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Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows
Theriogenology 63 (2005) 2382–2394 www.journals.elsevierhealth.com/periodicals/the
Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows Evandro S. Sartorellia, Luciano M. Carvalhoa, D.R. Bergfeltb, O.J. Gintherb, Ciro M. Barrosa,* a
Department of Pharmacology, Institute of Bioscience, University of Sa˜o Paulo State (UNESP), 18618-000 Botucatu, Sa˜o Paulo, Brazil b Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin USA Received 20 April 2004; accepted 16 August 2004
Abstract Follicle diameter deviation is defined as the beginning of the differential change in growth rates between the largest and next largest follicles subsequent to wave emergence and is considered a key component of follicle selection. Follicle selection has been extensively studied in European breeds of cattle (Bos taurus) but has not been critically studied in Zebu breeds (Bos indicus). The objectives of the present study were to determine and compare the morphological characteristics of deviation associated with the first post-ovulatory wave (Wave 1) of the estrous cycle in Nelore heifers (n = 8) and nonlactating cows (n = 11). Beginning on the day of ovulation (day 0), the three largest follicles (F1–F3, respectively) were individually tracked every 12 h for 6 d using transrectal ultrasonography. In individual animals, deviation was determined graphically using visual inspection of the diameter profiles of F1, F2 and sometimes F3 (observed deviation) and mathematically using segmented regression analysis of the diameter differences between F1 and F2 or sometimes F3 (calculated deviation). Mean day of emergence of Wave 1 when F1 reached >3 mm (approximately 1 d after ovulation) and growth rate of F1 during deviation (approximately 1.4 mm/d) were not significantly different between heifers and cows. The results of determining the beginning of deviation within heifers and cows using the observed and calculated methods were not significantly different. Averaged over both methods, diameter deviation occurred 2.8 d after ovulation when F1 reached 5.7 mm in heifers, and 2.4 d after ovulation when F1 reached 6.1 mm in cows. In conclusion, the emergence of Wave 1 and growth rates and diameters of the future dominant follicles at the beginning of deviation were similar in Nelore heifers and nonlactating cows, regardless of the methods used to * Corresponding author. Tel.: +55 14 3811 6253; fax: +55 14 3815 3744. E-mail address: [email protected] (C.M. Barros). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.08.017
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determine deviation. Relative to Holstein cattle, emergence of Wave 1 appeared to occur about 1 d later and diameter of the future dominant follicle at the beginning of deviation was about 2 mm smaller in Nelore. # 2004 Elsevier Inc. All rights reserved. Keywords: Nelore; Follicle; Cattle; Bos indicus; Follicle selection
1. Introduction Nelore, and cross-breeds thereof, is the predominant Zebu breed of cattle (Bos indicus) in Brazil and is becoming more popular in other tropical and subtropical countries of South America and around the world [1,2]. Many of the hormonal regimens used to synchronize and stimulate ovarian function for embryo transfer in Nelore have been adopted from studies done in European breeds of cattle (Bos taurus). Reportedly [1], gonadotropin superstimulation regimens are more variable in Zebu breeds of cattle compared to European breeds. The nature of the apparent difference in gonadotropin response between Bos indicus and Bos taurus is not known since few studies are available that have directly or simultaneously compared ovarian function between species. Ovarian follicle dynamics during the estrous cycle in Zebu breeds of cattle has been reviewed [1,2] and indirectly compared with that in European breeds of cattle. Apparently, there are no documented studies that have directly compared follicular wave characteristics between Nelore and European breeds of dairy or beef cattle simultaneously in one study. In this regard, the limited amount of follicle data available for Nelore have been compared with that in Holsteins, most likely because more studies have been done to examine the temporal and functional characteristics of follicular wave dynamics in Holstein cattle than in any other dairy or beef breed of Bos taurus [3–5]. The most distinctive difference between these species of cattle appears to be that the maximum diameter of the dominant follicle is about 11 mm in Nelore [6] compared with about 16 mm in Holsteins [3]. Evidently, more detailed studies are required to compare the morphological and physiological aspects of follicle development between Zebu and European cattle so that the apparent difference in the ovarian response to gonadotropin stimulation between these two species can be adequately addressed. Numerous studies [1] have reported that superovulation in response to exogenous gonadotropins in cattle is reduced if treatment is initiated in the presence of a dominant follicle. Hence, gonadotropin superstimulation regimens are devised to avoid treatment during dominance. The temporal and functional characteristics associated with establishing dominance have been extensively studied in European breeds of cattle, especially Holsteins [3–5]. In Nelore, morphological characteristics of follicle development throughout the estrous cycle have been documented [6]; however, assessments of the timing and follicle diameter changes associated with the establishment of dominance during follicular wave development have not been critically determined. Follicle dominance is a consequence of a differential change in growth rates between the future dominant and subordinate follicles of a wave at the end of the common growth phase and is considered a key component of follicle selection in monovular species [7]. The
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beginning of the departure in growth rates between the two largest follicles is defined as follicle deviation [8]. In individual animals, the beginning of deviation has been determined visually using graphical profiles of the two largest follicles (observed deviation) as well as mathematically by applying segmented regression analysis to the differences in diameter between the largest and second largest follicles (calculated deviation; [9]). In Holstein heifers, the mean interval from wave emergence to the beginning of observed and calculated deviation was not significantly different. Combined, therefore, mean deviation and subsequent dominance occurred 2.6 d after wave emergence when the future dominant follicle was 8.6 mm. These results were comparable to those in other Holstein heifers in which observed deviation occurred about 2.8 d after wave emergence when the future dominant follicle reached a mean 8.5 mm [3]. The objectives of the present study were to determine and compare the morphological characteristics associated with follicle diameter deviation within (observed versus calculated deviation) and between Nelore heifers and nonlactating cows during development of the first post-ovulatory wave of the estrous cycle. It is expected that the determination of the beginning of follicle diameter deviation will serve as a key point of reference to study the physiological basis of follicle selection in future studies.
2. Materials and methods 2.1. Animals The experiments were conducted with Nelore cattle in a subtropical environment at the University of Sa˜ o Paulo State (UNESP) experimental farm located in Botucatu, Sa˜ o Paulo, Brazil (latitude 228510 S, longitude 488260 W) during the summer (December–February). Pure-bred nulliparous heifers (n = 11) ranged in age, body weight and body condition from 2.5 to 3 yr, 325 to 394 kg and 2.5 to 3.5 condition score (0 = thin to 5 = obese; [10]), respectively. Pure- and cross-bred multiparous cows (n = 23) were nonlactating and ranged in age, body weight and body condition from 5 to 10 yr, 379 to 423 kg and 2.5 to 3.5 condition score, respectively. The Nelore cross-bred cows (Nelore Gir, n = 6) were considered indistinct from the pure-bred cows since they were cross-bred within species (Bos indicus Bos indicus) and that the genetic make up was primarily Nelore (>80%). Heifers and cows were confined to a dry lot and fed a ration of 72% tifton hay, 19% citrus pulp, 10% cotton bran and 0.5% mineral mix daily. Water was available ad libitum. 2.2. Ovulation synchronization Ovulation (day 0) was synchronized to optimize the homogeneity of ovulations among heifers and cows and maximizes the number of animals at similar stages of follicular development during the periovulatory period. Heifers were synchronized with a prostaglandin analog (D-cloprostenol, 150 mg, im; Prolise1, ARSA S.R.L., Buenos Aires, Argentina) to regress an existing CL. Eight days later, all heifers received an intravaginal progesterone releasing device (CIDR-B1, InterAg, Hamilton, New Zealand) for 7 d, combined with estradiol benzoate (EB, 2.5 mg, im; Estrogin1, Farmavet, Sa˜ o Paulo,
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Brazil) at CIDR insertion, a second cloprostenol treatment at CIDR removal and 1 mg EB 24 h later. Cows were also synchronized with a CIDR plus EB combination but did not receive an initial dose of cloprostenol. Additionally, all cows received a GnRH agonist (Liserelin, 50 mg, im; Gestran Plus1, ARSA S.R.L., Buenos Aires, Argentina) at CIDR insertion, cloprostenol at CIDR removal and EB 24 h later. To minimize the handling of a few animals that may have an extended interval to ovulation after the end of the synchronization regimen, heifers and cows were excluded from the study if ovulation was delayed >54 h after the last EB treatment. 2.3. Ultrasonography Ultrasonography (Aloka SSD 500 with a 7.5 MHZ transrectal probe, Tokyo, Japan) was used to determine the presence of a CL, detect ovulation and monitor follicular development pre- and post-ovulation. Subsequent to EB treatment, only the largest follicle was monitored every 12 h to detect ovulation. Beginning on the day of ovulation and for 6 d thereafter, the two or three largest follicles/ovary were tracked individually at 12-h intervals and measured using the built-in electronic calipers of the scanner [11] from at least two separate images per follicle at each examination. The accuracy of the electronic measurements were verified in a water bath by taking the measurements of a sphere with a known cross-sectional diameter of 8.4 mm; a diameter representative of the expected time of deviation as reported in Holstein heifers [3]. The water-bath diameter of the sphere was determined from 10 separate images by calculating the average of two lines of measurement approximately perpendicular to one another, similar to that done in vivo. The mean S.D. was 8.3 0.1 mm, with a range of 8.2–8.4 mm. The three largest follicles/animal were designated Follicle 1 (F1, largest), Follicle 2 (F2, second largest) and Follicle 3 (F3, third largest) according to their hierarchal position on day 6. Emergence of the post-ovulatory wave (Wave 1) was defined according to first detection of F1 when it reached >3.0 mm. Only waves with a single dominant follicle were assessed for deviation; waves with co-dominant follicles (two follicles >10 mm [12]) were not considered. To determine the time of observed deviation, the sequential changes in diameter of F1 and F2 from first detection of F1 to day 6 were inspected. In some instances the change in growth of F3 was used to support that of F2 for determining deviation [13]. The last examination before the decrease in growth of F2 or F3 was designated the beginning of observed deviation [3]. To determine the time of calculated deviation, a segmented regression model was used as previously described [9]. Briefly, to prepare the follicle data for analysis, the differences in diameter between F1 and F2 or, in some instances F3, were determined. The diameter differences were truncated before the examination when F1 reached >4 mm and after the examination when F1 reached >10 mm or at its maximum diameter if F1 attained <10 mm by day 6. Although an attempt was made to standardize the truncated period, it was sometimes necessary to truncate the upper limit based on inspection of the diameter profiles of the three largest follicles [9]. The truncation of the follicle data was done to exclude or minimize the extent of the variability associated with follicle measurements around the time of detection of F1 and the beginning of the static phase [14] and focus the segmented regression on a period critical to calculating deviation. For each animal, the truncated period was fit to the diameter
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differences in a successive manner beginning with the earliest time. Segment 1 represented the common growth phase before deviation [7], Segment 2 represented the period of dominance thereafter, and a Join Point connecting the two segments represented the end of the common growth phase and the beginning of deviation. The optimal Join Point value that corresponded to the maximum coefficient of determination (R2) was designated the calculated day of deviation provided the slope of Segment 2 was greater than the slope of Segment 1. The diameter of F1 corresponding to the calculated day of deviation was estimated using a simple linear regression of the diameters of F1 during the truncated period for each animal. The slope of the regression equation was also used to represent the growth rate of F1 during the corresponding period. 2.4. Statistical analysis Prior to statistical analyses, the data were examined for normality using Kolmogorov– Smirnov test and suspected extreme values were challenged using Dixon’s outlier test [15]. If the normality test was significant (P < 0.05), the data were transformed by either natural logarithm or square root. If neither transformation satisfied normality, the data were ranked according to the Kruskal. Wallis test. The SAS MIXED procedure with a repeated statement to account for the autocorrelation between sequential follicle measurements was used to determine the main effects of heifers versus cows and day and the corresponding interaction. If significant main effects or interactions were detected, unpaired t-tests were used to determine mean differences between heifers and cows within days and paired t-tests were used to determine mean differences between days within heifers and cows. The observed and calculated methods to determine the time to deviation and the diameter of F1 at deviation were compared within and between heifers and cows using t-tests. Number of days from ovulation to first detection of F1, diameter of F1 when first detected and growth rate of F1 between heifers and cows were also examined using t-tests. A probability of P < 0.05 indicated that a difference was significant and probabilities between P > 0.05 and P < 0.1 indicated that a difference approached significance. The original data are presented as the mean S.E.M. unless otherwise indicated.
3. Results 3.1. Heifers Two of 11 heifers were excluded from the study because they failed to ovulate within 54 h after the last hormonal treatment of the synchronization regimen and lost 10–20 kg in body weight during the corresponding time. The remaining nine heifers ovulated a mean 43.5 h following the last hormonal treatment and after the largest follicle reached a mean 11.4 mm. A third heifer was excluded because of poor demeanor and the difficulty to consistently collect adequate follicle data. Days from ovulation to the beginning of observed and calculated deviation and diameters of F1 at deviation for eight individual heifers are shown in Table 1. In Heifer A,
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Table 1 Days from ovulation (day 0) to the beginning of observed and calculated deviation and diameter (mm) of Follicle 1 (largest follicle) at deviation in Nelore heifers Day
Diameter of Follicle 1
Heifers
Observed
Calculated
Combined
Observed
Calculated
Combined
A B C D E F G H
ND 3.0 2.5 2.5 2.0 2.5 4.5 3.0
ND 3.0 2.5 2.2 2.3 2.1 4.4 2.2
– 3.0 2.5 2.4 2.2 2.3 4.4 2.6
– 6.0 5.1 6.4 6.3 5.5 5.6 5.0
– 5.9 6.1 6.0 6.6 4.8 6.0 4.5
– 6.0 5.6 6.2 6.4 5.2 5.8 4.8
Mean S.D. S.E.M.
2.9 0.8 0.3
2.7 0.8 0.3
2.8 0.8 0.3
5.7 0.6 0.2
5.7 0.8 0.3
5.7 0.6 0.2
No significant differences for the mean number of days and diameters of Follicle 1 between observed and calculated deviation; therefore, the results were combined for each method. ND (not determined) because observed deviation could not be readily identified and the requirement for calculated deviation was not satisfied (i.e., slope of Segment 2 must be greater than the slope of Segment 1).
observed deviation could not be readily identified. Subsequent to wave emergence, the increase in diameter of F1 and F2 occurred in parallel until the growth rate of F2 began to wane after day 1.5 (Fig. 1). After day 2, however, the growth rate of F2 resurged until a more abrupt and sustained cessation of growth occurred after day 4. Consequently, calculated deviation could not be estimated because the slope of the regression line for the diameter differences for Segment 2 was less than that for Segment 1 which did not satisfy the condition that the slope of Segment 2 must be greater than that of Segment 1. Alternatively, F3 was inspected to determine if it could be used to assist in the determination of deviation but the amount of available data was insufficient. For the remainder of the animals, observed and calculated deviation was readily determined using F1 and F2 (Heifers D–H) or F1 and F3 (Heifers B and C). There were no significant differences between the mean days and diameters of F1 for observed and calculated deviation (Table 1). The diameter profiles for F1 and F3 are depicted for Heifer B (Fig. 1) to illustrate the beginning of observed deviation on day 3 when F1 reached 6 mm. 3.2. Cows Eleven of 23 cows were excluded from the study because they failed to ovulate within 54 h after the last hormonal treatment of the synchronization regimen. For the other 12 cows (six pure- and six cross-bred animals), ovulation occurred a mean 42.5 h following the last hormonal treatment and after the largest follicle reached a mean 12.3 mm. One of the 12 cross-bred cows was not included in the determination of deviation because F1 and F2 appeared as co-dominant follicles (11.6 and 10.7 mm, respectively). Days from ovulation to the beginning of observed and calculated deviation and diameters of F1 at deviation for 11 individual cows are shown in Table 2. Observed and
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Fig. 1. Diameter profiles of the three largest follicles (Follicle 1, 2 or 3) of the first post-ovulatory wave beginning when Follicle 1 was first detected at >3 mm in individual Nelore heifers and nonlactating cows. In Heifer A, observed and calculated deviation could not be determined because of the atypical growth profile of Follicle 2; there was not an adequate amount of data to consider using Follicle 3. In Heifer B, observed deviation was readily determined based on the departure in growth between Follicles 1 and 3 beginning on day 3 when Follicle 1 reached 6 mm. In cow A, observed deviation was readily determined based on the departure in growth between Follicles 1 and 2 beginning on day 3.5 when Follicle 1 reached 6.6 mm.
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Table 2 Days from ovulation (day 0) to the beginning of observed and calculated deviation and diameter (mm) of Follicle 1 (largest follicle) at deviation in nonlactating Nelore cows Day
Diameter of Follicle 1
Cows
Observed
Calculated
Combined
Observed
Calculated
Combined
A B C D E F G H I J K
3.5 2.0 1.5 2.0 3.0 1.5 2.0 3.5 3.0 1.5 1.5
3.4 2.4 1.5 2.0 3.0 1.0 2.4 3.9 3.0 2.2 2.1
3.5 2.2 1.5 2.0 3.0 1.2 2.2 3.7 3.0 1.8 1.8
6.6 6.8 5.8 6.0 5.7 6.5 5.4 7.9 5.3 5.9 4.2
6.3 6.9 5.8 6.4 5.7 4.8 6.3 7.9 5.5 6.9 5.8
6.4 6.8 5.8 6.2 5.7 5.6 5.8 7.9 5.4 6.4 5.0
Mean S.D. S.E.M.
2.3 0.8 0.2
2.4 0.8 0.2
2.4 0.8 0.2
6.0 1.0 0.3
6.1 1.0 0.2
6.1 0.8 0.2
No significant differences for the mean number of days and diameters of Follicle 1 between observed and calculated deviation; therefore, the results were combined for each method.
calculated deviations were determined using F1 and F2 (Cows A–D and F–K) or F1 and F3 (Cow E). There were no significant differences between the mean days and diameters of F1 for observed and calculated deviation (Table 2). The diameter profiles for F1 and F2 are depicted for Cow A (Fig. 1) to illustrate the beginning of observed deviation on day 3.5 when F1 reached 6.6 mm. 3.3. Heifers versus cows There were no significant differences between heifers and cows for the number of days from ovulation to first detection of F1, diameters of F1 at first detection and growth rates of F1 during deviation (Table 3). Similarly, there were no significant differences between heifers and cows for the day of observed and calculated deviation and diameter of F1 at observed and calculated deviation as presented and compared between Tables 1 and 2.
Table 3 Mean S.E.M. characteristics of Follicle 1 (largest follicle) of the first follicular wave following ovulation (day 0) in Nelore heifers (n = 8) and nonlactating cows (n = 11) Follicle 1
Heifers
Cows
Combined
First detection after ovulation (d) Diameter at first detection (mm) Growth rate during deviation (mm/d)
1.0 + 0.2 4.3 + 0.2 1.3 + 0.2
0.8 + 0.2 4.3 + 0.2 1.4 + 0.5
0.9 + 0.2 4.3 + 0.1 1.4 + 0.1
No significant differences between Nelore heifers and cows for each characteristic of Follicle 1; therefore, the results were combined.
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Fig. 2. Mean S.E.M. diameters of the largest (Follicle 1) and second largest (Follicle 2) follicles at 12-h interval in Nelore heifers and nonlactating cows after adjusting the follicle data for each heifer and cow to the beginning of observed deviation (0 h). The left set of figures illustrates the diameter changes between Follicles 1 and 2 within heifers and cows while the other set indicates the statistical comparisons between heifers and cows for each of Follicles 1 and 2. Changes in diameters of Follicles 1 and 2 were not significantly different between heifers and cows (no main effect of heifers vs. cows or interaction with hour). The main effect of hour for both follicles was attributed to the progressive increases in diameters encompassing deviation for Follicle 1 (36 to 36 h) and before deviation for Follicle 2 (36 to 0 h).
The diameters of F1 and F2 at 12-h interval were adjusted to the beginning of observed deviation for each heifer and cow, averaged and then profiled from 36 h before to 36 h after deviation (Fig. 2). The left set of figures illustrates the mean follicle changes within heifers and cows while the other set indicates the statistical comparisons between heifers and cows for each follicle. Changes in diameters of F1 and F2 were not significantly different between heifers and cows (no main effect of heifers versus cows or interaction with hour). Averaged over all animals, the diameters of F1 and F2 at the beginning of observed deviation were 5.9 and 5.4 mm, respectively. There was a significant main effect of hour for both follicles that was attributed to the progressive increases in diameters encompassing deviation for F1 (36 to 36 h) and before deviation for F2 (36 to 0 h). Subsequent to deviation (0 to 36 h), there were no further significant increases in diameter of F2.
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4. Discussion The beginning of the differential change in growth rates between the largest (F1) and second largest (F2) follicles (i.e., diameter deviation) associated with the first postovulatory wave during the estrous cycle (Wave 1) was not significantly different between Nelore heifers and nonlactating cows, regardless of the methods (graphical versus mathematical) used to determine deviation. Averaged over both methods of determination, deviation occurred 2.8 d after ovulation when diameter of F1 reached 5.7 mm in heifers and 2.4 d after ovulation when F1 reached 6.1 mm in cows. The interval from ovulation to the beginning of diameter deviation and growth rate of F1 during deviation were not different between Nelore heifers and nonlactating cows and appeared comparable to that in Holstein heifers during deviation [3–5]. The emergence of Wave 1 and the interval from emergence to the beginning of deviation were also similar between Nelore heifers and cows but, compared to Holstein heifers [7,8], wave emergence occurred later and the interval from emergence to deviation occurred earlier. The emergence of Wave 1 approximately 1 d after ovulation followed by deviation approximately 1.6 d later when F1 reached approximately 6 mm in Nelore may be reflective of relatively low concentrations of FSH and relatively high concentrations of IGF-1 in Bos indicus as compared to Bos taurus cattle. In this regard, temporal changes in mean maximal concentrations of circulating FSH associated with the periovulatory FSH surge were about 48% less, while circulating concentrations of total IGF-1 were about 129% more in Brahman cows than in Angus cows during the estrous cycle [16]. In Holstein heifers, a recent functional study [17] indicated that IGF-1 might be a prominent factor involved in facilitating continued development of the dominant follicle in the presence of low concentrations of FSH during deviation. That IGF-1 may enhance follicle responsiveness to FSH in Zebu cattle is speculated on the basis of numerous summary reports [1] that lower doses of FSH induced a superovulatory response that appeared comparable to that in European breeds of cattle using higher doses of FSH. Together, the previous and present results indicate that the timing of gonadotropic and somatotropic effects on follicular wave emergence and deviation may be different between some breeds of Bos indicus and Bos taurus. The present study was not designed to examine the nature of follicle selection, but to characterize the beginning of diameter deviation as a key morphological event of selection so that it may serve as a reference point, as it has in Holsteins [8], to address the physiological basis of follicle selection in future studies in Nelore. The beginning of diameter deviation between F1 and F2 was determined in individual Nelore heifers and nonlactating cows using graphical (observed deviation) and mathematical (calculated deviation) methods as previously documented [9]. The latter method is a more objective approach that was used, in part, to substantiate the graphical method. In some instances (16%), the third largest follicle (F3) was used in addition or in place of F2 to assist in determining the day that observed and calculated deviation began. This approach seemed appropriate especially since it has been documented [13] that the beginning of deviation occurs, on average, simultaneously between the dominant follicle and the next three largest subordinate follicles of a wave. The diameter of F1 corresponding to the beginning of observed deviation was determined directly from the diameter data that
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was collected at 12-h interval for each heifer and cow, whereas that for calculated deviation was estimated using a simple linear regression of the follicle data during deviation with the calculated day of deviation included in the regression equation. The results of determining the beginning of follicle diameter deviation within heifers and cows using the observed and calculated methods were not significantly different. Thus, the mathematical method of determining diameter deviation substantiated the graphical method as a valid approach to determine follicle deviation in Nelore heifers and nonlactating cows. Combined, the beginning of deviation was determined 95% of the time as previously reported in Holstein heifers [9]. In one Nelore heifer (Heifer A), what appeared to be the beginning of observed deviation on day 1.5 was followed by resurgence in growth of F2 after day 2. A similar morphological event with a similar outcome has been reported in Holstein heifers [9]. Perhaps, the resurgence in the growth of F2 may have represented a delayed response to elevated gonadotropins as a result of a deficiency of F1 to adequately suppress FSH during deviation. Although the nature of this event is not known, a similar concept has been proposed for multiple deviations occurring within a follicular wave having co-dominant follicles in Holstein heifers [12]. Nonetheless, this relatively rare occurrence in the growth profiles of the two largest follicles during wave development negated the use of the graphical and mathematical methods to determine deviation. The present study provided new information that the timing of follicular wave emergence and diameter changes associated with the beginning of deviation is similar between Nelore heifers and nonlactating cows. It is not likely that the Gir component of the five cross-bred cows (<20% for each animal) had a major influence on the results since the crosses were within-species and the genetic make up of the pure-bred Nelore cows and the larger Nelore component of the cross-bred animals predominated. In a previous study [6] comparing the same types of animals, follicle development well after diameter deviation was not different between Nelore heifers and cows. Although the present and past studies indicated that follicular wave development (i.e., follicle emergence, growth, regression and dominance) is comparable in nulliparous and multiparous nonlactating Nelore cattle, follicular wave development in lactating animals may be different. The effect of lactation on follicle emergence, growth, regression and dominance is not known in Zebu cattle, but recently the results of a simultaneous comparison of ovarian function during the estrous cycle in Holstein heifers and lactating cows were reported [18]. Multiple ovulations and larger-sized ovulatory follicles were detected in cows than in heifers. Although the study was not designed to examine the morphological characteristics of the dominant and subordinate follicles during deviation, a retrospective assessment indicated that the mean diameters of the dominant and largest subordinate follicles were larger at the beginning of observed deviation in lactating cows (9.8 and 9.0 mm) than in heifers (8.3 and 7.5 mm). The mechanism that seems to allow dominant follicles to reach larger diameters in lactating Holstein cows than in heifers is not thoroughly known and whether a similar event occurs in lactating Bos indicus cattle, especially during deviation, awaits to be addressed. In conclusion, there were no significant differences between Nelore heifers and nonlactating cows regarding the initiation of follicle diameter deviation irrespective of the methods used to determine deviation. Hence, the morphological results were combined and summarized in relation to comparable characteristics reported in Holstein heifers [3–5]:
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(1) Emergence of the first post-ovulatory wave of the estrous cycle occurred 0.9 d after ovulation (approximately 1 d later than in Holsteins). (2) Diameter deviation occurred 2.6 d after ovulation (comparable to that in Holsteins) or about 1.6 d after wave emergence (approximately 1 d earlier than in Holsteins). (3) Growth rate of the largest follicle during deviation was 1.4 mm/d (comparable to that in Holsteins). (4) Diameter of the largest follicle at the beginning of deviation was 5.9 mm (approximately 2 mm smaller than in Holsteins). The impact that these results will have on current superstimulation regimens in Nelore is not known. Future studies are necessary to examine simultaneously the morphological and physiological characteristics during follicle deviation in Nelore and to determine whether these apparent differences between Bos indicus and Bos taurus inferred herein are related to species, breeds within a species (beef versus dairy), reproductive status (nonlacating versus lactating), climate (tropical versus temperate), nutritional management (pastured versus feedlot), or other factors. Acknowledgements The authors wish to thank Drs. Antonio C. Silveira, Mario B. Arrigoni and Francisco S. Wechsler, for lending the animals to conduct this study at Lageado Experimental Farm, University of Sa˜ o Paulo State (UNESP), Botucatu, Sa˜ o Paulo, Brazil. Evandro S. Sartorelli was supported by a CAPES fellowship for graduate work at the Institute of Bioscience (IBUNESP) and Luciano M. Carvalho was supported by a FAPESP fellowship for graduate work at the College of Veterinary Medicine (FMVZ-UNESP).
References [1] Barros CM, Nogueira MFG. Embryo transfer in Bos indicus cattle. Theriogenology 2001;56:1483–96. [2] Bo GA, Baruselli PS, Martinez MF. Pattern and manipulation of follicular development in Bos indicus cattle. Anim Reprod Sci 2003;78:307–26. [3] Ginther OJ, Wiltbank MC, Fricke PM, Gibbons JR, Kot K. Selection of the dominant follicle in cattle. Biol Reprod 1996;55:1187–94. [4] Fortune JE, Rivera GM, Evans ACO, Turzillo AM. Differentiation of dominant versus subordinate follicles in cattle. Biol Reprod 2001;65:648–54. [5] Mihm M, Crowe MA, Knight PG, Austin EJ. Follicle wave growth in cattle. Reprod Domest Anim 2002;37:191–200. [6] Figueiredo RA, Barros CM, Pinheiro OL, Soler JMP. Ovarian follicular dynamics in Nelore breed (Bos indicus). Theriogenology 1997;47:1489–505. [7] Ginther OJ, Beg MA, Bergfelt DR, Donadeu FX, Kot K. Follicle selection in monovular species. Biol Reprod 2001;65:638–47. [8] Ginther OJ, Beg MA, Donadeu FX, Bergfelt DR. Mechanism of follicle deviation in monovular species. Anim Reprod Sci 2003;78:239–57. [9] Bergfelt DR, Sego LH, Beg MA, Ginther OJ. Calculated follicle deviation using segmented regression for modeling diameter differences in cattle. Theriogenology 2003;59:1811–25. [10] Lowman BG, Scott NA, Somervalle SH. Condition scoring of cattle, Bulletin 6. Edinburgh: The East of Scotland College of Agriculture; 1976. p. 1–31.
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[11] Ginther OJ. Ultrasonic imaging and animal reproduction: fundamentals. Book 1. Cross Plains, WI: Equiservices Publishing; 1995. [12] Kulick LJ, Bergfelt DR, Kot K, Ginther OJ. Follicle selection in cattle: follicle deviation and codominance within sequential waves. Biol Reprod 2001;65:839–46. [13] Ginther OJ, Bergfelt DR, Beg MA, Kot K. Follicle selection in cattle: relationships among growth rate, diameter ranking, and capacity for dominance. Biol Reprod 2001;65:345–50. [14] Ginther OJ, Knopf L, Kastelic JP. Temporal associations among ovarian events in cattle during oestrous cycles with two and three follicular waves. J Reprod Fertil 1989;887:223–30. [15] Kanji GK. 100 Statistical tests, vol. 45. London, England: Sage Publications; 1993. p. 171. [16] Alvarez P, Spicer LJ, Chase Jr CC, Payton ME, Hamilton TD, Stewart RE, et al. Ovarian and endocrine characteristics during an estrous cycle in Angus, Brahman, and Senepol cows in subtropical environment. J Anim Sci 2000;78:1291–302. [17] Ginther OJ, Bergfelt DR, Beg MA, Meira C, Kot K. In vivo effects of an intrafollicular injection of insulinlike growth factor 1 on the mechanism of follicle deviation in heifers and mares. Biol Reprod 2004;70:99– 105. [18] Sartori R, Haughian JM, Shaver RD, Rosa GJM, Wiltbank MC. Comparison of ovarian function and circulating steroids in estrous cycles of Holstein heifers and lactating cows. J Dairy Sci 2004;87:905–20.
Morphological characterization of follicle deviation in Nelore (Bos indicus) heifers and cows Evandro S. Sartorellia, Luciano M. Carvalhoa, D.R. Bergfeltb, O.J. Gintherb, Ciro M. Barrosa,* a
Department of Pharmacology, Institute of Bioscience, University of Sa˜o Paulo State (UNESP), 18618-000 Botucatu, Sa˜o Paulo, Brazil b Animal Health and Biomedical Sciences, University of Wisconsin, Madison, Wisconsin USA Received 20 April 2004; accepted 16 August 2004
Abstract Follicle diameter deviation is defined as the beginning of the differential change in growth rates between the largest and next largest follicles subsequent to wave emergence and is considered a key component of follicle selection. Follicle selection has been extensively studied in European breeds of cattle (Bos taurus) but has not been critically studied in Zebu breeds (Bos indicus). The objectives of the present study were to determine and compare the morphological characteristics of deviation associated with the first post-ovulatory wave (Wave 1) of the estrous cycle in Nelore heifers (n = 8) and nonlactating cows (n = 11). Beginning on the day of ovulation (day 0), the three largest follicles (F1–F3, respectively) were individually tracked every 12 h for 6 d using transrectal ultrasonography. In individual animals, deviation was determined graphically using visual inspection of the diameter profiles of F1, F2 and sometimes F3 (observed deviation) and mathematically using segmented regression analysis of the diameter differences between F1 and F2 or sometimes F3 (calculated deviation). Mean day of emergence of Wave 1 when F1 reached >3 mm (approximately 1 d after ovulation) and growth rate of F1 during deviation (approximately 1.4 mm/d) were not significantly different between heifers and cows. The results of determining the beginning of deviation within heifers and cows using the observed and calculated methods were not significantly different. Averaged over both methods, diameter deviation occurred 2.8 d after ovulation when F1 reached 5.7 mm in heifers, and 2.4 d after ovulation when F1 reached 6.1 mm in cows. In conclusion, the emergence of Wave 1 and growth rates and diameters of the future dominant follicles at the beginning of deviation were similar in Nelore heifers and nonlactating cows, regardless of the methods used to * Corresponding author. Tel.: +55 14 3811 6253; fax: +55 14 3815 3744. E-mail address: [email protected] (C.M. Barros). 0093-691X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2004.08.017
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determine deviation. Relative to Holstein cattle, emergence of Wave 1 appeared to occur about 1 d later and diameter of the future dominant follicle at the beginning of deviation was about 2 mm smaller in Nelore. # 2004 Elsevier Inc. All rights reserved. Keywords: Nelore; Follicle; Cattle; Bos indicus; Follicle selection
1. Introduction Nelore, and cross-breeds thereof, is the predominant Zebu breed of cattle (Bos indicus) in Brazil and is becoming more popular in other tropical and subtropical countries of South America and around the world [1,2]. Many of the hormonal regimens used to synchronize and stimulate ovarian function for embryo transfer in Nelore have been adopted from studies done in European breeds of cattle (Bos taurus). Reportedly [1], gonadotropin superstimulation regimens are more variable in Zebu breeds of cattle compared to European breeds. The nature of the apparent difference in gonadotropin response between Bos indicus and Bos taurus is not known since few studies are available that have directly or simultaneously compared ovarian function between species. Ovarian follicle dynamics during the estrous cycle in Zebu breeds of cattle has been reviewed [1,2] and indirectly compared with that in European breeds of cattle. Apparently, there are no documented studies that have directly compared follicular wave characteristics between Nelore and European breeds of dairy or beef cattle simultaneously in one study. In this regard, the limited amount of follicle data available for Nelore have been compared with that in Holsteins, most likely because more studies have been done to examine the temporal and functional characteristics of follicular wave dynamics in Holstein cattle than in any other dairy or beef breed of Bos taurus [3–5]. The most distinctive difference between these species of cattle appears to be that the maximum diameter of the dominant follicle is about 11 mm in Nelore [6] compared with about 16 mm in Holsteins [3]. Evidently, more detailed studies are required to compare the morphological and physiological aspects of follicle development between Zebu and European cattle so that the apparent difference in the ovarian response to gonadotropin stimulation between these two species can be adequately addressed. Numerous studies [1] have reported that superovulation in response to exogenous gonadotropins in cattle is reduced if treatment is initiated in the presence of a dominant follicle. Hence, gonadotropin superstimulation regimens are devised to avoid treatment during dominance. The temporal and functional characteristics associated with establishing dominance have been extensively studied in European breeds of cattle, especially Holsteins [3–5]. In Nelore, morphological characteristics of follicle development throughout the estrous cycle have been documented [6]; however, assessments of the timing and follicle diameter changes associated with the establishment of dominance during follicular wave development have not been critically determined. Follicle dominance is a consequence of a differential change in growth rates between the future dominant and subordinate follicles of a wave at the end of the common growth phase and is considered a key component of follicle selection in monovular species [7]. The
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beginning of the departure in growth rates between the two largest follicles is defined as follicle deviation [8]. In individual animals, the beginning of deviation has been determined visually using graphical profiles of the two largest follicles (observed deviation) as well as mathematically by applying segmented regression analysis to the differences in diameter between the largest and second largest follicles (calculated deviation; [9]). In Holstein heifers, the mean interval from wave emergence to the beginning of observed and calculated deviation was not significantly different. Combined, therefore, mean deviation and subsequent dominance occurred 2.6 d after wave emergence when the future dominant follicle was 8.6 mm. These results were comparable to those in other Holstein heifers in which observed deviation occurred about 2.8 d after wave emergence when the future dominant follicle reached a mean 8.5 mm [3]. The objectives of the present study were to determine and compare the morphological characteristics associated with follicle diameter deviation within (observed versus calculated deviation) and between Nelore heifers and nonlactating cows during development of the first post-ovulatory wave of the estrous cycle. It is expected that the determination of the beginning of follicle diameter deviation will serve as a key point of reference to study the physiological basis of follicle selection in future studies.
2. Materials and methods 2.1. Animals The experiments were conducted with Nelore cattle in a subtropical environment at the University of Sa˜ o Paulo State (UNESP) experimental farm located in Botucatu, Sa˜ o Paulo, Brazil (latitude 228510 S, longitude 488260 W) during the summer (December–February). Pure-bred nulliparous heifers (n = 11) ranged in age, body weight and body condition from 2.5 to 3 yr, 325 to 394 kg and 2.5 to 3.5 condition score (0 = thin to 5 = obese; [10]), respectively. Pure- and cross-bred multiparous cows (n = 23) were nonlactating and ranged in age, body weight and body condition from 5 to 10 yr, 379 to 423 kg and 2.5 to 3.5 condition score, respectively. The Nelore cross-bred cows (Nelore Gir, n = 6) were considered indistinct from the pure-bred cows since they were cross-bred within species (Bos indicus Bos indicus) and that the genetic make up was primarily Nelore (>80%). Heifers and cows were confined to a dry lot and fed a ration of 72% tifton hay, 19% citrus pulp, 10% cotton bran and 0.5% mineral mix daily. Water was available ad libitum. 2.2. Ovulation synchronization Ovulation (day 0) was synchronized to optimize the homogeneity of ovulations among heifers and cows and maximizes the number of animals at similar stages of follicular development during the periovulatory period. Heifers were synchronized with a prostaglandin analog (D-cloprostenol, 150 mg, im; Prolise1, ARSA S.R.L., Buenos Aires, Argentina) to regress an existing CL. Eight days later, all heifers received an intravaginal progesterone releasing device (CIDR-B1, InterAg, Hamilton, New Zealand) for 7 d, combined with estradiol benzoate (EB, 2.5 mg, im; Estrogin1, Farmavet, Sa˜ o Paulo,
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Brazil) at CIDR insertion, a second cloprostenol treatment at CIDR removal and 1 mg EB 24 h later. Cows were also synchronized with a CIDR plus EB combination but did not receive an initial dose of cloprostenol. Additionally, all cows received a GnRH agonist (Liserelin, 50 mg, im; Gestran Plus1, ARSA S.R.L., Buenos Aires, Argentina) at CIDR insertion, cloprostenol at CIDR removal and EB 24 h later. To minimize the handling of a few animals that may have an extended interval to ovulation after the end of the synchronization regimen, heifers and cows were excluded from the study if ovulation was delayed >54 h after the last EB treatment. 2.3. Ultrasonography Ultrasonography (Aloka SSD 500 with a 7.5 MHZ transrectal probe, Tokyo, Japan) was used to determine the presence of a CL, detect ovulation and monitor follicular development pre- and post-ovulation. Subsequent to EB treatment, only the largest follicle was monitored every 12 h to detect ovulation. Beginning on the day of ovulation and for 6 d thereafter, the two or three largest follicles/ovary were tracked individually at 12-h intervals and measured using the built-in electronic calipers of the scanner [11] from at least two separate images per follicle at each examination. The accuracy of the electronic measurements were verified in a water bath by taking the measurements of a sphere with a known cross-sectional diameter of 8.4 mm; a diameter representative of the expected time of deviation as reported in Holstein heifers [3]. The water-bath diameter of the sphere was determined from 10 separate images by calculating the average of two lines of measurement approximately perpendicular to one another, similar to that done in vivo. The mean S.D. was 8.3 0.1 mm, with a range of 8.2–8.4 mm. The three largest follicles/animal were designated Follicle 1 (F1, largest), Follicle 2 (F2, second largest) and Follicle 3 (F3, third largest) according to their hierarchal position on day 6. Emergence of the post-ovulatory wave (Wave 1) was defined according to first detection of F1 when it reached >3.0 mm. Only waves with a single dominant follicle were assessed for deviation; waves with co-dominant follicles (two follicles >10 mm [12]) were not considered. To determine the time of observed deviation, the sequential changes in diameter of F1 and F2 from first detection of F1 to day 6 were inspected. In some instances the change in growth of F3 was used to support that of F2 for determining deviation [13]. The last examination before the decrease in growth of F2 or F3 was designated the beginning of observed deviation [3]. To determine the time of calculated deviation, a segmented regression model was used as previously described [9]. Briefly, to prepare the follicle data for analysis, the differences in diameter between F1 and F2 or, in some instances F3, were determined. The diameter differences were truncated before the examination when F1 reached >4 mm and after the examination when F1 reached >10 mm or at its maximum diameter if F1 attained <10 mm by day 6. Although an attempt was made to standardize the truncated period, it was sometimes necessary to truncate the upper limit based on inspection of the diameter profiles of the three largest follicles [9]. The truncation of the follicle data was done to exclude or minimize the extent of the variability associated with follicle measurements around the time of detection of F1 and the beginning of the static phase [14] and focus the segmented regression on a period critical to calculating deviation. For each animal, the truncated period was fit to the diameter
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differences in a successive manner beginning with the earliest time. Segment 1 represented the common growth phase before deviation [7], Segment 2 represented the period of dominance thereafter, and a Join Point connecting the two segments represented the end of the common growth phase and the beginning of deviation. The optimal Join Point value that corresponded to the maximum coefficient of determination (R2) was designated the calculated day of deviation provided the slope of Segment 2 was greater than the slope of Segment 1. The diameter of F1 corresponding to the calculated day of deviation was estimated using a simple linear regression of the diameters of F1 during the truncated period for each animal. The slope of the regression equation was also used to represent the growth rate of F1 during the corresponding period. 2.4. Statistical analysis Prior to statistical analyses, the data were examined for normality using Kolmogorov– Smirnov test and suspected extreme values were challenged using Dixon’s outlier test [15]. If the normality test was significant (P < 0.05), the data were transformed by either natural logarithm or square root. If neither transformation satisfied normality, the data were ranked according to the Kruskal. Wallis test. The SAS MIXED procedure with a repeated statement to account for the autocorrelation between sequential follicle measurements was used to determine the main effects of heifers versus cows and day and the corresponding interaction. If significant main effects or interactions were detected, unpaired t-tests were used to determine mean differences between heifers and cows within days and paired t-tests were used to determine mean differences between days within heifers and cows. The observed and calculated methods to determine the time to deviation and the diameter of F1 at deviation were compared within and between heifers and cows using t-tests. Number of days from ovulation to first detection of F1, diameter of F1 when first detected and growth rate of F1 between heifers and cows were also examined using t-tests. A probability of P < 0.05 indicated that a difference was significant and probabilities between P > 0.05 and P < 0.1 indicated that a difference approached significance. The original data are presented as the mean S.E.M. unless otherwise indicated.
3. Results 3.1. Heifers Two of 11 heifers were excluded from the study because they failed to ovulate within 54 h after the last hormonal treatment of the synchronization regimen and lost 10–20 kg in body weight during the corresponding time. The remaining nine heifers ovulated a mean 43.5 h following the last hormonal treatment and after the largest follicle reached a mean 11.4 mm. A third heifer was excluded because of poor demeanor and the difficulty to consistently collect adequate follicle data. Days from ovulation to the beginning of observed and calculated deviation and diameters of F1 at deviation for eight individual heifers are shown in Table 1. In Heifer A,
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Table 1 Days from ovulation (day 0) to the beginning of observed and calculated deviation and diameter (mm) of Follicle 1 (largest follicle) at deviation in Nelore heifers Day
Diameter of Follicle 1
Heifers
Observed
Calculated
Combined
Observed
Calculated
Combined
A B C D E F G H
ND 3.0 2.5 2.5 2.0 2.5 4.5 3.0
ND 3.0 2.5 2.2 2.3 2.1 4.4 2.2
– 3.0 2.5 2.4 2.2 2.3 4.4 2.6
– 6.0 5.1 6.4 6.3 5.5 5.6 5.0
– 5.9 6.1 6.0 6.6 4.8 6.0 4.5
– 6.0 5.6 6.2 6.4 5.2 5.8 4.8
Mean S.D. S.E.M.
2.9 0.8 0.3
2.7 0.8 0.3
2.8 0.8 0.3
5.7 0.6 0.2
5.7 0.8 0.3
5.7 0.6 0.2
No significant differences for the mean number of days and diameters of Follicle 1 between observed and calculated deviation; therefore, the results were combined for each method. ND (not determined) because observed deviation could not be readily identified and the requirement for calculated deviation was not satisfied (i.e., slope of Segment 2 must be greater than the slope of Segment 1).
observed deviation could not be readily identified. Subsequent to wave emergence, the increase in diameter of F1 and F2 occurred in parallel until the growth rate of F2 began to wane after day 1.5 (Fig. 1). After day 2, however, the growth rate of F2 resurged until a more abrupt and sustained cessation of growth occurred after day 4. Consequently, calculated deviation could not be estimated because the slope of the regression line for the diameter differences for Segment 2 was less than that for Segment 1 which did not satisfy the condition that the slope of Segment 2 must be greater than that of Segment 1. Alternatively, F3 was inspected to determine if it could be used to assist in the determination of deviation but the amount of available data was insufficient. For the remainder of the animals, observed and calculated deviation was readily determined using F1 and F2 (Heifers D–H) or F1 and F3 (Heifers B and C). There were no significant differences between the mean days and diameters of F1 for observed and calculated deviation (Table 1). The diameter profiles for F1 and F3 are depicted for Heifer B (Fig. 1) to illustrate the beginning of observed deviation on day 3 when F1 reached 6 mm. 3.2. Cows Eleven of 23 cows were excluded from the study because they failed to ovulate within 54 h after the last hormonal treatment of the synchronization regimen. For the other 12 cows (six pure- and six cross-bred animals), ovulation occurred a mean 42.5 h following the last hormonal treatment and after the largest follicle reached a mean 12.3 mm. One of the 12 cross-bred cows was not included in the determination of deviation because F1 and F2 appeared as co-dominant follicles (11.6 and 10.7 mm, respectively). Days from ovulation to the beginning of observed and calculated deviation and diameters of F1 at deviation for 11 individual cows are shown in Table 2. Observed and
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Fig. 1. Diameter profiles of the three largest follicles (Follicle 1, 2 or 3) of the first post-ovulatory wave beginning when Follicle 1 was first detected at >3 mm in individual Nelore heifers and nonlactating cows. In Heifer A, observed and calculated deviation could not be determined because of the atypical growth profile of Follicle 2; there was not an adequate amount of data to consider using Follicle 3. In Heifer B, observed deviation was readily determined based on the departure in growth between Follicles 1 and 3 beginning on day 3 when Follicle 1 reached 6 mm. In cow A, observed deviation was readily determined based on the departure in growth between Follicles 1 and 2 beginning on day 3.5 when Follicle 1 reached 6.6 mm.
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Table 2 Days from ovulation (day 0) to the beginning of observed and calculated deviation and diameter (mm) of Follicle 1 (largest follicle) at deviation in nonlactating Nelore cows Day
Diameter of Follicle 1
Cows
Observed
Calculated
Combined
Observed
Calculated
Combined
A B C D E F G H I J K
3.5 2.0 1.5 2.0 3.0 1.5 2.0 3.5 3.0 1.5 1.5
3.4 2.4 1.5 2.0 3.0 1.0 2.4 3.9 3.0 2.2 2.1
3.5 2.2 1.5 2.0 3.0 1.2 2.2 3.7 3.0 1.8 1.8
6.6 6.8 5.8 6.0 5.7 6.5 5.4 7.9 5.3 5.9 4.2
6.3 6.9 5.8 6.4 5.7 4.8 6.3 7.9 5.5 6.9 5.8
6.4 6.8 5.8 6.2 5.7 5.6 5.8 7.9 5.4 6.4 5.0
Mean S.D. S.E.M.
2.3 0.8 0.2
2.4 0.8 0.2
2.4 0.8 0.2
6.0 1.0 0.3
6.1 1.0 0.2
6.1 0.8 0.2
No significant differences for the mean number of days and diameters of Follicle 1 between observed and calculated deviation; therefore, the results were combined for each method.
calculated deviations were determined using F1 and F2 (Cows A–D and F–K) or F1 and F3 (Cow E). There were no significant differences between the mean days and diameters of F1 for observed and calculated deviation (Table 2). The diameter profiles for F1 and F2 are depicted for Cow A (Fig. 1) to illustrate the beginning of observed deviation on day 3.5 when F1 reached 6.6 mm. 3.3. Heifers versus cows There were no significant differences between heifers and cows for the number of days from ovulation to first detection of F1, diameters of F1 at first detection and growth rates of F1 during deviation (Table 3). Similarly, there were no significant differences between heifers and cows for the day of observed and calculated deviation and diameter of F1 at observed and calculated deviation as presented and compared between Tables 1 and 2.
Table 3 Mean S.E.M. characteristics of Follicle 1 (largest follicle) of the first follicular wave following ovulation (day 0) in Nelore heifers (n = 8) and nonlactating cows (n = 11) Follicle 1
Heifers
Cows
Combined
First detection after ovulation (d) Diameter at first detection (mm) Growth rate during deviation (mm/d)
1.0 + 0.2 4.3 + 0.2 1.3 + 0.2
0.8 + 0.2 4.3 + 0.2 1.4 + 0.5
0.9 + 0.2 4.3 + 0.1 1.4 + 0.1
No significant differences between Nelore heifers and cows for each characteristic of Follicle 1; therefore, the results were combined.
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Fig. 2. Mean S.E.M. diameters of the largest (Follicle 1) and second largest (Follicle 2) follicles at 12-h interval in Nelore heifers and nonlactating cows after adjusting the follicle data for each heifer and cow to the beginning of observed deviation (0 h). The left set of figures illustrates the diameter changes between Follicles 1 and 2 within heifers and cows while the other set indicates the statistical comparisons between heifers and cows for each of Follicles 1 and 2. Changes in diameters of Follicles 1 and 2 were not significantly different between heifers and cows (no main effect of heifers vs. cows or interaction with hour). The main effect of hour for both follicles was attributed to the progressive increases in diameters encompassing deviation for Follicle 1 (36 to 36 h) and before deviation for Follicle 2 (36 to 0 h).
The diameters of F1 and F2 at 12-h interval were adjusted to the beginning of observed deviation for each heifer and cow, averaged and then profiled from 36 h before to 36 h after deviation (Fig. 2). The left set of figures illustrates the mean follicle changes within heifers and cows while the other set indicates the statistical comparisons between heifers and cows for each follicle. Changes in diameters of F1 and F2 were not significantly different between heifers and cows (no main effect of heifers versus cows or interaction with hour). Averaged over all animals, the diameters of F1 and F2 at the beginning of observed deviation were 5.9 and 5.4 mm, respectively. There was a significant main effect of hour for both follicles that was attributed to the progressive increases in diameters encompassing deviation for F1 (36 to 36 h) and before deviation for F2 (36 to 0 h). Subsequent to deviation (0 to 36 h), there were no further significant increases in diameter of F2.
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4. Discussion The beginning of the differential change in growth rates between the largest (F1) and second largest (F2) follicles (i.e., diameter deviation) associated with the first postovulatory wave during the estrous cycle (Wave 1) was not significantly different between Nelore heifers and nonlactating cows, regardless of the methods (graphical versus mathematical) used to determine deviation. Averaged over both methods of determination, deviation occurred 2.8 d after ovulation when diameter of F1 reached 5.7 mm in heifers and 2.4 d after ovulation when F1 reached 6.1 mm in cows. The interval from ovulation to the beginning of diameter deviation and growth rate of F1 during deviation were not different between Nelore heifers and nonlactating cows and appeared comparable to that in Holstein heifers during deviation [3–5]. The emergence of Wave 1 and the interval from emergence to the beginning of deviation were also similar between Nelore heifers and cows but, compared to Holstein heifers [7,8], wave emergence occurred later and the interval from emergence to deviation occurred earlier. The emergence of Wave 1 approximately 1 d after ovulation followed by deviation approximately 1.6 d later when F1 reached approximately 6 mm in Nelore may be reflective of relatively low concentrations of FSH and relatively high concentrations of IGF-1 in Bos indicus as compared to Bos taurus cattle. In this regard, temporal changes in mean maximal concentrations of circulating FSH associated with the periovulatory FSH surge were about 48% less, while circulating concentrations of total IGF-1 were about 129% more in Brahman cows than in Angus cows during the estrous cycle [16]. In Holstein heifers, a recent functional study [17] indicated that IGF-1 might be a prominent factor involved in facilitating continued development of the dominant follicle in the presence of low concentrations of FSH during deviation. That IGF-1 may enhance follicle responsiveness to FSH in Zebu cattle is speculated on the basis of numerous summary reports [1] that lower doses of FSH induced a superovulatory response that appeared comparable to that in European breeds of cattle using higher doses of FSH. Together, the previous and present results indicate that the timing of gonadotropic and somatotropic effects on follicular wave emergence and deviation may be different between some breeds of Bos indicus and Bos taurus. The present study was not designed to examine the nature of follicle selection, but to characterize the beginning of diameter deviation as a key morphological event of selection so that it may serve as a reference point, as it has in Holsteins [8], to address the physiological basis of follicle selection in future studies in Nelore. The beginning of diameter deviation between F1 and F2 was determined in individual Nelore heifers and nonlactating cows using graphical (observed deviation) and mathematical (calculated deviation) methods as previously documented [9]. The latter method is a more objective approach that was used, in part, to substantiate the graphical method. In some instances (16%), the third largest follicle (F3) was used in addition or in place of F2 to assist in determining the day that observed and calculated deviation began. This approach seemed appropriate especially since it has been documented [13] that the beginning of deviation occurs, on average, simultaneously between the dominant follicle and the next three largest subordinate follicles of a wave. The diameter of F1 corresponding to the beginning of observed deviation was determined directly from the diameter data that
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was collected at 12-h interval for each heifer and cow, whereas that for calculated deviation was estimated using a simple linear regression of the follicle data during deviation with the calculated day of deviation included in the regression equation. The results of determining the beginning of follicle diameter deviation within heifers and cows using the observed and calculated methods were not significantly different. Thus, the mathematical method of determining diameter deviation substantiated the graphical method as a valid approach to determine follicle deviation in Nelore heifers and nonlactating cows. Combined, the beginning of deviation was determined 95% of the time as previously reported in Holstein heifers [9]. In one Nelore heifer (Heifer A), what appeared to be the beginning of observed deviation on day 1.5 was followed by resurgence in growth of F2 after day 2. A similar morphological event with a similar outcome has been reported in Holstein heifers [9]. Perhaps, the resurgence in the growth of F2 may have represented a delayed response to elevated gonadotropins as a result of a deficiency of F1 to adequately suppress FSH during deviation. Although the nature of this event is not known, a similar concept has been proposed for multiple deviations occurring within a follicular wave having co-dominant follicles in Holstein heifers [12]. Nonetheless, this relatively rare occurrence in the growth profiles of the two largest follicles during wave development negated the use of the graphical and mathematical methods to determine deviation. The present study provided new information that the timing of follicular wave emergence and diameter changes associated with the beginning of deviation is similar between Nelore heifers and nonlactating cows. It is not likely that the Gir component of the five cross-bred cows (<20% for each animal) had a major influence on the results since the crosses were within-species and the genetic make up of the pure-bred Nelore cows and the larger Nelore component of the cross-bred animals predominated. In a previous study [6] comparing the same types of animals, follicle development well after diameter deviation was not different between Nelore heifers and cows. Although the present and past studies indicated that follicular wave development (i.e., follicle emergence, growth, regression and dominance) is comparable in nulliparous and multiparous nonlactating Nelore cattle, follicular wave development in lactating animals may be different. The effect of lactation on follicle emergence, growth, regression and dominance is not known in Zebu cattle, but recently the results of a simultaneous comparison of ovarian function during the estrous cycle in Holstein heifers and lactating cows were reported [18]. Multiple ovulations and larger-sized ovulatory follicles were detected in cows than in heifers. Although the study was not designed to examine the morphological characteristics of the dominant and subordinate follicles during deviation, a retrospective assessment indicated that the mean diameters of the dominant and largest subordinate follicles were larger at the beginning of observed deviation in lactating cows (9.8 and 9.0 mm) than in heifers (8.3 and 7.5 mm). The mechanism that seems to allow dominant follicles to reach larger diameters in lactating Holstein cows than in heifers is not thoroughly known and whether a similar event occurs in lactating Bos indicus cattle, especially during deviation, awaits to be addressed. In conclusion, there were no significant differences between Nelore heifers and nonlactating cows regarding the initiation of follicle diameter deviation irrespective of the methods used to determine deviation. Hence, the morphological results were combined and summarized in relation to comparable characteristics reported in Holstein heifers [3–5]:
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(1) Emergence of the first post-ovulatory wave of the estrous cycle occurred 0.9 d after ovulation (approximately 1 d later than in Holsteins). (2) Diameter deviation occurred 2.6 d after ovulation (comparable to that in Holsteins) or about 1.6 d after wave emergence (approximately 1 d earlier than in Holsteins). (3) Growth rate of the largest follicle during deviation was 1.4 mm/d (comparable to that in Holsteins). (4) Diameter of the largest follicle at the beginning of deviation was 5.9 mm (approximately 2 mm smaller than in Holsteins). The impact that these results will have on current superstimulation regimens in Nelore is not known. Future studies are necessary to examine simultaneously the morphological and physiological characteristics during follicle deviation in Nelore and to determine whether these apparent differences between Bos indicus and Bos taurus inferred herein are related to species, breeds within a species (beef versus dairy), reproductive status (nonlacating versus lactating), climate (tropical versus temperate), nutritional management (pastured versus feedlot), or other factors. Acknowledgements The authors wish to thank Drs. Antonio C. Silveira, Mario B. Arrigoni and Francisco S. Wechsler, for lending the animals to conduct this study at Lageado Experimental Farm, University of Sa˜ o Paulo State (UNESP), Botucatu, Sa˜ o Paulo, Brazil. Evandro S. Sartorelli was supported by a CAPES fellowship for graduate work at the Institute of Bioscience (IBUNESP) and Luciano M. Carvalho was supported by a FAPESP fellowship for graduate work at the College of Veterinary Medicine (FMVZ-UNESP).
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