and 70:30) on OM intake in both goats and sheep fed above maintenance. The differences in CF of the diets were mainly due to the variance in CF concentrations. However, Allen (2000) noted that no effect of CF ranging from 25-40% was found on DM intake in dairy cows, although feed intake generally decreases with increasing CF. The CF in concentrate was high (coffee husks and corn tumpi), hence the need/requirement of fiber still can be fulfilled. This means that the intake of forages was low, the negative effect in the digestive process was not happened since the concentrates given still have high structural carbohydrates. This condition could be used as a reference by farmers, especially in the dry season where the forages were difficult to get and relatively expensive. Miller (1979) reported that the energy could affect the efficiency of the ration used, excess energy in ration and also caused decrease ration efficiency used and tended to be accumulated in the body fat. One of the disadvantage was the excess of the amino acids is increased further and these need to be deamination and excreted, with consequent reduction in the energy value of the diet and increased pollution.

There were not just low energy and high protein content in the ration caused low protein efficiency but the ratio of energy : protein should be considered to get better protein efficiency. Thus, the condition should be considers for protein protection to avoid rumen degradation. Protein and energy intake from control and HRUP rations were more than enough to fulfill the need of maintenance and production. This wasn’t similar to the results of research by Encinias et al. (2005), that there was no difference in intake of DM on dairy cows fed Brome grass hay (9.6% CP) which is supplemented by undegraded protein. However, most research suggests that increasing energy intake will increase both content and production of protein in milk and milk production is improved by increasing CP intake (De Peters and Cant, 1992).

Table 3:	Average of reproduction performance in dairy cows
ns: Non significant (p>0.05)

Performance reproduction: Success of reproduction performance is mainly determined by days open, the longer days open means the longer calving interval which affects the lowering amount of milk production throughout the cow life. The cow pregnancy detection was carried out or checked by artificial inseminator from Department of Agriculture, Daerah Istimewa Yogyakarta by performing a detection of pregnancy. The results overall of reproduction performance was presented in Table 3.

Postpartum mating (PPM): The result shows that the shortest average PPM found in the HRUP group is 81.33±19.83 days and control group is 91.83±31.16 days. Based on the PPM data obtained, the HRUP group still in normal range of the first time cows mating after parturition. It’s higher than, Aboozar et al. (2012) results about HRUP value 57.50±7.30. This range confirms, Salisbury and Vandemark (1993) opinion that first mating is the best performed within 60-90 days after parturition cows. The lower of PPM value, it will be more efficient the reproduction performance of dairy cows. The difference in PPM on each cow is caused by differences in the occurrence of estrous as the result of milk production difference which will cause weight loss. Therefore the time to achieve the normal body weight for reproduction will be varies. These variations are caused by differing negative energy, which triggers reproductive problems such as sub estrous and silent heat or quiet ovulation, as stated by Peters and Ball (1995), that very low nutrition quality and quantity are the main causes of anestrous and the prolonging of estrous period after parturition. Berry et al. (2003) adds that the PPM normal results of optimal (60-90 days) and this could be attributed to different factors. Most researchers suggest that the reason for the delay in interval to first service is greater negative energy balance in modern dairy cows. Negative energy balance delays the continuation of ovarian activity.

Service per conception (S/C): The result shows that the shortest average S/C found in the HRUP group is 1.17±0.41 times and control group is 1.50±0.55 times. It’s lower than, Aboozar et al. (2012) research about HRUP value 1.76±0.34. The average estrous cycle in each group could be calculated because there were dairy cows with service per conception score 1, therefore upon these dairy cows the estrous cycle could not be determined. This long interval is due to silent estrous caused by negative nutrient imbalance. This is parallel with Perry (1984) opinion, who said that lack of energy after giving birth will extend estrous interval and will delay ovulation. Yifat et al. (2009) stated that appropriate and in time heat detection and insemination could be attributed to lower or higher number of service of per conception. The findings of the present study on services per conception suggested comparatively better insemination services at the herds during the period of the study, because S/C value is normal and noting >2.00.

Days Open (DO): The result shows that the shortest average S/C found in the HRUP group is 97.33±41.52 days and control group is 142.33±66.87 days. It’s higher than Aboozar et al. (2012) study about HRUP value 90.38±1.53. The control group has long days open, this not in line with Antiyatmi (2009) that the optimal days open or the safest time to bring a cow into mating again is between 60-90 days after parturition, if it is too long, production during the next lactation period will decrease between 75-110 days. The long days open period is caused by the decrease of PPM and by the high service per conception. The long days open period will extend calving interval, which will decrease the number of calves produced throughout the cow lifetime. This in line with the opinion of Foley et al. (1972) that the long days open will lower the cows productivity during its lifetime.

Blood Urea Nitrogen (BUN): The average BUN value between controls and HRUP were significant (p<0.05). The average BUN value of each group is 15.58±1.99 vs. 18.09±1.50 mg dL^–1. BUN concentrations are presented in Table 4. The BUN value of the control group fed with CP content of 18.55% and RUP 27.47% lower than the BUN group HRUP with CP feed containing 18.83 and 32.78% RUP. The BUN values will decrease in animals offered the high synchrony index diets. The results disagreed with Sinclair et al. (2000), who found that plasma urea levels were unaffected by a synchronous treatments in temperate fed basal diet. Urea is a small molecule that equilibrates between the reproductive tract and plasma (Duby and Trischler, 1986). Deleterious effects of high blood urea have been demonstrated on sperm viability (Duby and Trischler, 1986) and embryo survival both in vitro (Dasgupta et al., 1971) and in vivo (Saitoh and Takahashi, 1977).

Table 4:	Average concentration of blood urea nitrogen
*Significant (p<0.05)

In addition, feeding high rumen degradable protein diets altered uterine pH in heifers (Elrod and Butler, 1993), hormonal patterns during the estrous cycle and early pregnancy (Berardinelli et al., 2001) and thus have a direct effect on gametes (Canfield et al., 1990) and embryo survival (Berardinelli et al., 2001). Recently, in this experiment it was found that beef cattle fed a diet containing a higher synchrony index had a lower BUN, this would indicate that a synchronous diet increased N-utilization efficiency. Synchronizing the rate of dietary energy and nitrogen release is a possible way to avoid excess blood urea nitrogen and excessively high levels of plasma ammonia, leading to improved reproductive efficiency. However, Ferguson et al. (1993) reported that a blood urea nitrogen concentrations exceeding 20 mg% was associated with a reduced conception rate in lactating cows. Since the BUN concentration in this experiment was lower than 20 mg%, BUN would not affect the fertility in dairy cows.

CONCLUSION

The conclusion is HRUP supplementation to early lactation dairy cows would be impact on BUN value, but no impact on reproduction performance of dairy cows.

ACKNOWLEDGMENTS

Authors are thankful to the Dairy Cow Units, Department of Agriculture, Daerah Istimewa Yogyakarta for providing experimental facilities, materials and technical support throughout this experiment. Thanks are also extended to Dirjen Dikti Ministry of Education and Culture, Indonesia for Beasiswa Unggulan (Superior Scholarship).