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Research Article

Effect of High Rumen Undegraded Protein (HRUP) Supplementation on Estrous Response and Progesterone Hormone Profile in Dairy Cows Raised Under Indonesia Tropical Environmental Conditions

Rochijan , Budi Prasetyo Widyobroto and Ismaya
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This study was designed to determine the effect of High Rumen Undegraded Protein (HRUP) supplementation on estrous response and profile progesterone hormone in early lactation dairy cows under Indonesia tropical environment. The experiment was conducted with twelve early lactation Friesian Holstein cows divided into two groups. Six cows were used as a control group and other six 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. Rumen undegraded protein for control group was 27.47% and HRUP group was 32.78%. Feeding twice daily, morning and afternoon. Water was given by ad libitum. The observed variable were feed intake, estrous response and concentration of progesterone hormone. Data obtained were examined by t-test analysis, except estrous response analyzed descriptively. The result showed that 100% estrous response value was normal and the comparison of progesterone hormone on 1st, 5th, 10th, 15th, 17th, 21st and 26th day between control and HURP group was 0.13±0.02 vs. 0.13±0.04, 0.26±0.24 vs. 0.20±0.08, 0.13±0.03 vs. 1.72±2.23, 0.10±0.04 vs. 1.72±3.77, 0.09±0.02 vs. 3.66±3.76, 0.09±0.01 vs. 0.66±0.80 and 0.12±0.03 vs. 1.20±1.08 ng mL–1. It was concluded that HRUP supplementation on early lactation dairy cows could not increase the concentration of profile progesterone hormone. However estrous response value as well as estrous indication and behavior was normal.

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Rochijan , Budi Prasetyo Widyobroto and Ismaya , 2016. Effect of High Rumen Undegraded Protein (HRUP) Supplementation on Estrous Response and Progesterone Hormone Profile in Dairy Cows Raised Under Indonesia Tropical Environmental Conditions. Asian Journal of Animal Sciences, 10: 175-181.

DOI: 10.3923/ajas.2016.175.181

Received: November 25, 2015; Accepted: February 23, 2016; Published: April 15, 2016


The increasing of productivity on dairy cows is an effort should be made to improve the population in order to fulfill the need of cow protein that improving, especially milk. Livestock productivity in general involves various aspects, such as the production of calves, meat, eggs and milk. From the aspect of reproduction, improved reproducibility is a very necessary thing for the breeding, because this condition is a series that should not be broken in order to improve the reproductive efficiency, starting from cows born, puberty, mating and maternity. The efforts have been improved the reproductive efficiency, the role of feed is very high for subsistence in productivity during lactation cow. For lactating cows in the three semester especially early lactation, many cows require large DM intake to maintain milk production and initiate repair the reproductive tract (Rochijan et al., 2014). Therefore, the need for dairy cow must meet their needs and produce normally.

The process in reproduction related to mechanism of hormonal system, between the hypothalamic hormones gonadotropin releasing hormone (GnRH), Follicle Stimulating Hormone (FSH) and Luteinizing Hormone (LH), ovarian hormones (estrogen and progesterone) and uterine hormones (prostaglandin) (Hafez, 2000). Hormone ovary which has a big role on reproduction are estrogen and progesterone. Hartantyo (1995) report that the concentration of blood plasma progesterone can determine the state of the animal in a state of infertility, normal, estrous and pregnant so it can use to detection of estrous, pregnancy examinations and knowing other pathological conditions and Gunn et al. (2014) also said that feeding increased dietary Crude Protein (CP) abundant in Rumen Undegraded Protein (RUP) fraction impacted reproductive parameters prior to ovulation that would be indicative of improved fertility in cattle (impact in ovarian follicular growth patterns and affected follicular estradiol production and circulating concentrations of progesterone in the subsequent estrous cycle). Diets high in CP support high milk yield, but are also associated with lower reproductive performance. High protein can result in elevated plasma urea concentrations that affect the uterine environment and fertility. Nutritional interactions resulting in poor fertility of high producing dairy cows include the antecedent effects of negative energy balance and effects of high dietary protein (Litoi, 2014).

Indonesia is a tropical country and the climate is fairly even all year round. The climate and weather of Indonesia is characterized by two tropical seasons, which vary with the equatorial air circulation (the Walker circulation) and the meridian air circulation (the Hardley circulation), so that weather usually has high temperatures and humidity (Thayeb, 2015). The weather in Indonesia is characterized by high temperature and humidity, which ranged between 27.7-34.6°C and humidity ranged between 55.8 and 86.8% (BPS., 2010; Hernawan, 2014) and temperature humanity index value in Indonesia is 78-80. Productivity of dairy cows in tropical environment, which are generally thought to result from the lower lack of management that is not in accordance with the demands of livestock to produce optimally. The efforts have been made to improve the performance of dairy cows to increase the quality and quantity of rations and understanding the to fulfill the nutrients of rumen microbes and host cow, such as feeding the Rumen Undegraded Protein (RUP) which can prevent protein degradation in the rumen and can be used directly by the animal host by absorption in the small intestine. So from that the results of this study will influence the data obtained with feed high rumen undegraded protein on estrous response and profile progesterone hormone of early lactation dairy cows.


Time and place of experiment: This experiment was conducted in Dairy Cow Units, Department of Agriculture, Daerah Istimewa Yogyakarta. Weather in Indonesia is generally hot (23-34°C) and humid (73-87% relative humidity) (Thayeb, 2015). 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, Gadjah Mada Universities.

Animals and diets: The experiment was conducted with twelve early lactation Friesian Holstein cows divided into two groups. Cows housed in pens models stanchion barn, equipped with a feed and drink water. Six cows were used as a control group and other six cows as HURP group. The diets used for control and HRUP group were forages and concentrate with a ratio of 60:40 (DM basis), but different concentrate composition for each groups (Table 1). 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, distilled water and a set of progesterone ELISA analysis.

Table 1:Concentrate formulation ingredients and nutrient compositions 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, TDN: Total digestible nutrient, HRUP: High rumen undrgraded protein, aResults of the analysis in sacco degradation and formulas Widyobroto et al. (1997) and bResults of the formula of Hartadi et al. (2005)

Estrous response indication and behavior: This observation, in the form of changes in behavior and reproductive organs that occur during estrus include: behavior and quietly climbed up the other fellow cows, cattle restless, mooing cow-bellow, decreased appetite and changes in the condition of the vulva include: the presence of the vulva red (red, swollen, warm), clear mucus, thick and hang (Putro, 2008).

Methods blood sampling and hormone analysis: Blood sampling performed on cows (control and HRUP) whose have as many as six head estrous through artery caudals of 3 mL vacutainer K3 Ethyline Diamine Tetraacetic Acid (EDTA) tube, 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 is stored at a temperature of -20°C freezer until ELISA analysis.

Measurement of kit progesterone hormone using Enzyme-Linked Immunosorbent Assay (ELISA) (DRG., 2007). A total of 25 mL of solution, 25 mL of standards, controls 25, 25 and 200 mL sample of enzyme conjugate was added to each microtiter plate wells using a micro pipette and wait for 10 sec until fully mixed. Afterwards, sample incubated for 60 min without a cover plate at room temperature. The next process is done washing 3 times with the addition of a solution of 400 mL wash solution for each microtiter plate wells using ELISA tool. Furthermore, the residual droplets attached to the microtiter plate removed. A total of 200 mL substrate solution to each wells were incubated for 15 min at room temperature. Stop solution was then added 100 mL at any pitting. Determination of Optical Density (OD) enzyme conjugate is done after 10 min of adding stop solution, using a microtiter plate reader at a wavelength of 450±10 nm.

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


Intake of forage and 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. Content ingredients and nutrient compositions concentrates formulation of experimental diet was presented in Table 1.

The average intake of dairy cows during the experiment was presented in Table 2. 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 and 6.67 kg DM/head/day, respectively. This was similar to the study 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. These observations were in accordance with Kiran and Mutsvangwa (2007) who found that increasing dietary RDP from 60-70% of CP increased OM intake (ranging from 1.2-1.4 kg day–1) in growing lambs. It has been reported that dietary RDP can promote intake by enhancing gastrointestinal motility (Egan and Moir, 1965).

There is many information on the effects of Forages:Concentrates (F:C) ratio on DMI, growth performance and production in housing-feeding cows.

Table 2: Nutrient intake of dairy cows receiving control and HRUP
DM: Dry matter, OM: Organic matter, CP: Crude protein, CF: Crude fibre, TDN: Total digestible nutrient and HRUP: High rumen undrgraded protein

In this study the ratio of F:C is 60:40, with the difference RUP value of the control group 27.47% and the HRUP group 32.78%, still showed a normal cow performance. Cantalapiedra-Hijar et al. (2009) observed DMI was not affected with the percentage of concentrate increasing in the diet from 30-70% in goats. Aguerre et al. (2011) found that increasing F:C ratios (47:53, 54:46, 61:39 and 68:32) in the diet had no effect on DMI of Holstein cows. A similar result was observed by Agle et al. (2010), who reported no change in DMI of lactating dairy cows fed diets contained 52 and 72% concentrate feeds. In contrast, Desnoyers et al. (2008) reported that DMI of dairy goats was increased (2.69-2.88 kg day–1) with increasing the percentage of concentrate in the diet from 30-60%. Similarly, Murphy et al. (2000) noted that cows fed 30:70 diets of F:C ratio had a significantly higher DMI than cows fed 50:50 diets.

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.

The high protein intake was caused by concentrates in ration has high protein content. 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 treated 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 studies 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).

Estrous response indication and behavior: The observation result of estrous response indication and behavior of dairy cows supplementation with HRUP show symptoms similar to those reported by Siregar et al. (2004) which showed symptoms of vulvar mucus and hang white, red and swollen vulva, as well as behavioral changes such as aggressive, bellowing-bellow and decreased appetite. Symptoms and estrous behavior cows in this study also supported by reported by Toelihere (2003) which mean the strain in Indonesia showed the same symptoms of estrous with cows in general, transparent white mucus from the vagina, vulva change into warm, red and wet and the cow became agitated, bellowing-bellow and decreased appetite. The cows wich used in the study showed that aggressive changes, trumpeted-bellow, decreased appetite and cows that secrete mucus indicates the cow estrous.

Dairy cows showed symptoms of quiet estrous (silent heat), where cows are asymptomatic clinically estrous but the ovaries ovulation and decreased appetite. Cows that showed decreased appetite and less aggressive, is likely to be caused due to estrogen levels under the threshold value that could not stimulate the clinical estrous (Karmita et al., 2001). Dairy cows that have estrogen levels above a threshold value able to displaying symptoms and estrous behavior. The threshold value of each individual animal is different, according to the physiological conditions. The presence of inhibition of LH receptors in the corpus luteum, resulting in LH does not activate the enzyme adenylate cyclase that functions in the reaction step for the sustainability of the life of the corpus luteum. The enzyme is not active cause lysis of the corpus luteum and is unable to secrete the hormone progesterone. The process also followed by the secretion of Follicle Stimulating Hormone (FSH) causing estrous (Kaltenbach and Dunn, 2001).

Changes in the external genitalia dairy cows observed when estrous was the vulva look wet because the mucus which come from vagina and vulva were look reddish and swollen. Toelihere (2003) reported that when the vulva was touched by the hands will relatively feel warmer. Changes in the vulva and vaginal mucosa anestrous in dairy cows did not look slimy or dry and pale and no swelling. It agrees with report of Putro (2008) that the symptoms when the cows were in anestrous condition could be visible from the outside was vulvar not mucus or dry and pale and no swelling.

Progesterone hormone profile: Based on the statistical analysis shows that cow which is feeding with a HRUP indicated no significant effect on the concentration of progesterone hormone (Table 3). The average concentration of progesterone hormone in the control group 1, 5, 10, 15, 17, 21 and 26 days was 0.13±0.02, 0.26±0.24, 0.13±0.03, 0.10±0.04, 0.09±0.02, 0.09±0.01, respectively and 0.12±0.03 ng mL–1 with a range of 0.06-0.54 ng mL–1. The treatment group was 0.13±0.04, 0.20±0.08, 1.72±2.23, 1.72±3.77, 3.66±3.76, 0.66±0.80 and 1.20±1.08 ng mL–1 with a range of 0.10-7.66 ng mL–1.

The ovary’s activity after giving birth is a very important consideration in order to improve reproductive performance. Developing and functioning of reproductive organs after birth is depend on the level of FSH and LH from the anterior pituitary is controlled by GnRH secreted by the hypothalamus. Hana (2004) reporting that the range of the progesterone hormone during estrous between 0.83±0.14-1.04±0.11 ng mL–1. The similar report by Valdez et al. (2005) mention that the new progesterone hormone levels could be detect since four day after estrous. The low levels of the progesterone hormone during the estrous were due to a follicular phase and high levels of estrogen (Hafez, 2000). High progesterone concentrations probably decrease LH pulsatility and the early decrease of estradiol allows an early rebound of FSH concentrations, promoting the emergence of the second follicular wave. Thereafter, the emergence of the subsequent wave is also advanced (Pang et al., 2010). This is in agreement with pharmacological studies performed in the cow (Adams et al., 1992).

Changes in the concentration of the progesterone hormone could be seen in Fig. 1. It could be shown that feeding with a HRUP capable of affecting the function of the corpus luteum to produce high amount of progesterone. The highest concentration of progesterone (3.12-7.66 ng mL–1) occurred on the 17th day of estrous cycle. Although, progesterone concentration indicating no significant differences between the two conditions, but the average treatment on 17th day was higher than the controls variable (3.66 vs. 0.09 ng mL–1). These results were contrary with the research by Astuti (2000) that a cow will show the highest concentrations of progesterone on 16th day after estrous.

The concentration of progesterone in Fig. 1 shows that the HRUP group began to increase graph in 5th day. Furthermore, in 10th, 15th-17th day, the concentration of progesterone hormone appeared to increase significantly and decreased in 17th-21st day.

Table 3:Average concentration of progesterone hormone profile in day of estrous cycle
HRUP: High rumen undrgraded protein

Fig. 1:Curves concentration of progesterone hormone profile in day of estrous cycle

On 21st day, the concentration of the progesterone hormone is relatively low due to the corpus luteum regression and estrous imminent. Hafez (2000) reported that the corpus luteum regresses from 16th day of the estrous cycle, thus resulting in lower concentrations of the progesterone hormone. In the control group also began to increase in the 5th day, but in the next day decreased until 21st day. This indicates that the cows in the control group did not undergo fertilization.


It was concluded that HRUP supplementation on early lactation dairy cows could not increase the concentration of profile progesterone hormone. However estrous response value as well as estrous indication and behavior was normal.


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).

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