Effect of Biostimulation on Some Behavioral Aspects and Energy Balance of Sahiwal Cows during Peri-estrual Period

Prolonged inter-calving period due to delayed in onset of post-partum ovarian function and poor estrus detection is one of the major reproductive problems especially in native breeds of cattle in India (Dhami et al., 2015). The hormonal treatment is effective but it goes against the practices of “clean, green and ethical” production (Patterson et al., 2003). Therefore, more natural approaches such as biostimulation (male effect) have been attempted in some of the livestock species to reduce the use of hormonal treatments (Mat et al., 2014; Fiol and Ungerfeld, 2010).
               
Biostimulation is the term referred to the stimulatory effect of a male on estrus and ovulation through genital stimulation, olfactory pheromones, or other less well-defined external cues such as tactile, visual, olfactory and auditory (Chenoweth.1983; Fiol and Ungerfeld, 2010). In cattle, biostimulation can be achieved by direct bull exposure, fence-line bull exposure, exposure to the urine of bulls. It has been observed that the post-partum anestrus interval is reduced in direct bull-exposed cows (Zalesky et al., 1984) and also in fence-line bull exposed cows (Fike et al., 1996) in comparison to non-bull exposed cows. Further, the presence of bulls shortened the interval between parturition and initiation of estrous cycles in cows that gained weight but not in cows that lost weight during postpartum period. This indicated that a positive energy balance and an optimum body condition is a pre-requisite for obtaining effective results due to biostimulation after calving (Monje et al., 1983). The behaviour of cows around estrus plays an important role in accurately, timely and effective detection of estrus (Zin et al., 2018). The ingestive and resting behaviours of the cows are mainly affected by estrus modulated by hormonal changes before, during and after estrus. The effect of biostimulation of the ingestive and resting behaviours and the energy balance before, during and after estrus have not been investigated. The present study therefore aims to investigate the effect of biostimulation by use of an intact bull by direct and fence-line contact with cows on the ingestive, resting behaviour and energy balance of periestrual Sahiwal cows.

Location
 
The experiment was conducted at the Livestock Research Centre, National Dairy Research Institute, Karnal, Haryana which is situated at 29°43" N Latitude and 77°2" Longitude, at an altitude of 227 m above mean sea level respectively. The maximum ambient temperature in summer goes up to 45°C and minimum temperature in winter comes down to 4°C with a diurnal variation in the order of 15-20°C. The annual rainfall is 70 cm, most of which is received from July to mid-September. The experiment was started in the month of October 2018 and lasted up to May 2019. An adjustment period of one month was given to the animals before the actual start of experiment.
 
Experimental animals and housing
 
Twenty four freshly calved healthy Sahiwal cows were selected from the herd of Sahiwal cows maintained at the institute farm. These cows were divided in to 3 groups of 8 animal in each based on their yield in previous lactation in case of pleuriparous cows and expected producing ability (EPA) in case of primiparous cows. These cows were allotted at random to 3 treatments by exposing them to 2 types of contacts to intact bulls for biostimulation. In T₁ the cows were exposed and allowed to contact with the fence-line bull round-the-clock throughout the length of the covered area as well as open paddock of the cows shed. In T₂ the cows were given a direct bull contact twice a day for a total duration of 12 hours from 6.30 am to 12.30 pm and again from 3:30 pm to 10.30 pm (excluding about 1.0 hour of time spent on evening milking). In T0 the cows were not exposed to any bull contact. All the experimental animals were housed under loose housing system and floor space (i.e. 3.5 m² covered area, 7 m² open area) and other housing conditions were provided according to BIS standard recommendations. The animals were milked twice daily at 5:00 a.m. and again at 5:00 p.m. The housing of 3 groups of animals were at a distance of about 0.5 km from each other in order to avoid the effect of air borne bull pheromones on control group of cows.
 
Feeding management
 
The feeding followed a similar pattern among the 3 groups, comprising of ad lib amounts of seasonal green fodder (Table 1) and concentrate mixtureare given in Table 2 (as per ICAR, 2013 recommendations). The animals were provided with farm grown green fodder of all seasons and dry roughage ad libitum quantity. The available concentrate mixture with 16-18% DCP and 70% TDN with the ingredients was offered to animals as per their milk yield. The dry matter intake of the cows was estimated at fortnightly intervals.
 

 

 
Collection of blood samples
 
The blood samples of experimental animals were collected at fortnightly from 15 days after parturition and were immediately centrifuged for separation of blood serum. The serum samples were preserved at -8°C till these were put to bioassay using ELISA.
 
Recording of behavioural parameters.
 
The general cow behaviours were observed through direct visual observation and video cameras which were installed to record the activity of cows for 24 hours daily three days before, on the day and three days after onset of estrus. The cameras had 8x digital zoom for closer viewing and were enabled with array infrared technology for best night vision. The images and video were stored in 16 channel digital video recorder (DVR) having hard disk of 2 TB space.
 
Data analysis
 
The significance of differences among the means of different parameters of 3 treatment groups of experimental animals was analysed by subjecting the data to Duncan Multiple Range Test (DMRT) with one-way ANOVA as per (Snedecor and Cochran, 1994) using SPSS computer software version 20.

Feed intake
 
The daily feed intake of the cows in terms of mean dry matter intake (DMI) per 100 kg body weight in T₀, T₁ and T₂ was 3.05±0.58, 3.12±0.67 and 3.18±0.85 kg respectively (Fig 1) which did not differ significantly among the 3 groups of cows. These values of mean DMI per 100 kg body weight in all the 3 groups of cows meet the recommended levels of daily DMI (2.5 to 3% of body weight) for the feeding of lactating cows as per ICAR (2013). Choudhary et al., (2020) also reported similar findings in Sahiwal heifers. Nutrition play a direct role both in the hypothalamus by synthesizing and releasing GnRH, as well as on the anterior pituitary by controlling the synthesis and release of Leptin, FSH, LH and Estrogen (Laksmi et al., 2019).
 

 
Time spent on feeding
 
During first post-partum estrus the feeding time on the day of estrus (d-0) in T₀, T₁ and T₂ was 4.63±0.15, 4.31±0.18, 4.09±0.09 hours, respectively, did not differ significantly among the 3 groups of cows. However, the mean time spent on feeding was found to be lower than on the reference day in all 3 groups of cows (Table 3). The percent reduction in feeding times in T₀, T₁ and T₂ on the day of estrus as compared to reference day was 23.97, 31.00 and 31.37, respectively. The mean feeding time witnessed a consistent decline in all 3 treatment groups of cows from d-3 of estrus and was the lowest on d-0 and thereafter it rose gradually and approached the reference values on d+3. In second estrus, the mean time spent on feeding (hours) on d-0 in T₁ and T₂ was 3.89±0.13 and 3.81±0.11, respectively, which was significantly (p<0.05) lower than in T₀ (4.29±0.03) (Table 4). As in case of first estrus, the mean feeding time had a consistent decline in all 3 groups of cows from d-3 of estrus, being the lowest on d-0 and then it increased gradually and was almost similar to the reference values on d+3. The percentage reduction in feeding times of T₀, T₁ and T₂ on the day of estrus as compared to reference day was 30.24, 35.70 and 37.54, respectively. On the day of estrus, the cows in T₁ and T₂ spent significantly lower time on feeding than T0 cows which might be due to the contact of bulls with cows that kept the estrus cows engaged in mounting and other estrus behavioural activities. Lyimo et al., (2000) and Lopez et al., (2004) found that estrus being strongly correlated with estradiol concentration reaching peak level on the day before estrus ensuing higher activity and thus results in the reduction of time spent on eating (Uphouse and Maswood, 1998). These findings are in agreement with Diskin and Sreenan (2000) and Pahl et al., (2015) who reported reduced feeding time on d-1 and d-0 of estrus in Holstein cows and Choudhary et al., (2020) in case of Sahiwal heifers.
 

 

 
Time spent on rumination
 
During first post-partum estrus, the mean rumination time on the day of estrus (d-0) in T₀, T₁ and T₂ was 6.36±0.15, 5.19±0.16, 5.01±0.09 hours, respectively, which was significantly (p<0.01) lower in T₁ and T₂ cows as compared to T₀ cows (Fig 3). This may be attributed to the fact that presence of bulls in direct contact of in fence-line contact may have engaged in higher physical activity and other estrus behaviours such as mounting. Consistent with time spent on eating, the time spent on rumination also showed a declining trend from d-1 and was the least on d-0 followed by a steady increase till d+3. Mean daily time spent on rumination (hours) on the reference day in T₀, T₁ and T₂ was 9.20±0.15, 9.05±0.13, 9.00±0.12 hours and the percent reduction in this on the day of estrus was 28.33, 46.18 and 48.57, respectively. The average rumination time on d-0 in second estrus in T₁ and T₂ was, 4.23±0.15 and 4.05±0.0.16 hours, respectively, which was found to be significantly (p<0.05) lower than the T₀ (5.88±0.23 hours). In second estrus also, the average time spent on rumination started declining from d-1 of estrus, was the lowest on d-0 and resumed to normal by d +3 in all 3 groups of cows. A pronounced decline in mean rumination time in both groups of bull exposed cows (T₁=53.9%; T₂=55.6%) was noticed on the day of estrus as compared to non-bull exposed cows (T₀=37.7%). These findings are in agreement with Sveberg et al., (2011); Pahl et al., (2015) and Roelofs et al., (2005) who reported that rumination time was obviously reduced in a wider period around estrus due to involvement in the sexually active groups’ activity. Similar findings were also reported by Choudhary et al., (2020) in Sahiwal heifers.
 
Time spent on resting
 
During first post-partum estrus, the mean resting times on d-0 in T₀, T₁ and T₂ were 6.88±0.21, 5.22±0.53, 5.50±0.11 hour, respectively, The resting times in T₁ and T₂ on the day of estrus were found to be significantly lower (p<0.05) than in T₀. However, no significant difference was observed between the resting times of T₁ and T₂ on the day of estrus (Fig 4). Mean daily times spent on resting on reference day in T₀, T₁ and T₂ were 9.60±0.20, 9.70±0.28 and 9.82 ±0.19 hours and the percent reduction from the these values on reference day was 28.33, 46.18 and 48.57, respectively. The average resting time in second post-partum estrus period on d-0 of estrus in T₁ and T₂ was 4.98±0.13 and 4.62±0.17 hour respectively, which was found to be significantly (p<0.05) lower than the T₀ (6.15±0.10 hour) (Fig 3) whereas no significant difference was observed between the resting times of T₁ and T₂ on the day of estrus. The percent reduction in feeding times in T₀, T₁ and T₂ on the day of estrus as compared to reference day was 38.50, 49.18 and 50.56, respectively. These results are in agreement with Kerbrat and Disenhaus (2004) and Dolecheck et al., (2015) who reported a decrease in the total daily resting time during estrus in Holstein cows. Choudhary et al., (2020) also observed similar findings in Sahiwal heifers.
 

 

 
Plasma beta-hydroxybutyric acid (BHBA) concentration
 
The mean concentration of BHBA was found to be highest in the first fortnight after calving in all the 3 groups of cows and these levels decreased consistently thereafter as the lactation progressed. There was no significant difference in the mean levels of BHBA among 3 groups of cows throughout the experimental period. Nonetheless, these levels of BHBA were within the normal physiological ranges of the animals in all 3 groups indicating that the animals of all animals were in positive energy balance. Earlier, the presence of bulls in contact with cows have been reported to shorten the interval between parturition and initiation of estrous cycles in cows that gained weight but not in cows that lost weight during postpartum period. This indicated that a positive energy balance and an optimum body condition is a pre-requisite for obtaining effective results due to biostimulation after calving (Monje et al., 1983). The levels of plasma concentration of BHBA (482.573±1.48) in postpartum sahiwal cows were similar decline to our findigs (Yadav et al., 2015).
 
Plasma non-esterified fatty acid (NEFA) concentration
 
The mean concentration of NEFA was found to be highest in the first fortnight after calving in all 3 groups of cows and the levels decreased subsequently until the first estrus. However, the differences in the mean values of NEFA among three groups were not statistically significant throughout the observation period. These values were within the normal physiological range indicating that the animals of all 3 groups were in positive energy balance. The level of plasma concentration of NEFA (399.316±14.2) in postpartum sahiwal cows were similar decline with our findings (Yadav et al., 2015). Similar decline in NEFA concentrations over postpartum in CB cows was observed. (Singh et al., 2020; Selvaraj et al., 2019 and Khune et al., 2019).

It was concluded that biostimulation of Sahiwal cows by exposing them to intact bulls either direct contact or through fence-line contact during post-partum period highly reduced their feeding time, rumination time and resting time during peri-estrus period as compared to non-bull exposed cows which may improve the efficiency of estrus detection. This practice of bull exposure, however, did not affect the energy levels of these animals.

Authors have sincere gratitude toward the Director, ICAR-National Dairy Research Institute, Karnal for providing all facilities for this study and all who helped in direct /indirect ways.