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Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle
Animal Reproduction Science 129 (2011) 140–145
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle S.S. Layek ∗ , T.K. Mohanty, A. Kumaresan, K. Behera, S. Chand Livestock Production & Management Section, Cattle Yard, National Dairy Research Institute, Karnal, Haryana 132001, India
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Article history: Received 6 July 2011 Received in revised form 5 November 2011 Accepted 19 November 2011 Available online 26 November 2011 Keywords: Estrus Ovulation Behavioural signs Zebu cattle
a b s t r a c t The present study reports the behavioural signs of estrus, their temporal distribution and duration of expression and their relationship with the time of ovulation in zebu cattle in order to identify the reliable sign(s) of estrus that could fairly predict the ovulation time. The onset, intensity and expression of various signs of estrus were continuously recorded till ovulation in 60 Sahiwal cows. Time of ovulation was determined by ultrasound examinations at 2 h interval. Estruses were mostly of moderate (52%) or weak (34%) intensity. Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane appeared early in relation to the ovulation time (31.27 ± 1.97, 31.05 ± 2.98 and 30.79 ± 2.53 h, respectively) in comparison to mounting (27.67 ± 2.33 h) and standing to be mounted (25.37 ± 2.11 h). Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane persisted significantly more duration (P < 0.01) than mounting and standing to be mounted. Further these cardinal signs appeared early in relation to time of ovulation, persisted for longer duration and expressed intensely. We conclude that mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane can be good predictor ovulation in this breed of cattle. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fertility over the past few decades is of serious concern in the dairy industry (Royal et al., 2000; López-Gatius, 2003). It is determined by very closely related composite events such as attainment of sexual maturity at an early age, cycling at regular interval, inseminated with fertile semen at proper time, conceive, calve normally, resume reproductive cyclicity and conceive well in time. Estrus detection and proper timing of insemination are the biggest bottleneck in achieving high conception rate in dairy animals (Roelofs et al., 2010). More commonly, animals are diagnosed to be in estrus based on the mounting or standing to be mounted, appearance of mucus discharge, and other physical activities (van Eerdenburg et al., 1996).
∗ Corresponding author. Tel.: +91 9896092516/9541471105. E-mail address: [email protected] (S.S. Layek). 0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2011.11.006
Different scoring systems have been developed and tested to identify proper estrus in Bos taurus cattle (van Eerdenburg et al., 1996, 2002; Van Vliet and van Eerdenburg, 1996). Those scoring system were developed on the basis of observed estrus signs and majority of them has taken mounting and standing to be mounted behaviour as the most reliable signs to predict ovulation time in Bos taurus cows (Roelofs et al., 2005). However, it is now well accepted that the expression of estrus behaviour varies with breed of cows (Venkata Naidu and Rao, 2006). Striking differences exist between Bos taurus and Bos indicus in terms of estrus duration and intensity of expression of different estrus signs (Mattoni and Ouedraogo, 2000; Walker et al., 2008). The routine AI program involves AM/PM schedule for insemination, i.e. cows found in estrus in the morning are inseminated in the same day evening and those in estrus in the evening are inseminated next morning (Trimberger and Davis, 1943). This protocol is recommended based on the fact that the interval
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between onset of estrus and ovulation ranges from 25.69 to 38.5 h (Saumande and Humblot, 2005). But the relationship between cardinal signs of estrus and ovulation is not common to all the cows and varies with breed and environment (Van Vliet and van Eerdenburg, 1996; Heres et al., 2000; Lyimo et al., 2000). As far as the indigenous cattle (Bos indicus) are concerned the estrous signs and their expression intensity has not been studied in detail. Further, the relationship of a particular estrus sign with the ovulation time is not known (Galina and Orihuela, 2007). It is in this regard the present study was undertaken to (i) identify the most reliable sign(s) to predict the time of onset of estrus and (ii) to find the relationship between estrous signs and time of ovulation so that time of ovulation could be predicted based on the estrous behaviour. 2. Materials and methods 2.1. Experimental animals The present study was conducted on Sahiwal cows maintained at Cattle Yard of National Dairy Research Institute (NDRI), Karnal, Haryana which is located at 29◦ 42 N latitude and 72◦ 02 E longitudes at an altitude of 250 m above the mean sea level in the bed of Indo-Gangetic alluvial plain. A subtropical climate prevails in the area. Maximum temperature during summer in the area is near about 45 ◦ C and minimum temperature during winter is near to 1.4 ◦ C. Relative humidity of the study area varies between 41–85% and rainfall between 760 and 960 mm. The day length in summer varies from 13 to 14 h whereas in winter it is 10–11 h. The experiment was approved by the Institutional Animal Ethics Committee (IAEC No. 2/09-21/2009). Sixty cows (between 1st and 5th parity) were observed to study the temporal pattern of estrus expression and to correlate the signs with ovulation. The duration of the experiment was seven months starting from September to March. The cows included in the study were more than 60 days postpartum and had already shown at least one spontaneous estrus before the commencement of the study. The average body weight of the experimental cows was 384.67 ± 5.26 kg (354–430 kg) and the body condition score of the cows were 3–3.5 (6 point scale, Prasad, 1994). The milk production of the cows varied from 8 to 16 l (9.95 ± 0.74 l) per day and all the animals were in the early stage of lactation. They were free from any anatomical and reproductive disabilities and also free from diseases. The experimental cows were kept in loose housing system under group management practice. The size of the paddock was 600 m2 and 80 cows were housed at a time in the paddock during the period of the study. The floor of the paddock was covered with brick on edges flooring. The paddock was surrounded by metal pipes in three sides and one side was guarded by feeding mangers and standing pipes. The feeding mangers were covered with asbestos sheets at moderate height with low slope inclination. At one corner of the paddock, there was provision of drinking water trough with running tap water. There were 6 canopy type tree plantations within the paddock for shelter of the cows during day time. The space in the paddock, feeding manger and watering trough was as per BIS standard.
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This system of housing allowed free movement of cows and easy identification during estrus detection activities. The cows were tagged with plastic ear tag, mentioning the animal number. The cows were fed with ad lib green fodder and measured amount of concentrate. The concentrate was fed at the rate of 1.5 kg per animal for body maintenance in general and additional concentrate was given at the rate of 1.0 kg for every 2.5 kg milk production, above 5.0 kg milk yield. The concentrate to the milking cows was fed in divided allowances during three times milking. The cows were milked in machine milking system. 2.2. Observation of estrus behaviour All the experimental cows were observed round the clock by trained personnel. The observers recorded the onset of estrus based on the behavioural signs (Restlessness, sniffing/licking of vulva, frequent micturition, tail raising, bellowing, mounted but not standing, chin resting, mounting other cows, mounting head side, standing to be mounted) and the frequency of a particular sign over different time periods. In order to confirm the visual detection of estrus the cows were examined for tumefaction of vulva, reddening of vulvar mucus membrane and uterine tonicity. The onset and the frequency of the expression of estrus signs were observed and recorded in the pre-tested estrus symptom recording chart designed for the purpose (Mangal, 2009). The midpoint time between the first observation of the cardinal signs of estrus and the previous check without any behavioural signs was considered as the time of onset of estrus. Similarly the end of the estrus was considered as the midpoint between first finding of an animal to be off estrus and the previous check in estrus. Estrus duration (in hour) was calculated from the onset and the cessation of estrus. 2.3. Scoring of estrus signs The scoring of the estrus behaviour was done following a scoring system developed by Dash (1980) for zebu cattle. In this scoring system, different behavioural signs observed during estrus were assigned with a score depending upon their frequency or intensity of expression (Table 2.1). All the frequencies or intensities of each behavioural signs were converted into a score of 1, 2 or 3. The scores obtained for each of the behavioural signs for an individual animal were added to obtain a total score. Total score for each animal observed was classified into three classes such as weak (1–11), moderate (12–22) and intense (23–33). 2.4. Detection of ovulation time The ovulation time was determined by using a linear array ultrasound scanner equipped with a multi-frequency transducer probe (Prosound 2, Aloka Ltd., Japan). The cows detected in estrus were subjected to ultrasound examination every two hour to check the ovarian follicle status. The ultrasound was performed in the temporary trevis designed within the paddock so that the disturbance to the cows was less. The midpoint between first finding of the
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Table 2.1 Scales for scoring estrus symptoms. Estrus symptoms
Standing heat Mounting on others Chin resting Sniffing/licking Tail raising Micturition Restlessness Aggression Bellowing Mucus discharge Tumefaction of vulva
Frequency of expression during an estrus period Weak (score-1)
Medium (score-2)
Strong (score-3)
<9 <9 <5 <6 <3 <3 Mild Mild Very seldom Absent Weak
9–14 9–14 5–7 6–8 3–4 3–4 Moderate Moderate Occasional Slight Moderate
>14 >14 >7 >8 >4 >4 Pronounced Pronounced Often Copious Intense
ruptured follicle and the previous check with intact follicle was considered ovulation time.
2.5. Statistical analysis Total Intensity of each estrus was calculated by summing up scores assigned to each value and they were classified into 3 classes viz. weak, moderate and strong. The cows in the first parity were taken in the primiparous group and rest of the cows (2nd–5th parity) were taken in the pluriparous group. Chi-square test was performed to find out the difference between intensity of expression between primiparous and pluriparous animals. Descriptive statistics was calculated for different duration of estrus, estrus to ovulation duration and end of estrus to ovulation duration and all means are represented as mean ± SE. Duration of estrus was grouped into 3 classes such as <12 h, 12–18 h and >18 h and estrus to ovulation duration was grouped into 3 classes (<28 h, 28–36 h and >36 h) and Chi-square test was used to find out difference between primiparous and pluriparous animals. The behaviours that were evident in more than 80% of the estruses such as standing to be mounted, mounting on herdmates, chin resting, sniffing/licking, mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane, were considered predominant estrus signs in Sahiwal breed. These signs were used for estimating the relationship of onset and duration of expression of a particular estrus sign with
time of ovulation. One-way ANOVA to find out difference between different cardinal signs in duration between onset of the cardinal signs to ovulation and duration of expression. All the analyses were performed using was performed using SigmPlot 11® software package (Systat software Inc., USA).
3. Results 3.1. Individual cardinal signs Among the sixty estrus periods observed, a majority of estrus periods in Sahiwal cows were of weak intensity (52%) followed by moderate intensity (34%). Expressions of individual cardinal signs are depicted in Table 3.1. Reddening of vulvar mucus membrane, tumefaction of vulva, mucus discharge and uterine tonicity appeared early and evident in all the estrous periods with intense expression intensity. Higher proportion of pluriparous cows showed intense reddening of mucus membrane (90.24%) and mucus discharge (75.61%) than the primiparous cows (70.59% and 56.25%, respectively). The chin resting and sniffing/licking the vulva region of other cows appeared next to above cited signs and was also evident in majority (89.65% and 91.38%, respectively) of the cows during the estrus. Sniffing/licking behaviour was shown by higher proportion of cows in primiparous group (100%) in comparison to pluriparous (87.80%) and higher proportion
Table 3.1 Intensity (%) of different cardinal signs of estrus in Sahiwal cows. Cardinal signs
Standing to be mounted Mounting on herdmates Chin resting Sniffing/licking Tail raising Bellowing Micturition Restlessness Aggression Reddening of vulva Tumefaction of vulva Amount of mucus discharge Uterine tone
Percent of animals
98.27 96.49 89.65 91.38 41.38 18.96 17.24 68.96 50 100 100 100 100
Degree of expression Weak
Moderate
Intense
35.09 56.37 40.38 47.17 25 45.45 90 50 51.72 3.45 1.73 7.02 3.46
40.35 27.27 42.31 39.62 62.5 45.45 10 30 27.59 12.07 13.79 22.81 5.18
24.56 16.36 17.31 13.21 12.5 9.1 – 20 20.69 84.48 84.34 70.17 91.36
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Table 3.2 Mean (±SE) duration of estrus and onset of estrus to ovulation in different parity of Sahiwal cows.
Primiparous (n = 17) Pluriparous (n = 43) Overall (n = 60)
Duration of estrus (h)
Interval from onset of estrus to ovulation (h)
Interval from end of estrus to ovulation (h)
14.35 ± 1.35 (9–28) 14.61 ± 0.99 (6–31) 14.53 ± 0.80 (6–31)
30.62 ± 1.96 (14.5–48) 32.51 ± 1.39 (16–56) 31.96 ± 1.13 (14.5–56)
16.32 ± 1.25 (1.5–22) 17.9 ± 0.74 (8–27) 17.47 ± 0.64 (8–27)
Fig. 3.1. Distribution of estrus duration (A) and onset of estrus to ovulation duration (B) in Sahiwal cows.
showed weak intensity of expression in pluriparous cows (52.78%) than primiparous cows (35.29%). Standing to be mounted and mounting behaviours were evident in 98.27% and 96.49% estrus periods, respectively but the signs were weak or moderate in expression. Weak expression intensity was higher in the pluriparous cows in comparison to primiparous cows. Micturition (17.24%) and bellowing (18.96%) was shown by very few cows with low expression intensity.
We observed that estrus duration was <18 h in majority (84.48%) of the cows. Low proportion of cows showed estrus of more than 18 h duration (15.52%). The estrus to ovulation duration was also in similar pattern. In the present study, 79.31% of the cows ovulated in <36 h after the onset of the estrus and low proportion (20.69%) ovulated >36 h after the onset of the estrus (Fig. 3.1). There was no significant variation in between primiparous and pluriparous cows in this distribution.
3.2. Estrus duration and ovulation time
3.3. Timing of ovulation in relation to cardinal signs of estrus
The duration of estrus in Sahiwal cows ranged from 6 to 31 h with an average of 14.53 ± 0.80 h. There was no significant difference in the duration of estrus between pluriparous and primiparous cows (Table 3.2). On an average, the experimental cows ovulated 31.96 ± 1.13 h after the onset of estrus. Like in case of the estrus duration, there was no significant difference between pluriparous and primiparous (32.51 ± 1.39 h) and primiparous (30.62 ± 1.96 h) cows regarding the ovulation time in relation to the onset of estrus. The time interval between the end of estrus and ovulation ranged 1.5–22 h in primiparous and 8–27 h in pluriparous cows. The average time interval between the end of estrus and ovulation was 17.47 ± 0.64 h.
Among the different signs of estrus, tumefaction of vulva, reddening of vulvar mucus membrane and the mucus discharge appeared at earliest in relation to the ovulation time, followed by sniffing or licking of vulva and chin resting. Mounting herdmates was the next behaviour to be displayed followed by the standing to be mounted behaviour appeared at the last in relation to ovulation time. The onset, end and duration of expression of different signs are represented in Table 3.3. Tumefaction of vulva, reddening of vulvar mucus membrane and the mucus discharge were observed in all the cows and these behaviours were exclusively found during the estrus period and their expression intensity was also
Table 3.3 Ovulation time in relation to onset and end of different signs of estrus in zebu cattle. Onset to ovulation duration (h) Mucus discharge Tumefaction of vulva Reddening of vulva Chin resting Sniffing/licking Mounting on herdmates Standing to be mounted
31.27 31.05 30.79 30.09 29.67 27.67 25.37
± ± ± ± ± ± ±
1.97 2.98 2.53 3.79 2.35 2.33 2.11
Values containing different superscript in column vary significantly at P < 0.01.
End to ovulation duration (h) 18.65 11.77 12.59 18.63 18.45 21.55 20.17
± ± ± ± ± ± ±
1.56 1.29 1.76 1.81 1.59 2.01 2.32
Duration of expression (h) 12.89 20.55 20.25 11.73 11.32 6.17 5.15
± ± ± ± ± ± ±
1.79b 2.14a 1.63a 2.56b 1.69b 2.17c 2.05c
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higher in case of Sahiwal animals. These behaviours were displayed at an average 30 h (31.05 ± 2.98 h, 30.79 ± 2.53 h, 31.27 ± 1.97 h, respectively) before the ovulation. Although sniffing and licking of vulva (91.38%) and chin resting (89.65%) behaviour was displayed by most of the cows during the estrus and they appeared early (29.67 ± 2.35 h and 30.09 ± 3.79 h) before ovulation, these behaviours were not always exclusive to estrus. Mounting (27.67 ± 2.33 h) and standing to be mounted (25.37 ± 2.11 h) behaviour were late in appearance in relation to the ovulation time. Though these behaviours were evident in majority of (mounting on herdmates in 94.12% and Standing to be mounted in 97.12%) the estrous period in the present study, their intensity of expression was weak and the duration of the expression was significantly (P < 0.01) lower (6.17 ± 2.17 h and 5.15 ± 2.05 h, respectively) than other signs like tumefaction of vulva (20.55 ± 2.14), reddening of vulvar mucus membrane (20.25 ± 1.63 h) and mucus discharge (12.89 ± 1.79 h) (Table 3.3). Again a single mounting lasted for no more than 4–6 s, making the behaviour tough to be observed if not monitored continuously. 4. Discussion For a parameter to be useful as a predictor of ovulation time, the parameter have to meet several pre-requisites, such as it should have a small variation in time to ovulation, the measurements should be easy to carry out, repeatable and preferable to be automated. It should also be present in the high proportion of cows (van Eerdenburg et al., 1996). We, in the present study, studied the behavioural signs of estrus and their relationship with ovulation in zebu cattle. The cardinal signs taken into consideration were found in more than 80% of estrous periods. Along with the most conclusive behaviours like mounting herdmates and standing to be mounted other behaviours like tumefaction of vulva, reddening of vulvar mucus membrane, mucus discharge, sniffing/licking and chin resting were also taken into consideration for calculating their duration from the ovulation time. The present study observed that a majority of the cows showed standing to be mounted and mounting on herdmates activity during estrus, which is in agreement with those reported by Negussie et al. (2002) on Fogera cows (Bos indicus). However these two estrus signs were expressed for short period of time in majority of the cows. Further the expression intensity of these two parameters was also weak and a single mounting lasted for not more than 4–6 s. Thus detection of estrus based on these signs could be difficult unless observed continuously. Similar type of weaker expression of mounting behaviour (1.3 mounts/h) was reported by Orihuela (2000). Mounting behaviour is highly dependent upon the number of cows in the sexually active group and their social hierarchy (Hurnik et al., 1975), thus number of cows in a particular group could also influence the expression intensity of a particular behaviour besides the breed specific characteristics. In addition to this the endocrine profile during the periestrual period of an individual animal has also been reported to affect the estrus behaviour expression
(Roelofs et al., 2005). It has been reported that mounting on herdmates behaviour could be used as a good predictor of ovulation in Holstein Friesian cows (Roelofs et al., 2005) but the same may not be applicable in Sahiwal cows as it is expressed in less intensity and for shorter duration as compared to Holstein Friesian. There is every chance that these behaviours go unnoticed if a continuous observation for estrus detection is not followed which is not a feasible estrus detection option thus restricting its use as a good predictor of ovulation in Sahiwal cattle. Although sniffing and licking of vulva (91.38%) and chin resting (89.65%) behaviour was displayed by most of the cows during estrus but these behaviours was not exclusive to the estrus period. These behaviours were also displayed by the cows when they were not in estrus, which is in consonance with the findings of earlier workers (van Eerdenburg et al., 2002; Roelofs et al., 2005). But the expression of these behaviours during estrus was always accompanied by segregation of the cows from the herd which was not evident in non estrus animals. The cows showing sniffing or licking behaviour in the small sexually active group can therefore be easily identified and the ovulation time can be predicted based upon these behaviours. Tumefaction of vulva, reddening of vulvar mucus membrane and the mucus discharge were observed in all the cows and these behaviours were exclusively found during the estrus period and their expression intensity was also higher in case of Sahiwal cows. Also these signs can easily be identified from distance as the vulva region of the Sahiwal cows is large and generally the cows in estrus have a characteristics tail raising pattern. Uniform red colour of the breed makes it easier to identify the dried cervical mucus secretion which takes a typical white colour after get dried. The estrus duration recorded in the present study is in consonance with those reported by earlier workers (13.6 ± 1.0 h, Galina et al., 1987; 15.54 ± 0.28, Venkata Naidu and Rao, 2006). However the duration is higher than the value observed by Pinheiro et al. (1998) in Nelore cattle (10.5 ± 1.0 h). The reason for the difference in the duration between the studies may be due to different criteria, used to describe the onset and the end of estrus. The observation schedule followed by different workers may also be the reason for the difference. 5. Conclusion From the present study it can be concluded that in Sahiwal cows the mounting behaviour is less intense and expressed for shorter duration making these behaviour less conclusive in detection of estrus cows. Tumefaction of vulva, reddening of vulvar mucus membrane, mucus discharge, chin resting and sniffing/licking could be better predictor of ovulation time in this breed of cattle. Acknowledgements The authors express their sincere gratitude to Dr. A.K. Srivastava, Director and Vice Chancellor, National Dairy Research Institute, Karnal and Dr. Shiv Prasad, in charge,
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Cattle yard, National Dairy Research Institute, Karnal for providing all research facilities for the successful completion of this study. The work was partly funded by the World bank supported NAIP project (NAIP/C4/C2008/032). The authors are also thankful to Mr. Jita Ram for his technical assistance during the study. References Dash, P.C., 1980. Reproductive behaviour of dairy cows (Bos indicus, Bos taurus and Bos taurus × Bos indicus). Ph.D. Thesis. (NDRI) Panjab University Chandigarh. Galina, C.S., Orihuela, A., 2007. The detection of estrus in cattle raised under tropical conditions: what we know and what we need to know. Horm. Behav. 52, 32–38. Galina, C.S., Orihuela, A., Duchateau, A., 1987. Reproductive physiology in Zebu cattle: unique reproductive aspects that affect their performance. Food Anim. Pract. 3, 619–633. Heres, L., Dieleman, S.J., van Eerdenburg, F.J.C.M., 2000. Validation of a new method of visual oestrus detection on the farm. Vet. Quart. 22, 50–55. Hurnik, J.F., King, G.J., Robertson, H.A., 1975. Estrus and related behaviour in postpartum Holstein cows. Appl. Anim. Ethol. 2, 55–65. López-Gatius, F., 2003. Is fertility declining in dairy cattle? A retrospective study in north-eastern Spain. Theriogenology 60, 89–99. Lyimo, Z.C., Nielen, M., Ouweltjes, W., Kruip, T.A.M., van Eerdenburg, F.J.C.M., 2000. Relationship among estradiol, cortisol and intensity of estrous behavior in dairy cattle. Theriogenology 53 (9), 1783–1795. Mangal, V., 2009. Study on Estrous Behaviour, Cervical Mucus Characteristics and Fertility in Sahiwal Cattle. M.V. Sc. Thesis. NDRI, Karnal, Haryana, India. Mattoni, M., Ouedraogo, A., 2000. A comparative study on the estrus response to PGF2 ␣ analogue treatment, and conception rates according to time of AI, in Zebu (Bos indicus) and Baoule (Bos taurus) cattle. Trop. Anim. Health Prod. 32, 127–134. Negussie, F., Kassa, T., Tibbo, M., 2002. Behavioural and physical signs associated with estrus and some aspects of reproductive performance in Fogera cows and heifers. Trop. Anim. Health Prod. 34, 319–328.
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Orihuela, A., 2000. Some factors affecting the behavioural manifestation of oestrus in cattle: a review. Appl. Anim. Behav. Sci. 70, 1–16. Pinheiro, O.L., Barros, C.M., Figueiredo, R.A., Valle, E.R., Encamacao, R.O., Padovani, C.R., 1998. Estrus behaviour and the estrus-to-ovulation interval in Nellore cattle (Bos indicus) with natural estrus or estrus induced with prostaglandin F2 ␣ or norgestomet and estradiol valerate. Theriogenology 49, 667–681. Prasad, S., 1994. Body condition scoring and feeding management in relation to production performance of cross bred dairy cattle. Ph.D. Thesis. National Dairy Research Institute, Karnal, India. Roelofs, J., López–Gatius, F., Hunter, R.H.F., van Eerdenburg, F.J.C.M., Hanzen, C., 2010. When is a cow in estrus? Clinical and practical aspects. Theriogenology 74 (3), 327–344. Roelofs, J.B., van Eerdenburg, F.J.C.M., Soede, N.M., Kemp, B., 2005. Various behavioural signs of estrus and their relationship with time of ovulation in dairy cattle. Theriogenology 63, 1366–1377. Royal, M.D., Darwash, A.O., Flint, A.P.F., Webb, R., Wooliams, J.A., Lamming, G.E., 2000. Declining fertility in dairy cattle: changes in traditional and endocrine parameters of fertility. Anim. Sci. 70, 487–501. Saumande, J., Humblot, P., 2005. The variability in the interval between estrus and ovulation in cattle and its determinants. Anim. Reprod. Sci. 85, 171–182. Trimberger, G.W., Davis, H.P., 1943. Conception rate in dairy cattle from artificial insemination at various stages of estrus. Nebraska Agric. Exp. Stn. Res. Bull. 129, 1–14. van Eerdenburg, F.J.C.M., Karthaus, D., Taverne, M.A.M., Merics, I., Szenci, O., 2002. The relationship between estrus behavioral score and time of ovulation in dairy cattle. J. Dairy Sci. 85, 1150–1156. van Eerdenburg, F.J.C.M., Loeffler, S.H., Van Vliet, J.H., 1996. Detection of oestrus in dairy cows: a new approach to an old problem. Vet. Quart. 18, 52–54. Van Vliet, J.H., van Eerdenburg, F.J.C.M., 1996. Sexual activities of oestrus detection in lactating Holstein cows. Appl. Anim. Behav. Sci. 50, 57–69. Venkata Naidu, G., Rao, K.B., 2006. Estrus pattern and conception rate in postpartum lactating Ongole (Zebu) cows. Indian J. Anim. Reprod. 27 (1), 14–17. Walker, S.L., Smith, R.F., Routly, J.E., Jones, D.N., Morris, M.J., Dobson, H., 2008. Lameness, activity time–budgets and estrus expression in dairy cattle. J. Dairy Sci. 91, 4552–4559.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Behavioural signs of estrus and their relationship to time of ovulation in Zebu (Sahiwal) cattle S.S. Layek ∗ , T.K. Mohanty, A. Kumaresan, K. Behera, S. Chand Livestock Production & Management Section, Cattle Yard, National Dairy Research Institute, Karnal, Haryana 132001, India
a r t i c l e
i n f o
Article history: Received 6 July 2011 Received in revised form 5 November 2011 Accepted 19 November 2011 Available online 26 November 2011 Keywords: Estrus Ovulation Behavioural signs Zebu cattle
a b s t r a c t The present study reports the behavioural signs of estrus, their temporal distribution and duration of expression and their relationship with the time of ovulation in zebu cattle in order to identify the reliable sign(s) of estrus that could fairly predict the ovulation time. The onset, intensity and expression of various signs of estrus were continuously recorded till ovulation in 60 Sahiwal cows. Time of ovulation was determined by ultrasound examinations at 2 h interval. Estruses were mostly of moderate (52%) or weak (34%) intensity. Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane appeared early in relation to the ovulation time (31.27 ± 1.97, 31.05 ± 2.98 and 30.79 ± 2.53 h, respectively) in comparison to mounting (27.67 ± 2.33 h) and standing to be mounted (25.37 ± 2.11 h). Mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane persisted significantly more duration (P < 0.01) than mounting and standing to be mounted. Further these cardinal signs appeared early in relation to time of ovulation, persisted for longer duration and expressed intensely. We conclude that mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane can be good predictor ovulation in this breed of cattle. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Fertility over the past few decades is of serious concern in the dairy industry (Royal et al., 2000; López-Gatius, 2003). It is determined by very closely related composite events such as attainment of sexual maturity at an early age, cycling at regular interval, inseminated with fertile semen at proper time, conceive, calve normally, resume reproductive cyclicity and conceive well in time. Estrus detection and proper timing of insemination are the biggest bottleneck in achieving high conception rate in dairy animals (Roelofs et al., 2010). More commonly, animals are diagnosed to be in estrus based on the mounting or standing to be mounted, appearance of mucus discharge, and other physical activities (van Eerdenburg et al., 1996).
∗ Corresponding author. Tel.: +91 9896092516/9541471105. E-mail address: [email protected] (S.S. Layek). 0378-4320/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2011.11.006
Different scoring systems have been developed and tested to identify proper estrus in Bos taurus cattle (van Eerdenburg et al., 1996, 2002; Van Vliet and van Eerdenburg, 1996). Those scoring system were developed on the basis of observed estrus signs and majority of them has taken mounting and standing to be mounted behaviour as the most reliable signs to predict ovulation time in Bos taurus cows (Roelofs et al., 2005). However, it is now well accepted that the expression of estrus behaviour varies with breed of cows (Venkata Naidu and Rao, 2006). Striking differences exist between Bos taurus and Bos indicus in terms of estrus duration and intensity of expression of different estrus signs (Mattoni and Ouedraogo, 2000; Walker et al., 2008). The routine AI program involves AM/PM schedule for insemination, i.e. cows found in estrus in the morning are inseminated in the same day evening and those in estrus in the evening are inseminated next morning (Trimberger and Davis, 1943). This protocol is recommended based on the fact that the interval
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between onset of estrus and ovulation ranges from 25.69 to 38.5 h (Saumande and Humblot, 2005). But the relationship between cardinal signs of estrus and ovulation is not common to all the cows and varies with breed and environment (Van Vliet and van Eerdenburg, 1996; Heres et al., 2000; Lyimo et al., 2000). As far as the indigenous cattle (Bos indicus) are concerned the estrous signs and their expression intensity has not been studied in detail. Further, the relationship of a particular estrus sign with the ovulation time is not known (Galina and Orihuela, 2007). It is in this regard the present study was undertaken to (i) identify the most reliable sign(s) to predict the time of onset of estrus and (ii) to find the relationship between estrous signs and time of ovulation so that time of ovulation could be predicted based on the estrous behaviour. 2. Materials and methods 2.1. Experimental animals The present study was conducted on Sahiwal cows maintained at Cattle Yard of National Dairy Research Institute (NDRI), Karnal, Haryana which is located at 29◦ 42 N latitude and 72◦ 02 E longitudes at an altitude of 250 m above the mean sea level in the bed of Indo-Gangetic alluvial plain. A subtropical climate prevails in the area. Maximum temperature during summer in the area is near about 45 ◦ C and minimum temperature during winter is near to 1.4 ◦ C. Relative humidity of the study area varies between 41–85% and rainfall between 760 and 960 mm. The day length in summer varies from 13 to 14 h whereas in winter it is 10–11 h. The experiment was approved by the Institutional Animal Ethics Committee (IAEC No. 2/09-21/2009). Sixty cows (between 1st and 5th parity) were observed to study the temporal pattern of estrus expression and to correlate the signs with ovulation. The duration of the experiment was seven months starting from September to March. The cows included in the study were more than 60 days postpartum and had already shown at least one spontaneous estrus before the commencement of the study. The average body weight of the experimental cows was 384.67 ± 5.26 kg (354–430 kg) and the body condition score of the cows were 3–3.5 (6 point scale, Prasad, 1994). The milk production of the cows varied from 8 to 16 l (9.95 ± 0.74 l) per day and all the animals were in the early stage of lactation. They were free from any anatomical and reproductive disabilities and also free from diseases. The experimental cows were kept in loose housing system under group management practice. The size of the paddock was 600 m2 and 80 cows were housed at a time in the paddock during the period of the study. The floor of the paddock was covered with brick on edges flooring. The paddock was surrounded by metal pipes in three sides and one side was guarded by feeding mangers and standing pipes. The feeding mangers were covered with asbestos sheets at moderate height with low slope inclination. At one corner of the paddock, there was provision of drinking water trough with running tap water. There were 6 canopy type tree plantations within the paddock for shelter of the cows during day time. The space in the paddock, feeding manger and watering trough was as per BIS standard.
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This system of housing allowed free movement of cows and easy identification during estrus detection activities. The cows were tagged with plastic ear tag, mentioning the animal number. The cows were fed with ad lib green fodder and measured amount of concentrate. The concentrate was fed at the rate of 1.5 kg per animal for body maintenance in general and additional concentrate was given at the rate of 1.0 kg for every 2.5 kg milk production, above 5.0 kg milk yield. The concentrate to the milking cows was fed in divided allowances during three times milking. The cows were milked in machine milking system. 2.2. Observation of estrus behaviour All the experimental cows were observed round the clock by trained personnel. The observers recorded the onset of estrus based on the behavioural signs (Restlessness, sniffing/licking of vulva, frequent micturition, tail raising, bellowing, mounted but not standing, chin resting, mounting other cows, mounting head side, standing to be mounted) and the frequency of a particular sign over different time periods. In order to confirm the visual detection of estrus the cows were examined for tumefaction of vulva, reddening of vulvar mucus membrane and uterine tonicity. The onset and the frequency of the expression of estrus signs were observed and recorded in the pre-tested estrus symptom recording chart designed for the purpose (Mangal, 2009). The midpoint time between the first observation of the cardinal signs of estrus and the previous check without any behavioural signs was considered as the time of onset of estrus. Similarly the end of the estrus was considered as the midpoint between first finding of an animal to be off estrus and the previous check in estrus. Estrus duration (in hour) was calculated from the onset and the cessation of estrus. 2.3. Scoring of estrus signs The scoring of the estrus behaviour was done following a scoring system developed by Dash (1980) for zebu cattle. In this scoring system, different behavioural signs observed during estrus were assigned with a score depending upon their frequency or intensity of expression (Table 2.1). All the frequencies or intensities of each behavioural signs were converted into a score of 1, 2 or 3. The scores obtained for each of the behavioural signs for an individual animal were added to obtain a total score. Total score for each animal observed was classified into three classes such as weak (1–11), moderate (12–22) and intense (23–33). 2.4. Detection of ovulation time The ovulation time was determined by using a linear array ultrasound scanner equipped with a multi-frequency transducer probe (Prosound 2, Aloka Ltd., Japan). The cows detected in estrus were subjected to ultrasound examination every two hour to check the ovarian follicle status. The ultrasound was performed in the temporary trevis designed within the paddock so that the disturbance to the cows was less. The midpoint between first finding of the
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Table 2.1 Scales for scoring estrus symptoms. Estrus symptoms
Standing heat Mounting on others Chin resting Sniffing/licking Tail raising Micturition Restlessness Aggression Bellowing Mucus discharge Tumefaction of vulva
Frequency of expression during an estrus period Weak (score-1)
Medium (score-2)
Strong (score-3)
<9 <9 <5 <6 <3 <3 Mild Mild Very seldom Absent Weak
9–14 9–14 5–7 6–8 3–4 3–4 Moderate Moderate Occasional Slight Moderate
>14 >14 >7 >8 >4 >4 Pronounced Pronounced Often Copious Intense
ruptured follicle and the previous check with intact follicle was considered ovulation time.
2.5. Statistical analysis Total Intensity of each estrus was calculated by summing up scores assigned to each value and they were classified into 3 classes viz. weak, moderate and strong. The cows in the first parity were taken in the primiparous group and rest of the cows (2nd–5th parity) were taken in the pluriparous group. Chi-square test was performed to find out the difference between intensity of expression between primiparous and pluriparous animals. Descriptive statistics was calculated for different duration of estrus, estrus to ovulation duration and end of estrus to ovulation duration and all means are represented as mean ± SE. Duration of estrus was grouped into 3 classes such as <12 h, 12–18 h and >18 h and estrus to ovulation duration was grouped into 3 classes (<28 h, 28–36 h and >36 h) and Chi-square test was used to find out difference between primiparous and pluriparous animals. The behaviours that were evident in more than 80% of the estruses such as standing to be mounted, mounting on herdmates, chin resting, sniffing/licking, mucus discharge, tumefaction of vulva and reddening of vulvar mucus membrane, were considered predominant estrus signs in Sahiwal breed. These signs were used for estimating the relationship of onset and duration of expression of a particular estrus sign with
time of ovulation. One-way ANOVA to find out difference between different cardinal signs in duration between onset of the cardinal signs to ovulation and duration of expression. All the analyses were performed using was performed using SigmPlot 11® software package (Systat software Inc., USA).
3. Results 3.1. Individual cardinal signs Among the sixty estrus periods observed, a majority of estrus periods in Sahiwal cows were of weak intensity (52%) followed by moderate intensity (34%). Expressions of individual cardinal signs are depicted in Table 3.1. Reddening of vulvar mucus membrane, tumefaction of vulva, mucus discharge and uterine tonicity appeared early and evident in all the estrous periods with intense expression intensity. Higher proportion of pluriparous cows showed intense reddening of mucus membrane (90.24%) and mucus discharge (75.61%) than the primiparous cows (70.59% and 56.25%, respectively). The chin resting and sniffing/licking the vulva region of other cows appeared next to above cited signs and was also evident in majority (89.65% and 91.38%, respectively) of the cows during the estrus. Sniffing/licking behaviour was shown by higher proportion of cows in primiparous group (100%) in comparison to pluriparous (87.80%) and higher proportion
Table 3.1 Intensity (%) of different cardinal signs of estrus in Sahiwal cows. Cardinal signs
Standing to be mounted Mounting on herdmates Chin resting Sniffing/licking Tail raising Bellowing Micturition Restlessness Aggression Reddening of vulva Tumefaction of vulva Amount of mucus discharge Uterine tone
Percent of animals
98.27 96.49 89.65 91.38 41.38 18.96 17.24 68.96 50 100 100 100 100
Degree of expression Weak
Moderate
Intense
35.09 56.37 40.38 47.17 25 45.45 90 50 51.72 3.45 1.73 7.02 3.46
40.35 27.27 42.31 39.62 62.5 45.45 10 30 27.59 12.07 13.79 22.81 5.18
24.56 16.36 17.31 13.21 12.5 9.1 – 20 20.69 84.48 84.34 70.17 91.36
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Table 3.2 Mean (±SE) duration of estrus and onset of estrus to ovulation in different parity of Sahiwal cows.
Primiparous (n = 17) Pluriparous (n = 43) Overall (n = 60)
Duration of estrus (h)
Interval from onset of estrus to ovulation (h)
Interval from end of estrus to ovulation (h)
14.35 ± 1.35 (9–28) 14.61 ± 0.99 (6–31) 14.53 ± 0.80 (6–31)
30.62 ± 1.96 (14.5–48) 32.51 ± 1.39 (16–56) 31.96 ± 1.13 (14.5–56)
16.32 ± 1.25 (1.5–22) 17.9 ± 0.74 (8–27) 17.47 ± 0.64 (8–27)
Fig. 3.1. Distribution of estrus duration (A) and onset of estrus to ovulation duration (B) in Sahiwal cows.
showed weak intensity of expression in pluriparous cows (52.78%) than primiparous cows (35.29%). Standing to be mounted and mounting behaviours were evident in 98.27% and 96.49% estrus periods, respectively but the signs were weak or moderate in expression. Weak expression intensity was higher in the pluriparous cows in comparison to primiparous cows. Micturition (17.24%) and bellowing (18.96%) was shown by very few cows with low expression intensity.
We observed that estrus duration was <18 h in majority (84.48%) of the cows. Low proportion of cows showed estrus of more than 18 h duration (15.52%). The estrus to ovulation duration was also in similar pattern. In the present study, 79.31% of the cows ovulated in <36 h after the onset of the estrus and low proportion (20.69%) ovulated >36 h after the onset of the estrus (Fig. 3.1). There was no significant variation in between primiparous and pluriparous cows in this distribution.
3.2. Estrus duration and ovulation time
3.3. Timing of ovulation in relation to cardinal signs of estrus
The duration of estrus in Sahiwal cows ranged from 6 to 31 h with an average of 14.53 ± 0.80 h. There was no significant difference in the duration of estrus between pluriparous and primiparous cows (Table 3.2). On an average, the experimental cows ovulated 31.96 ± 1.13 h after the onset of estrus. Like in case of the estrus duration, there was no significant difference between pluriparous and primiparous (32.51 ± 1.39 h) and primiparous (30.62 ± 1.96 h) cows regarding the ovulation time in relation to the onset of estrus. The time interval between the end of estrus and ovulation ranged 1.5–22 h in primiparous and 8–27 h in pluriparous cows. The average time interval between the end of estrus and ovulation was 17.47 ± 0.64 h.
Among the different signs of estrus, tumefaction of vulva, reddening of vulvar mucus membrane and the mucus discharge appeared at earliest in relation to the ovulation time, followed by sniffing or licking of vulva and chin resting. Mounting herdmates was the next behaviour to be displayed followed by the standing to be mounted behaviour appeared at the last in relation to ovulation time. The onset, end and duration of expression of different signs are represented in Table 3.3. Tumefaction of vulva, reddening of vulvar mucus membrane and the mucus discharge were observed in all the cows and these behaviours were exclusively found during the estrus period and their expression intensity was also
Table 3.3 Ovulation time in relation to onset and end of different signs of estrus in zebu cattle. Onset to ovulation duration (h) Mucus discharge Tumefaction of vulva Reddening of vulva Chin resting Sniffing/licking Mounting on herdmates Standing to be mounted
31.27 31.05 30.79 30.09 29.67 27.67 25.37
± ± ± ± ± ± ±
1.97 2.98 2.53 3.79 2.35 2.33 2.11
Values containing different superscript in column vary significantly at P < 0.01.
End to ovulation duration (h) 18.65 11.77 12.59 18.63 18.45 21.55 20.17
± ± ± ± ± ± ±
1.56 1.29 1.76 1.81 1.59 2.01 2.32
Duration of expression (h) 12.89 20.55 20.25 11.73 11.32 6.17 5.15
± ± ± ± ± ± ±
1.79b 2.14a 1.63a 2.56b 1.69b 2.17c 2.05c
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higher in case of Sahiwal animals. These behaviours were displayed at an average 30 h (31.05 ± 2.98 h, 30.79 ± 2.53 h, 31.27 ± 1.97 h, respectively) before the ovulation. Although sniffing and licking of vulva (91.38%) and chin resting (89.65%) behaviour was displayed by most of the cows during the estrus and they appeared early (29.67 ± 2.35 h and 30.09 ± 3.79 h) before ovulation, these behaviours were not always exclusive to estrus. Mounting (27.67 ± 2.33 h) and standing to be mounted (25.37 ± 2.11 h) behaviour were late in appearance in relation to the ovulation time. Though these behaviours were evident in majority of (mounting on herdmates in 94.12% and Standing to be mounted in 97.12%) the estrous period in the present study, their intensity of expression was weak and the duration of the expression was significantly (P < 0.01) lower (6.17 ± 2.17 h and 5.15 ± 2.05 h, respectively) than other signs like tumefaction of vulva (20.55 ± 2.14), reddening of vulvar mucus membrane (20.25 ± 1.63 h) and mucus discharge (12.89 ± 1.79 h) (Table 3.3). Again a single mounting lasted for no more than 4–6 s, making the behaviour tough to be observed if not monitored continuously. 4. Discussion For a parameter to be useful as a predictor of ovulation time, the parameter have to meet several pre-requisites, such as it should have a small variation in time to ovulation, the measurements should be easy to carry out, repeatable and preferable to be automated. It should also be present in the high proportion of cows (van Eerdenburg et al., 1996). We, in the present study, studied the behavioural signs of estrus and their relationship with ovulation in zebu cattle. The cardinal signs taken into consideration were found in more than 80% of estrous periods. Along with the most conclusive behaviours like mounting herdmates and standing to be mounted other behaviours like tumefaction of vulva, reddening of vulvar mucus membrane, mucus discharge, sniffing/licking and chin resting were also taken into consideration for calculating their duration from the ovulation time. The present study observed that a majority of the cows showed standing to be mounted and mounting on herdmates activity during estrus, which is in agreement with those reported by Negussie et al. (2002) on Fogera cows (Bos indicus). However these two estrus signs were expressed for short period of time in majority of the cows. Further the expression intensity of these two parameters was also weak and a single mounting lasted for not more than 4–6 s. Thus detection of estrus based on these signs could be difficult unless observed continuously. Similar type of weaker expression of mounting behaviour (1.3 mounts/h) was reported by Orihuela (2000). Mounting behaviour is highly dependent upon the number of cows in the sexually active group and their social hierarchy (Hurnik et al., 1975), thus number of cows in a particular group could also influence the expression intensity of a particular behaviour besides the breed specific characteristics. In addition to this the endocrine profile during the periestrual period of an individual animal has also been reported to affect the estrus behaviour expression
(Roelofs et al., 2005). It has been reported that mounting on herdmates behaviour could be used as a good predictor of ovulation in Holstein Friesian cows (Roelofs et al., 2005) but the same may not be applicable in Sahiwal cows as it is expressed in less intensity and for shorter duration as compared to Holstein Friesian. There is every chance that these behaviours go unnoticed if a continuous observation for estrus detection is not followed which is not a feasible estrus detection option thus restricting its use as a good predictor of ovulation in Sahiwal cattle. Although sniffing and licking of vulva (91.38%) and chin resting (89.65%) behaviour was displayed by most of the cows during estrus but these behaviours was not exclusive to the estrus period. These behaviours were also displayed by the cows when they were not in estrus, which is in consonance with the findings of earlier workers (van Eerdenburg et al., 2002; Roelofs et al., 2005). But the expression of these behaviours during estrus was always accompanied by segregation of the cows from the herd which was not evident in non estrus animals. The cows showing sniffing or licking behaviour in the small sexually active group can therefore be easily identified and the ovulation time can be predicted based upon these behaviours. Tumefaction of vulva, reddening of vulvar mucus membrane and the mucus discharge were observed in all the cows and these behaviours were exclusively found during the estrus period and their expression intensity was also higher in case of Sahiwal cows. Also these signs can easily be identified from distance as the vulva region of the Sahiwal cows is large and generally the cows in estrus have a characteristics tail raising pattern. Uniform red colour of the breed makes it easier to identify the dried cervical mucus secretion which takes a typical white colour after get dried. The estrus duration recorded in the present study is in consonance with those reported by earlier workers (13.6 ± 1.0 h, Galina et al., 1987; 15.54 ± 0.28, Venkata Naidu and Rao, 2006). However the duration is higher than the value observed by Pinheiro et al. (1998) in Nelore cattle (10.5 ± 1.0 h). The reason for the difference in the duration between the studies may be due to different criteria, used to describe the onset and the end of estrus. The observation schedule followed by different workers may also be the reason for the difference. 5. Conclusion From the present study it can be concluded that in Sahiwal cows the mounting behaviour is less intense and expressed for shorter duration making these behaviour less conclusive in detection of estrus cows. Tumefaction of vulva, reddening of vulvar mucus membrane, mucus discharge, chin resting and sniffing/licking could be better predictor of ovulation time in this breed of cattle. Acknowledgements The authors express their sincere gratitude to Dr. A.K. Srivastava, Director and Vice Chancellor, National Dairy Research Institute, Karnal and Dr. Shiv Prasad, in charge,
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Cattle yard, National Dairy Research Institute, Karnal for providing all research facilities for the successful completion of this study. The work was partly funded by the World bank supported NAIP project (NAIP/C4/C2008/032). The authors are also thankful to Mr. Jita Ram for his technical assistance during the study. References Dash, P.C., 1980. Reproductive behaviour of dairy cows (Bos indicus, Bos taurus and Bos taurus × Bos indicus). Ph.D. Thesis. (NDRI) Panjab University Chandigarh. Galina, C.S., Orihuela, A., 2007. The detection of estrus in cattle raised under tropical conditions: what we know and what we need to know. Horm. Behav. 52, 32–38. Galina, C.S., Orihuela, A., Duchateau, A., 1987. Reproductive physiology in Zebu cattle: unique reproductive aspects that affect their performance. Food Anim. Pract. 3, 619–633. Heres, L., Dieleman, S.J., van Eerdenburg, F.J.C.M., 2000. Validation of a new method of visual oestrus detection on the farm. Vet. Quart. 22, 50–55. Hurnik, J.F., King, G.J., Robertson, H.A., 1975. Estrus and related behaviour in postpartum Holstein cows. Appl. Anim. Ethol. 2, 55–65. López-Gatius, F., 2003. Is fertility declining in dairy cattle? A retrospective study in north-eastern Spain. Theriogenology 60, 89–99. Lyimo, Z.C., Nielen, M., Ouweltjes, W., Kruip, T.A.M., van Eerdenburg, F.J.C.M., 2000. Relationship among estradiol, cortisol and intensity of estrous behavior in dairy cattle. Theriogenology 53 (9), 1783–1795. Mangal, V., 2009. Study on Estrous Behaviour, Cervical Mucus Characteristics and Fertility in Sahiwal Cattle. M.V. Sc. Thesis. NDRI, Karnal, Haryana, India. Mattoni, M., Ouedraogo, A., 2000. A comparative study on the estrus response to PGF2 ␣ analogue treatment, and conception rates according to time of AI, in Zebu (Bos indicus) and Baoule (Bos taurus) cattle. Trop. Anim. Health Prod. 32, 127–134. Negussie, F., Kassa, T., Tibbo, M., 2002. Behavioural and physical signs associated with estrus and some aspects of reproductive performance in Fogera cows and heifers. Trop. Anim. Health Prod. 34, 319–328.
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