- Email: [email protected]
Relationship among estradiol, cortisol and intensity of estrous behavior in dairy cattle
ELSEVIER
Z.C. Lyimo,
RELATIONSHIP AMONG INTENSITY OF ESTROUS
ESTRADIOL, CORTISOL AND BEHAVIOR IN DAIRY CATTLE
la M. Nielen, ’ W. Ouweltjes,
’ T.A.M. Kruip 3 and F.J.C.M. van Eerdenburglb
’ Farm Animal Health, Faculty of Veterinary Medicine, University of Utrecht ’ Research Station for Cattle, Sheep and Horse Husbandry, Lelystad 3 ID-Lelystad, Lelystad, The Netherlands Received for publication: Accepted:
January
19,
November
19,
1999
1999
ABSTRACT Economic profitability of a dairy farm is based, in part, on the calving interval of the cows. The optimal interval is 365 d. To achieve this, the cow needs to be pregnant within 85 d post partum. The first and most problematic step in this process is the determination of the optimal time for insemination, which is based on estrous behavior. The expression of estrous behavior, however, is at a low level in modem dairy herds, resulting in low detection rates and longer calving intervals. In the present study, a point scale was used to monitor postpartum, nonpregnant cows for estrous symptoms. Frequent blood samples were taken around es&us, and the cows were fit with pedometers to measure their activity. Correlations between the occurrence of symptoms of estrus and levels of estradiol and cortisol were then analyzed. Standing heat, the standard symptom of estrus, was observed in only 53% of the cows. A high correlation of 0.7 was found between estradiol concentration and estrous behavior. This was empasized by the fact that the estradiol level reached its highest level of 7.76 + 2.39 (SD) pg/mL at the same time as the highest behavior score. The highest pedometer readout lagged 8 h behind this moment. Cortisol levels did not exceed the physiological levels in rest situations but showed an increase at the time estrous behavior was at its maximum. The present study showed that standing heat is not the primary symptom for detecting estrus in cows. Pedometers are a useful aid but they have to be read several times a day. The high correlation between the visual symptoms of estrus and estradiol concentrations indicates that visual estrus detection is an efficient, reliable way to determine the right time for insemination. Q 2000 by Elsevier SCienCe hc.
Key words: management
sexual
behavior,
hormonal
regulation,
stress, estrus
detection,
dairy
farm
Acknowledgements The authors thank: Masimba Ndengu and Cas Kruitwagen for discussing the statistical analysis; the staff of the Waiboerhoeve; Roe1 Withaar, Jan van Dieren, Albert Postma, Jan Buys, Hedwig Uylen and Pieter Lagendijk for their technical assistance; Annemarie van Bijnen and Elene Vos for the hormone assays and Ruth Zadoks for her direction and inspiration. Herman Kamminga is specially acknowledged for his assistance. Many thanks to NEDAP for the use of the pedometers. This study was supported by Holland Genetics. a Present address: box 5444, Tanga, Tanzania b Correspondence and reprint requests: F.J.C.M. van Eerdenburg, Farm Animal Health, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 7, 3584 CL Utrecht, The Netherlands. Email: [email protected] Therioaenoloqy
53:1783-1795,
2000
0093-691WOOi$-see
front matter
Theriogenology
1784 INTRODUCTION
Around 60 d post partum, a dairy cow produces milk at maximum level. After this time, the daily milk yield gradually declines (44). To get an optimal amount of milk from a cow, it is generally accepted that she should produce a calf within 365 d (10, 26). Recent publications, however, dispute this optimal calving interval. Better consistency of the milk yield indicate that extended lactations might be feasible in the future (4, 28). Besides reduced milk production, long calving intervals result in lower income due to fewer calves born and more semen used (32). Ruiz et al. (35) calculated that on a herd level 4 more open days resulted in a loss of 13 dollars per cow per year. The larger the number of days open, the larger the milk yield per day will be (10, 35). Therefore, estrus detection is of crucial importance on a dairy farm: one cycle missed means an increase in calving interval of 3 wk for a particular cow. Another important factor that influences the length of the calving interval is the conception rate (5, 1 S), which is influenced by the period between insemination and ovulation and thus also by the accuracy of the estrus detection. Nearly all estrus detection methods that are available to a farmer are based on the expression of certain behaviors by the cow and physiological components (Table 1; for a review see 2 1). Detection rates are sometimes only 38% (18). Coleman (6) attributes these low detection rates 90% to the farmer and only 10% to the cows. His ideas might have to be reconsidered as van Vliet and van Eerdenburg (42) reported a low intensity of estrous behavior in high yielding Holstein Friesian cattle. The cause of this low intensity is not yet known. Environmental factors such as housing conditions, temperature, stress and the like. might be of influence (2, 13, 15, 16, 17, 29, 36). Stress, which is reflected in corticosteroid levels in blood, can disrupt estrous behavior in cattle and other ruminant species (7, 13, 34, 38). However, under standard housing conditions in the Netherlands, stress levels are reported to be minimal (22). Table 1. Scoring scale for observed symptoms of estrus Symptoms
of estrus
Mucous vaginal discharge Cajoling Restlessness Sniffing the vagina of other cow Chin resting Mounted but not standing Mounting (or attempt) other cows Mounting headside of other cow Standing heat
Score
3 3 5 10 15 10
35 45 100
In this table the scoring system for estrous symptoms is summarized. The system is cumulative, each time a symptom is observed the number of points is added to the total. Based on the methods of van Eerdenburg et al. (4 1) Because estrus detection is time consuming, systems have been developed to detect cows in estrus automatically. One of these systems is the pedometer. This device counts the number of steps an animal takes during a certain period of time. When a cow is in estrus her walking activity is increased (27). The pedometer is ususally read during milking, twice daily.
1785
Theriogenology
Estrogens play a key role in the regulation of the endocrine and behavioral events associated with the estrous cycle. Estradiol-170 (E,) induces the preovulatory luteinizing hormone (LH) surge as an “all or nothing” event. After a certain threshold of E2 is reached there will be a LH surge, which will result in ovulation or not. No reports are available of a “low” or “minor” LH surge, which results in a “minor” ovulation. However, with respect to estrous behavior, which is also supposed to be induced by E,, a large variety exists. With the point scale for the different components of estrous behavior by van Eerdenburg et al. (41; Table l), it became evident that the intensity of estrous behavior varies among individuals and at herd level (42). The present study, therefore, focussed on the intensity of estrous behavior in dairy cattle in relation to the plasma level of E, around estrus. The relationship between different estrous symptoms and E, were evaluated under practical, small scale, farming conditions. The relationship between typical estrous behaviour and walking activity, as measured with a pedometer, was also investigated. Furthermore, an attempt was made to elucidate the role of environmental stress, as reflected in an increase in peripheral cortisol levels. MATERIALS Animals
AND METHODS
and Housing
The study was carried out between February 19 and May 1, 1997, at the Waiboerhoeve in Lelystad, the Netherlands. Data were collected from 14 Holstein Friesian cows that were housed in a free stall with slatted floor and cubicles. The total number of cows in the group was 37. The age of the animals varied between 2 and 8 yr. The rolling herd average milk yield was 7256 kg / 305 d. Cows were well identified by large painted numbers on the flanks in addition to the standard plastic ear tags and numbered collars. Most of the cows in the group were pregnant or had calved less than 30 d before the start of the study. Cows were not inseminated during the study. The animals were fed according to production level with a routine mixture of grass silage, corn silage, concentrates and mineral supplements. Milking was twice a day at 0645 h and 1700 h and lasted for about 1 h. All handling of the animals was performed with a minimal disruption of daily routine and with minimal stress induction. Visual Observation
of Estrous Behavior
Behavior was monitored by trained observers who had no other tasks at the farm. They stood or walked slowly in front of the feeding fence. The scale of van Eerdenburg et al. (41) was used to measure intensity of estrous behavior (see Table 1). The herd was observed over 30-min periods every 3 h for 24 h. After each observation period, the observer walked slowly through the herd to check for cows with vaginal mucous discharge. At night, lights were on. When a cow reached a score of 1100 points within a 24-h period, she was considered to be in estrus. For cows < 30 d post par-turn, a threshold of 50 points, achieved in one or two consecutive observation periods, was applied. The day of heat was considered to be Day 1 for that cycle. The observation period with the highest score was appointed as Time 0.
Theriogenology
1786 Pedometers
For activity measurement, pedometers (NEDAP, the Netherlands), were attached to a front leg. Every 12 h (at 1200 and 2400), after the visual observations, the activity of every cow was recorded using a hand held reader. The NEDAP pedometer counts the number of steps a cow makes and divides that by 10. In the analysis, the activity as read directly from the device was used. No calculations or algorithms have been made or used before the analysis. Blood Sampling After the first observed es&us, at Days 10 and 12 of the cycle, 10 mL of blood was taken by venipuncture of the coccygeal vein. At Day 15 of the cycle, the animals were fitted with an indwelling jugular catheter (Braun Cavafix Certo 255, 45 cm, 18G) in order to allow repeated blood sampling without stress. The catheters were firmly fixed to the neck in a plastic protective harness and with adhesive tape (Leucoplast (R)). The canulas were flushed and filled with saline containing 25 IUimL heparin after use. All cows were handled gently and adapted to the blood sampling with ease. From Day 16 of the cycle onwards, blood samples of 10 mL were collected 4 times a day into heparinized tubes, after the visual observations, at 0530, 1130, 1730 and 2330 h. Frequent sampling continued until 24 h after the last signs of the next es&us. A final sample was taken 48 h after the last heat signs and at that time the catheter was removed. If an animal did not show heat signs within 30 d after the last estrus, the ovaries were palpated per rectum. If the ovaries appeared to be normal and cyclic, blood sampling was continued. If not, the catheter was removed. Blood samples were immediately put on ice and centrifuged at 3500 rpm for 15 min at 4°C after the sampling. Plasma was decanted and stored at -20°C until assayed. Hormone
Assays
Estradiol-17 l3 and cortisol concentrations were determined in the samples that were collected around behavioral estrus, starting with the sample that was taken at Day 16 of the cycle and ending with the last sample taken (= 48 h post behavioral estrus). Progesterone levels were determined in the samples of Days 10 and 12 of the cycle. Progesterone was assayed as described by van de Wiel et al. (40). Mean values (ng/mL) + SD and their corresponding intra-assay coefficients of variation (CV) were 2.72 + 0.48 (n=l2, CV = 17.6%) 9.60 + 0.32 (n= 14, CV = 5.7%) and 32.4 + 4.28 (n = 20, CV = 13.2%) respectively. Mean values (+ SD) and inter-assay CVs were 2.15 + 0.56 (n = 10, CV = 26%) and 7.56 + 1.27 (n = 10, CV = 16.8%), respectively. Sensitivity was 0.4 pg per well. Estradiol-17 8 was determined by RIA as validated by van de Wiel et al. (39). The intra-assay CV for one control plasma sample was 4.5% (mean = 8.9 pg/mL, n = 8) and for another sample 4.8% (mean = 46.1 pg/mL, n = 8). The inter-assay CV for one control sample was 7.7% (mean = 18.1 pg/mL, n = 22) the sensitivity was 0.2 pg per well. Cortisol was assayed with a DELFIA as described by Erkens et al. (14). This is a time-resolved fluoroimmunoassay for cortisol in unextracted bovine plasma or serum with optimized procedures to eliminate steroid binding plasma protein interference and to minimize non specific Streptavidin - Europium binding. The intra-assay CVs for control samples with concentrations of 71.1, 39.2 and 10.3 ng/mL were 8.2, 7.9 and 11.3% (n = 16). The corresponding inter-assay CVs were 7.4, 8.8 and 19.5% (n=l3). The sensitivity was 0.5 ng/mL.
Theriogenology Statistical
i 787
Evaluation
Four indices were used to describe and compare the amount of estrous behavior: 1) the total estrus score, being the total number of points an animal acquired during 1 estrus, maximum estrus score, being the maximum number of points acquired by an animal during one single observation period, 3) average estrus score, being the mean number of points that was scored during all observation periods of one estrus. The length of time an animal showed estrous signs was included in the analysis as a fourth parameter. For E2 and progesterone,
the maximum
level of an estrous cycle was used in the statistical
analysis. Pearson correlations were computed for the above parameters on a within cow basis using the statistical package SPSS (SPSS Inc. USA). Furthermore, since EZ and progesterone both have an effect on estrous behaviour, also a partial Pearson correlation was calculated for Ez, correcting for progesterone (33). Also correlations were calculated between the E2 and cortisol levels and the occurrence of the different components of estrous behavior. To determine the relationship between the intensity of behavior, the time of rise in level of El and the increase in walking activity, values of the estrous periods of all cows were combined and averaged. For each cow the maximum level of each parameter was considered to be 100%. The other values are presented as a percentage of this maximum. In the graphs, the moment of maximum behavioural score is considered as Time 0. The other parameters are presented accordingly. RESULTS In the group of 37 animals, 14 had at least 2 estrous periods that were visually detected. Of these 14 animals, 4 had one more estrous period during the study. The results of the observations and hormone levels are presented in Table 2. The mean number of points scored during the recorded estrous periods was 1115 +/- 575 (SD). Mean duration of these estrous periods was 20.3 h +I- 10.4 (SD). In 53 % of the estrous periods standing heat was observed. Table 2. Results of the observations
Score per observation Total score Length (hours) E2 - max (pg/mL) Corns01 (ng/mL)
and levels of hormones
Mean
Median
208 1115 20.3 7.76 5.94
151 1006 5.95 5.96 4.27
(14 estrous periods in 11 cows)
Minimum 3 210 6 0.75 0.50
Maximum
SD
1085. 2388 33 13.86 25.04
212 575 10.4 2.39 4.85
Estradiol had a mean maximum of 7.76 +/- 2.39 (SD) pg/mL. Maximum cortisol during 24 h previous to maximal estrus score observation period was 10.59 +/- 5.40 (SD) ng/mL, whereas the mean maximum cortisol level during the 24 h after maximal estrus score was 6.88 +/- 5.71 (SD).
Theriogenology
1788
Correlations calculated are presented in Table 3. Maximum Ez level is positively correlated with total estrus score and length of the estrous period, but not with the maximum Table 3: (Partial)
Pearson Correlations
n= 14(11 cows)
between the studied parameter?
E2
(maximum) Score total Score max Average score Length No. of standing heat No. of mounted No. of mounting No. of mounting headside No. of chin resting No. of sniffing vulva No. of cajoling No. of unrest
0.563 0.134 0.362 0.449 -0.137 -0.103 0.318 0.079 0.294 0.018 -0.211 0.056
(0.023)b (0.620) (0.168) (0.081) (0.613) (0.704) (0.23 1) (0.770) (0.270) (0.948) (0.434) (0.838)
Pedometer
E2
(corrected 0.658 0.228 0.447 0.602 0.381 -0.166 0.555 0.155 0.673 0.324 -0.038 0.595
for P4) (0.006)b (0.395) (0.055) (0.014)b (0.146) (0.538) (0.026)b (0.567) (0.004)b (0.221) (0.890) (0.015)b
” Correlations between E2, without and after correction for diestral progesterone activity and the various behavioral indices and specified estrous symptoms. hStatistically significant (P < 0.05) correlations.
0.457 0.618 0.515 0.349 0.278 0.035 0.320 0.266 0.350 0.270 0.111 0.422
(0.439) (0.439) (0.485) (0.186) (0.015)b (0.766) (0.005)b (0.020)b (0.184) (0.019)b (0.341) (O.OOO)b
level, pedometer
number of points acquired during one observation period. Length and total estrus score are positively correlated. Mean cortisol level during the 24 h pre maximum estrus score was not correlated with any of the indices for estrus intensity. Mean cortisol level during the 24 h post maximum estrus score is correlated with the maximum score (r = 0.678; P = 0.005) but not with the length, total estrus score or average estrus score. Cortisol level around the maximum estrus score was not correlated with the heat score (r = 0.17). Figure 1 represents the mean behavioural score around estrus, presented as a percentage of the maximum level for each individual animal. For each individual animal the observation ‘period with maximum score is appointed as Time 0. In the upper panel, mean E2 (+ SD) levels are plotted. on the same time axis, as a percentage of individual maxima, in the lower panel, pedometer activity is plotted in a similar way. In Figure 2, the mean cortisol levels (+ SD) are plotted around the maximum behavior score. independent of the time of day. In Figure 3 the mean cortisol levels of all cows are plotted on a day time axis, independent of the time of the day estrus behavior is at its maximum. In summary, we observed visually the following trends in the graphs: El levels drop immediately after the behaviour score is at its maximum and pedometer activity peaks 6 to 12 h after the behaviour. Cortisol shows a slight increase during estrus and peaks at the same moment as El and behavior do (no statistical tests were performed due to the low number of animals).
Theriogenology
1789
40 -
20 -
-36
-30
-24
48
-12
-6
0
6
12
18
24
30
36 hours
tpedometer +
80
Behaviour
1
60
-36
-30
-24
-18
-12
-6
0
6
12
18
24
30
38 hour8
Figure 1. Upper and lower panel represent the mean behavioural score around estrus, presented as a percentage of the maximum level for each individual animal. For each animal, the observation period with maximum score is appointed as Time 0. In the upper panel, mean E2 (+SD) levels are plotted as a percentage of individual maxima; in the lower panel, pedometer activity is plotted in a similar way. The E2 levels drop after the behavior scoe is at its maximum; pedometer activity readings peak 6 to 12 hours after the behavior.
Theriogenology
1790
100
12
-Behaviour
ng/mL
-O-Cortisol
90
10
80 70
8 60 50
6
40 30 20 10 0 -132
-114
-96
-78
-60
-42
-24
-6
12
30
48
66
84
102 hours
Figure 2. Mean cortisol levels (+SD) are plotted around es&us. Mean behavior is represented as a percentage of the individual maximum point scores. The moment of maximum behavioral score is set to be time 0. Cortisol levels are of the corresponding time points and show a slight increase around the maximum behavioral activity.
nglml
10
8
6
1730
i ---ESTRUS-
I
I -4
-3
-2
-1
I
I
17:30 I
0
I
I
I,,,
1
,
,
2
,
,
,
,
3
,
DAY
Figure 3. Mean cortisol levels (+SD) around estrus. In this graph the levels are grouped at the same sampling time. The bar indicates the mean period of estrous behavior. The diurnal rhythm is visible and is disturbed on the day of estrus. Thus indicating that the rise in cortisol level is not due to the timing of estrus during the day.
Theriogenology
1791 DISCUSSION
The present study shows that maximum E, levels correlate with the total amount of estrous behavior shown by a cow during an estrous period, as is reflected in the total estrus score and length of the estrus. However, other ways of indicating the intensity of the behavior, like maximum score or average estrus score, are not correlated with E,. For a farmer who wishes to detect his cows in estrus, the total estrus score and length are important parameters, since he will not have much time to watch his cattle often and for long periods of time. If a cow is in estrus for a long period, the chances of being detected are higher. However, in certain herds the duration of estrus is rather short, sometimes only 4 h (42). Expression of estrous behavior is only clear after priming by progesterone during diestrus (1, 8). Without progesterone priming, ovulation will occur without clearly expressed behavior, which is generally addressed as “silent estrus.” This is physiological for the first postpartum estrus, which occurs around 17 days after calving (45). Because of this priming effect, the correlations of E, and the behavioral indices were corrected for progesterone in the statistical analysis (33). Progesterone levels are generally low during estrus in cattle, although a low level apparently is not a prerequisite since pregnant cows can also show marked estrous behavior, even standing heat (11). Because there is a high correlation between the drop in E, level and the occurrence of the preovulatory LH surge, which in turn is correlated with the moment of ovulation (9, 24, 37), this study shows that it is possible to determine the endocrinological status of a cow accurately on the basis of the behavior of the animal. The fact that the correlation factors for E, and some of the estrous symptoms differ can be used to determine the most important symptoms for detection by the farmer. Watching for mounting behavior, chin resting and unrest will result in the best detection rates. Standing heat, however, which is historically and by definition the most reliable symptom, does not occur in enough cows to be an accurate symptom to wait for. The pedometer score lags behind the behaviour score and E, maximum. This could be expected since the pedometer was read after the activity had increased. However, if a farmer relies on this device only, he will have a problem with inseminating his animals at the right time. The present study confirms that pedometers can only be seen as an aid in estrus detection and that one cannot solely rely on them. It can be a very useful device since it measures the activity 24 h a day, also at night. For this reason, it might detect cows in estrus that have short estrous periods. The correlations clearly show that behavior that is accompanied by moving around increased the pedometer score. Estrus detection is mainly performed in order to inseminate the cows. It is therefore important to know when the animals ovulate in relation to the symptoms shown. Due to time limitations, most farmers can not spend the time that is needed for accurate detection. The use of pedometers obviates need. Although pedometers have a problem with false positive attentions (20, 30) many farmers think that the detection rates are acceptable. However, most studies regarding detection rates do not include pregnancy rates in the results. It is therefore important to notice that in the present study the relation between the moment of detection is compared between Ez levels, visual detection and detection with pedometers. The optimal time for insemination is, according to several reports (17, 24, 25, 31) in the second half or at the end of observed estrus. The results presented in Figure 1 show that this occurs around the time that the pedometer shows activity. In daily practice, if the farmer has to rely on an inseminator, it is
1792 likely that the pregnancy rate will be low since a large number of inseminations will have occurred after ovulation. A more frequent read out of the devices, i.e., at the concentrate dispensers, might overcome the problem stated here, as has been shown by Maatje et al. (3 1). The fact that there was no negative correlation between pre estrus cortisol and any of the behavior indices does not necessarily mean that stress does not inhibit estrous behavior in dairy cattle as it does in other species such as sheep (13). The number of animals studied in the present study is limited and the variation in cortisol level is substantial. Furthermore, the circumstances for all animals were similar. The levels of cortisol in the present study are within the range of nonstressed animals in other studies (3, 43). It is therefore probable that there was no stressor active during the period studied. In his survey, Hopster (22) found no indications for high stress levels under Dutch husbandry conditions for dairy cattle. Estrus itself apparently causes some stress. As can be seen in Figures 2 and 3, cortisol levels are elevated during the period of estrus. The diurnal rhythm was disturbed and the maximum level was measured at the same moment maximum behavior scores were observed. There was also a positive correlation with the maximum behavior score. However, from the present data no indication can be derived for which components of the behavior are most stressful, since none of the recorded specified behaviors is correlated with cortisol. The slight increase in cortisol level as observed in the present study is also reported by Dieleman et al. (9). Many farmers restrain very active cows during estrus because of the disturbance of the herd. This will add more stress and might therefore result in a low pregnancy rate of these animals, since acute stress around this period might influence the timing and amplitude of the preovulatory LH surge (12, 13,23). The average length of estrus was approximately 20 h. When this length is compared with that in other herds in recent studies (19, 42), it is relatively long and in accordance with that in older studies, in which estrus was defined as the period from first standing heat until the last standing heat (21). Standing heat was observed in only 53% of the cows, thus confirming the findings of other studies in which a similar phenomenon was reported (19, 42). In our present study, the herd was observed frequently and for long periods, which is not a typical daily practice at dairies. This study confirms therefore the need to look for parameters other than standing heat, i.e., mounting, chin resting and restlessness, to detect cows in es&us. The results of the present study confirmed to look for when a farmer wants to detect his cows they need to be read several times a day. Since estrous symptoms and E, levels, visual estrus determine the optimal time for insemination.
that standing heat is not the primary symptom in estrus. Pedometers might be a useful aid but there is a high correlation between the visual detection is a reliable and efficient way to
1793
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Theriogenology Holdworth RJ, Markville NAR. Evaluation of pedometers for oestrus detection in dairy cows. Vet Ret 1982;lll: 116. Holtz W, Meinhardt H. Die Brunstdiagnose beim Rind. Reprod Dom Anim 1975;28: 315341. Hopster H. Effects of housing and management on welfare and health of cattle older than six months. IV0 report B-405. Zeist, The Netherlands. 1995. Hopster H, Blokhuis HJ. Consistent individual stress responses of dairy cows during social isolation. Appl Anim Behav Sci 1994;40: 83-84. Hunter RHF. Causes for failure of fertilisation in domestic species. In: Embryonic mortality in domestic species, Zavy MT, Geisert RD (eds), Boca Raton: CRC Press, 1994; l-22. Hunter RHF, Greve T. Could artificial insemination be more fruitful? Penalties associated with aging eggs. Reprod Dom Anim 1997;32: 137-141. Jalvingh AW, Dijkhuizen AA, van Arendonk JA. Optimizing the herd calving pattern with linear programming and dynamic probablistic simulation. J Dairy Sci 1994;77: 17191730. Kiddy CA. Variation of physical activity as an indication of estrus in dairy cows. J Dairy Sci 1977;60:235-243. Knight CH, Sorensen A, Muir DD. Biological control of lactation persistency and milk quality. 49th EAAP Mtg, Warsaw, Poland 1998. Lee JA, Rouse1 JD, Beatty JF. Effects of temperature and season on bovine adrenal cortical function, blood cell profile and milk production. J Dairy Sci 1976;59: 104-108. Liu X, Spahr SL. Automated electronic activity measurement for the detection of estrus in dairy cattle. J Dairy Sci 1993;76: 2906-2912. Maatje K, Loeffler SH, Engel B. Predicting optimal time of insemination in cows that show visual signs of estrus by estimating onset of estrus with pedometers. J Dairy Sci 1997;80: 1098-l 105. Marcoot RE, Garverick HA. Detecting heat in dairy cows. The National Dairy Database 1992. Neter J, Wasserman W, Kutner MH. Applied Linear Statistical Models. Boston, Irwin, 1990. Peter AT, Simon JE, Luker, CW, Bosu WTK. Site of action for endotoxin-induced cortisol release in the suppression of preovulatory luteinizing hormone surges. Theriogenology 1990;33: 637-643. Ruiz FJ, Oltenacu PA, Smith RD. Evaluation of on farm milk progesterone tests to determine non pregnant cows and to prevent insemination errors. J Dairy Sci 1989;72: 2718-27. Scott GH. What is animal stress and how is it measured? J Anim Sci 1981;52: 150-153. Shemesh M, Ayalon N, Linder HR. Oestradiol levels in the peripheral blood of cows during the oestrous cycle. J Endocrinol 1972;55: 73-78. Stoebel DP, Moberg DP. Effect of adrenocorticotropin and cortisol on luteinizing hormone surge and oestrous behavior of cows. J Dairy Sci 1982;65: 1016-1024. Van de Wiel DFM, Erkens J, Koops W, Vos E, Van Landenghem. Perioestrous and midluteal time courses of circulating LH, FSH, prolactin, E,-17beta and progesterone in the domestic pig. Biol Reprod 198 1;24: 223-233. Van de Wiel DFM, Koops W. Development and validation of an enzyme immunoassay for progesterone in bovine milk or blood plasma. Anim Reprod Sci 1986; 10: 201-2 13.
Theriogenology 41. 42. 43. 44. 45.
1795
Van Eerdenburg FJCM, Loefler HSH, Van Vliet JH. Detection of oestrous in dairy cows: A new approach to an old problem. Vet Quart 1996;18: 52-54. Van Vliet JH, Van Eerdenburg FJCM. Sexual activities and oestrous detection in lactating Holstein cows. Appl Anim Behav Sci 1996;50: 57-69. Wagner WC, Li PS. Influence of adrenal corticosteroids on postpartum pituitary and ovarian function. Cur Topics Vet Med Anim Sci 1982:20: 197-220. Wood PDP. The biometry of lactation. J Agric Sci 1977;88 333-339. Zurek E, Foxcroft GR, Kennelly JJ. Metabolic status and interval to first ovulation in postpartum dairy cows. J Dairy Sci 1995;78:1909-1920.
Z.C. Lyimo,
RELATIONSHIP AMONG INTENSITY OF ESTROUS
ESTRADIOL, CORTISOL AND BEHAVIOR IN DAIRY CATTLE
la M. Nielen, ’ W. Ouweltjes,
’ T.A.M. Kruip 3 and F.J.C.M. van Eerdenburglb
’ Farm Animal Health, Faculty of Veterinary Medicine, University of Utrecht ’ Research Station for Cattle, Sheep and Horse Husbandry, Lelystad 3 ID-Lelystad, Lelystad, The Netherlands Received for publication: Accepted:
January
19,
November
19,
1999
1999
ABSTRACT Economic profitability of a dairy farm is based, in part, on the calving interval of the cows. The optimal interval is 365 d. To achieve this, the cow needs to be pregnant within 85 d post partum. The first and most problematic step in this process is the determination of the optimal time for insemination, which is based on estrous behavior. The expression of estrous behavior, however, is at a low level in modem dairy herds, resulting in low detection rates and longer calving intervals. In the present study, a point scale was used to monitor postpartum, nonpregnant cows for estrous symptoms. Frequent blood samples were taken around es&us, and the cows were fit with pedometers to measure their activity. Correlations between the occurrence of symptoms of estrus and levels of estradiol and cortisol were then analyzed. Standing heat, the standard symptom of estrus, was observed in only 53% of the cows. A high correlation of 0.7 was found between estradiol concentration and estrous behavior. This was empasized by the fact that the estradiol level reached its highest level of 7.76 + 2.39 (SD) pg/mL at the same time as the highest behavior score. The highest pedometer readout lagged 8 h behind this moment. Cortisol levels did not exceed the physiological levels in rest situations but showed an increase at the time estrous behavior was at its maximum. The present study showed that standing heat is not the primary symptom for detecting estrus in cows. Pedometers are a useful aid but they have to be read several times a day. The high correlation between the visual symptoms of estrus and estradiol concentrations indicates that visual estrus detection is an efficient, reliable way to determine the right time for insemination. Q 2000 by Elsevier SCienCe hc.
Key words: management
sexual
behavior,
hormonal
regulation,
stress, estrus
detection,
dairy
farm
Acknowledgements The authors thank: Masimba Ndengu and Cas Kruitwagen for discussing the statistical analysis; the staff of the Waiboerhoeve; Roe1 Withaar, Jan van Dieren, Albert Postma, Jan Buys, Hedwig Uylen and Pieter Lagendijk for their technical assistance; Annemarie van Bijnen and Elene Vos for the hormone assays and Ruth Zadoks for her direction and inspiration. Herman Kamminga is specially acknowledged for his assistance. Many thanks to NEDAP for the use of the pedometers. This study was supported by Holland Genetics. a Present address: box 5444, Tanga, Tanzania b Correspondence and reprint requests: F.J.C.M. van Eerdenburg, Farm Animal Health, Faculty of Veterinary Medicine, University of Utrecht, Yalelaan 7, 3584 CL Utrecht, The Netherlands. Email: [email protected] Therioaenoloqy
53:1783-1795,
2000
0093-691WOOi$-see
front matter
Theriogenology
1784 INTRODUCTION
Around 60 d post partum, a dairy cow produces milk at maximum level. After this time, the daily milk yield gradually declines (44). To get an optimal amount of milk from a cow, it is generally accepted that she should produce a calf within 365 d (10, 26). Recent publications, however, dispute this optimal calving interval. Better consistency of the milk yield indicate that extended lactations might be feasible in the future (4, 28). Besides reduced milk production, long calving intervals result in lower income due to fewer calves born and more semen used (32). Ruiz et al. (35) calculated that on a herd level 4 more open days resulted in a loss of 13 dollars per cow per year. The larger the number of days open, the larger the milk yield per day will be (10, 35). Therefore, estrus detection is of crucial importance on a dairy farm: one cycle missed means an increase in calving interval of 3 wk for a particular cow. Another important factor that influences the length of the calving interval is the conception rate (5, 1 S), which is influenced by the period between insemination and ovulation and thus also by the accuracy of the estrus detection. Nearly all estrus detection methods that are available to a farmer are based on the expression of certain behaviors by the cow and physiological components (Table 1; for a review see 2 1). Detection rates are sometimes only 38% (18). Coleman (6) attributes these low detection rates 90% to the farmer and only 10% to the cows. His ideas might have to be reconsidered as van Vliet and van Eerdenburg (42) reported a low intensity of estrous behavior in high yielding Holstein Friesian cattle. The cause of this low intensity is not yet known. Environmental factors such as housing conditions, temperature, stress and the like. might be of influence (2, 13, 15, 16, 17, 29, 36). Stress, which is reflected in corticosteroid levels in blood, can disrupt estrous behavior in cattle and other ruminant species (7, 13, 34, 38). However, under standard housing conditions in the Netherlands, stress levels are reported to be minimal (22). Table 1. Scoring scale for observed symptoms of estrus Symptoms
of estrus
Mucous vaginal discharge Cajoling Restlessness Sniffing the vagina of other cow Chin resting Mounted but not standing Mounting (or attempt) other cows Mounting headside of other cow Standing heat
Score
3 3 5 10 15 10
35 45 100
In this table the scoring system for estrous symptoms is summarized. The system is cumulative, each time a symptom is observed the number of points is added to the total. Based on the methods of van Eerdenburg et al. (4 1) Because estrus detection is time consuming, systems have been developed to detect cows in estrus automatically. One of these systems is the pedometer. This device counts the number of steps an animal takes during a certain period of time. When a cow is in estrus her walking activity is increased (27). The pedometer is ususally read during milking, twice daily.
1785
Theriogenology
Estrogens play a key role in the regulation of the endocrine and behavioral events associated with the estrous cycle. Estradiol-170 (E,) induces the preovulatory luteinizing hormone (LH) surge as an “all or nothing” event. After a certain threshold of E2 is reached there will be a LH surge, which will result in ovulation or not. No reports are available of a “low” or “minor” LH surge, which results in a “minor” ovulation. However, with respect to estrous behavior, which is also supposed to be induced by E,, a large variety exists. With the point scale for the different components of estrous behavior by van Eerdenburg et al. (41; Table l), it became evident that the intensity of estrous behavior varies among individuals and at herd level (42). The present study, therefore, focussed on the intensity of estrous behavior in dairy cattle in relation to the plasma level of E, around estrus. The relationship between different estrous symptoms and E, were evaluated under practical, small scale, farming conditions. The relationship between typical estrous behaviour and walking activity, as measured with a pedometer, was also investigated. Furthermore, an attempt was made to elucidate the role of environmental stress, as reflected in an increase in peripheral cortisol levels. MATERIALS Animals
AND METHODS
and Housing
The study was carried out between February 19 and May 1, 1997, at the Waiboerhoeve in Lelystad, the Netherlands. Data were collected from 14 Holstein Friesian cows that were housed in a free stall with slatted floor and cubicles. The total number of cows in the group was 37. The age of the animals varied between 2 and 8 yr. The rolling herd average milk yield was 7256 kg / 305 d. Cows were well identified by large painted numbers on the flanks in addition to the standard plastic ear tags and numbered collars. Most of the cows in the group were pregnant or had calved less than 30 d before the start of the study. Cows were not inseminated during the study. The animals were fed according to production level with a routine mixture of grass silage, corn silage, concentrates and mineral supplements. Milking was twice a day at 0645 h and 1700 h and lasted for about 1 h. All handling of the animals was performed with a minimal disruption of daily routine and with minimal stress induction. Visual Observation
of Estrous Behavior
Behavior was monitored by trained observers who had no other tasks at the farm. They stood or walked slowly in front of the feeding fence. The scale of van Eerdenburg et al. (41) was used to measure intensity of estrous behavior (see Table 1). The herd was observed over 30-min periods every 3 h for 24 h. After each observation period, the observer walked slowly through the herd to check for cows with vaginal mucous discharge. At night, lights were on. When a cow reached a score of 1100 points within a 24-h period, she was considered to be in estrus. For cows < 30 d post par-turn, a threshold of 50 points, achieved in one or two consecutive observation periods, was applied. The day of heat was considered to be Day 1 for that cycle. The observation period with the highest score was appointed as Time 0.
Theriogenology
1786 Pedometers
For activity measurement, pedometers (NEDAP, the Netherlands), were attached to a front leg. Every 12 h (at 1200 and 2400), after the visual observations, the activity of every cow was recorded using a hand held reader. The NEDAP pedometer counts the number of steps a cow makes and divides that by 10. In the analysis, the activity as read directly from the device was used. No calculations or algorithms have been made or used before the analysis. Blood Sampling After the first observed es&us, at Days 10 and 12 of the cycle, 10 mL of blood was taken by venipuncture of the coccygeal vein. At Day 15 of the cycle, the animals were fitted with an indwelling jugular catheter (Braun Cavafix Certo 255, 45 cm, 18G) in order to allow repeated blood sampling without stress. The catheters were firmly fixed to the neck in a plastic protective harness and with adhesive tape (Leucoplast (R)). The canulas were flushed and filled with saline containing 25 IUimL heparin after use. All cows were handled gently and adapted to the blood sampling with ease. From Day 16 of the cycle onwards, blood samples of 10 mL were collected 4 times a day into heparinized tubes, after the visual observations, at 0530, 1130, 1730 and 2330 h. Frequent sampling continued until 24 h after the last signs of the next es&us. A final sample was taken 48 h after the last heat signs and at that time the catheter was removed. If an animal did not show heat signs within 30 d after the last estrus, the ovaries were palpated per rectum. If the ovaries appeared to be normal and cyclic, blood sampling was continued. If not, the catheter was removed. Blood samples were immediately put on ice and centrifuged at 3500 rpm for 15 min at 4°C after the sampling. Plasma was decanted and stored at -20°C until assayed. Hormone
Assays
Estradiol-17 l3 and cortisol concentrations were determined in the samples that were collected around behavioral estrus, starting with the sample that was taken at Day 16 of the cycle and ending with the last sample taken (= 48 h post behavioral estrus). Progesterone levels were determined in the samples of Days 10 and 12 of the cycle. Progesterone was assayed as described by van de Wiel et al. (40). Mean values (ng/mL) + SD and their corresponding intra-assay coefficients of variation (CV) were 2.72 + 0.48 (n=l2, CV = 17.6%) 9.60 + 0.32 (n= 14, CV = 5.7%) and 32.4 + 4.28 (n = 20, CV = 13.2%) respectively. Mean values (+ SD) and inter-assay CVs were 2.15 + 0.56 (n = 10, CV = 26%) and 7.56 + 1.27 (n = 10, CV = 16.8%), respectively. Sensitivity was 0.4 pg per well. Estradiol-17 8 was determined by RIA as validated by van de Wiel et al. (39). The intra-assay CV for one control plasma sample was 4.5% (mean = 8.9 pg/mL, n = 8) and for another sample 4.8% (mean = 46.1 pg/mL, n = 8). The inter-assay CV for one control sample was 7.7% (mean = 18.1 pg/mL, n = 22) the sensitivity was 0.2 pg per well. Cortisol was assayed with a DELFIA as described by Erkens et al. (14). This is a time-resolved fluoroimmunoassay for cortisol in unextracted bovine plasma or serum with optimized procedures to eliminate steroid binding plasma protein interference and to minimize non specific Streptavidin - Europium binding. The intra-assay CVs for control samples with concentrations of 71.1, 39.2 and 10.3 ng/mL were 8.2, 7.9 and 11.3% (n = 16). The corresponding inter-assay CVs were 7.4, 8.8 and 19.5% (n=l3). The sensitivity was 0.5 ng/mL.
Theriogenology Statistical
i 787
Evaluation
Four indices were used to describe and compare the amount of estrous behavior: 1) the total estrus score, being the total number of points an animal acquired during 1 estrus, maximum estrus score, being the maximum number of points acquired by an animal during one single observation period, 3) average estrus score, being the mean number of points that was scored during all observation periods of one estrus. The length of time an animal showed estrous signs was included in the analysis as a fourth parameter. For E2 and progesterone,
the maximum
level of an estrous cycle was used in the statistical
analysis. Pearson correlations were computed for the above parameters on a within cow basis using the statistical package SPSS (SPSS Inc. USA). Furthermore, since EZ and progesterone both have an effect on estrous behaviour, also a partial Pearson correlation was calculated for Ez, correcting for progesterone (33). Also correlations were calculated between the E2 and cortisol levels and the occurrence of the different components of estrous behavior. To determine the relationship between the intensity of behavior, the time of rise in level of El and the increase in walking activity, values of the estrous periods of all cows were combined and averaged. For each cow the maximum level of each parameter was considered to be 100%. The other values are presented as a percentage of this maximum. In the graphs, the moment of maximum behavioural score is considered as Time 0. The other parameters are presented accordingly. RESULTS In the group of 37 animals, 14 had at least 2 estrous periods that were visually detected. Of these 14 animals, 4 had one more estrous period during the study. The results of the observations and hormone levels are presented in Table 2. The mean number of points scored during the recorded estrous periods was 1115 +/- 575 (SD). Mean duration of these estrous periods was 20.3 h +I- 10.4 (SD). In 53 % of the estrous periods standing heat was observed. Table 2. Results of the observations
Score per observation Total score Length (hours) E2 - max (pg/mL) Corns01 (ng/mL)
and levels of hormones
Mean
Median
208 1115 20.3 7.76 5.94
151 1006 5.95 5.96 4.27
(14 estrous periods in 11 cows)
Minimum 3 210 6 0.75 0.50
Maximum
SD
1085. 2388 33 13.86 25.04
212 575 10.4 2.39 4.85
Estradiol had a mean maximum of 7.76 +/- 2.39 (SD) pg/mL. Maximum cortisol during 24 h previous to maximal estrus score observation period was 10.59 +/- 5.40 (SD) ng/mL, whereas the mean maximum cortisol level during the 24 h after maximal estrus score was 6.88 +/- 5.71 (SD).
Theriogenology
1788
Correlations calculated are presented in Table 3. Maximum Ez level is positively correlated with total estrus score and length of the estrous period, but not with the maximum Table 3: (Partial)
Pearson Correlations
n= 14(11 cows)
between the studied parameter?
E2
(maximum) Score total Score max Average score Length No. of standing heat No. of mounted No. of mounting No. of mounting headside No. of chin resting No. of sniffing vulva No. of cajoling No. of unrest
0.563 0.134 0.362 0.449 -0.137 -0.103 0.318 0.079 0.294 0.018 -0.211 0.056
(0.023)b (0.620) (0.168) (0.081) (0.613) (0.704) (0.23 1) (0.770) (0.270) (0.948) (0.434) (0.838)
Pedometer
E2
(corrected 0.658 0.228 0.447 0.602 0.381 -0.166 0.555 0.155 0.673 0.324 -0.038 0.595
for P4) (0.006)b (0.395) (0.055) (0.014)b (0.146) (0.538) (0.026)b (0.567) (0.004)b (0.221) (0.890) (0.015)b
” Correlations between E2, without and after correction for diestral progesterone activity and the various behavioral indices and specified estrous symptoms. hStatistically significant (P < 0.05) correlations.
0.457 0.618 0.515 0.349 0.278 0.035 0.320 0.266 0.350 0.270 0.111 0.422
(0.439) (0.439) (0.485) (0.186) (0.015)b (0.766) (0.005)b (0.020)b (0.184) (0.019)b (0.341) (O.OOO)b
level, pedometer
number of points acquired during one observation period. Length and total estrus score are positively correlated. Mean cortisol level during the 24 h pre maximum estrus score was not correlated with any of the indices for estrus intensity. Mean cortisol level during the 24 h post maximum estrus score is correlated with the maximum score (r = 0.678; P = 0.005) but not with the length, total estrus score or average estrus score. Cortisol level around the maximum estrus score was not correlated with the heat score (r = 0.17). Figure 1 represents the mean behavioural score around estrus, presented as a percentage of the maximum level for each individual animal. For each individual animal the observation ‘period with maximum score is appointed as Time 0. In the upper panel, mean E2 (+ SD) levels are plotted. on the same time axis, as a percentage of individual maxima, in the lower panel, pedometer activity is plotted in a similar way. In Figure 2, the mean cortisol levels (+ SD) are plotted around the maximum behavior score. independent of the time of day. In Figure 3 the mean cortisol levels of all cows are plotted on a day time axis, independent of the time of the day estrus behavior is at its maximum. In summary, we observed visually the following trends in the graphs: El levels drop immediately after the behaviour score is at its maximum and pedometer activity peaks 6 to 12 h after the behaviour. Cortisol shows a slight increase during estrus and peaks at the same moment as El and behavior do (no statistical tests were performed due to the low number of animals).
Theriogenology
1789
40 -
20 -
-36
-30
-24
48
-12
-6
0
6
12
18
24
30
36 hours
tpedometer +
80
Behaviour
1
60
-36
-30
-24
-18
-12
-6
0
6
12
18
24
30
38 hour8
Figure 1. Upper and lower panel represent the mean behavioural score around estrus, presented as a percentage of the maximum level for each individual animal. For each animal, the observation period with maximum score is appointed as Time 0. In the upper panel, mean E2 (+SD) levels are plotted as a percentage of individual maxima; in the lower panel, pedometer activity is plotted in a similar way. The E2 levels drop after the behavior scoe is at its maximum; pedometer activity readings peak 6 to 12 hours after the behavior.
Theriogenology
1790
100
12
-Behaviour
ng/mL
-O-Cortisol
90
10
80 70
8 60 50
6
40 30 20 10 0 -132
-114
-96
-78
-60
-42
-24
-6
12
30
48
66
84
102 hours
Figure 2. Mean cortisol levels (+SD) are plotted around es&us. Mean behavior is represented as a percentage of the individual maximum point scores. The moment of maximum behavioral score is set to be time 0. Cortisol levels are of the corresponding time points and show a slight increase around the maximum behavioral activity.
nglml
10
8
6
1730
i ---ESTRUS-
I
I -4
-3
-2
-1
I
I
17:30 I
0
I
I
I,,,
1
,
,
2
,
,
,
,
3
,
DAY
Figure 3. Mean cortisol levels (+SD) around estrus. In this graph the levels are grouped at the same sampling time. The bar indicates the mean period of estrous behavior. The diurnal rhythm is visible and is disturbed on the day of estrus. Thus indicating that the rise in cortisol level is not due to the timing of estrus during the day.
Theriogenology
1791 DISCUSSION
The present study shows that maximum E, levels correlate with the total amount of estrous behavior shown by a cow during an estrous period, as is reflected in the total estrus score and length of the estrus. However, other ways of indicating the intensity of the behavior, like maximum score or average estrus score, are not correlated with E,. For a farmer who wishes to detect his cows in estrus, the total estrus score and length are important parameters, since he will not have much time to watch his cattle often and for long periods of time. If a cow is in estrus for a long period, the chances of being detected are higher. However, in certain herds the duration of estrus is rather short, sometimes only 4 h (42). Expression of estrous behavior is only clear after priming by progesterone during diestrus (1, 8). Without progesterone priming, ovulation will occur without clearly expressed behavior, which is generally addressed as “silent estrus.” This is physiological for the first postpartum estrus, which occurs around 17 days after calving (45). Because of this priming effect, the correlations of E, and the behavioral indices were corrected for progesterone in the statistical analysis (33). Progesterone levels are generally low during estrus in cattle, although a low level apparently is not a prerequisite since pregnant cows can also show marked estrous behavior, even standing heat (11). Because there is a high correlation between the drop in E, level and the occurrence of the preovulatory LH surge, which in turn is correlated with the moment of ovulation (9, 24, 37), this study shows that it is possible to determine the endocrinological status of a cow accurately on the basis of the behavior of the animal. The fact that the correlation factors for E, and some of the estrous symptoms differ can be used to determine the most important symptoms for detection by the farmer. Watching for mounting behavior, chin resting and unrest will result in the best detection rates. Standing heat, however, which is historically and by definition the most reliable symptom, does not occur in enough cows to be an accurate symptom to wait for. The pedometer score lags behind the behaviour score and E, maximum. This could be expected since the pedometer was read after the activity had increased. However, if a farmer relies on this device only, he will have a problem with inseminating his animals at the right time. The present study confirms that pedometers can only be seen as an aid in estrus detection and that one cannot solely rely on them. It can be a very useful device since it measures the activity 24 h a day, also at night. For this reason, it might detect cows in estrus that have short estrous periods. The correlations clearly show that behavior that is accompanied by moving around increased the pedometer score. Estrus detection is mainly performed in order to inseminate the cows. It is therefore important to know when the animals ovulate in relation to the symptoms shown. Due to time limitations, most farmers can not spend the time that is needed for accurate detection. The use of pedometers obviates need. Although pedometers have a problem with false positive attentions (20, 30) many farmers think that the detection rates are acceptable. However, most studies regarding detection rates do not include pregnancy rates in the results. It is therefore important to notice that in the present study the relation between the moment of detection is compared between Ez levels, visual detection and detection with pedometers. The optimal time for insemination is, according to several reports (17, 24, 25, 31) in the second half or at the end of observed estrus. The results presented in Figure 1 show that this occurs around the time that the pedometer shows activity. In daily practice, if the farmer has to rely on an inseminator, it is
1792 likely that the pregnancy rate will be low since a large number of inseminations will have occurred after ovulation. A more frequent read out of the devices, i.e., at the concentrate dispensers, might overcome the problem stated here, as has been shown by Maatje et al. (3 1). The fact that there was no negative correlation between pre estrus cortisol and any of the behavior indices does not necessarily mean that stress does not inhibit estrous behavior in dairy cattle as it does in other species such as sheep (13). The number of animals studied in the present study is limited and the variation in cortisol level is substantial. Furthermore, the circumstances for all animals were similar. The levels of cortisol in the present study are within the range of nonstressed animals in other studies (3, 43). It is therefore probable that there was no stressor active during the period studied. In his survey, Hopster (22) found no indications for high stress levels under Dutch husbandry conditions for dairy cattle. Estrus itself apparently causes some stress. As can be seen in Figures 2 and 3, cortisol levels are elevated during the period of estrus. The diurnal rhythm was disturbed and the maximum level was measured at the same moment maximum behavior scores were observed. There was also a positive correlation with the maximum behavior score. However, from the present data no indication can be derived for which components of the behavior are most stressful, since none of the recorded specified behaviors is correlated with cortisol. The slight increase in cortisol level as observed in the present study is also reported by Dieleman et al. (9). Many farmers restrain very active cows during estrus because of the disturbance of the herd. This will add more stress and might therefore result in a low pregnancy rate of these animals, since acute stress around this period might influence the timing and amplitude of the preovulatory LH surge (12, 13,23). The average length of estrus was approximately 20 h. When this length is compared with that in other herds in recent studies (19, 42), it is relatively long and in accordance with that in older studies, in which estrus was defined as the period from first standing heat until the last standing heat (21). Standing heat was observed in only 53% of the cows, thus confirming the findings of other studies in which a similar phenomenon was reported (19, 42). In our present study, the herd was observed frequently and for long periods, which is not a typical daily practice at dairies. This study confirms therefore the need to look for parameters other than standing heat, i.e., mounting, chin resting and restlessness, to detect cows in es&us. The results of the present study confirmed to look for when a farmer wants to detect his cows they need to be read several times a day. Since estrous symptoms and E, levels, visual estrus determine the optimal time for insemination.
that standing heat is not the primary symptom in estrus. Pedometers might be a useful aid but there is a high correlation between the visual detection is a reliable and efficient way to
1793
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