Domestic Animal Endocrinology 46 (2014) 58–64
Contents lists available at ScienceDirect
Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com
Determination of anti-Müllerian hormone at estrus during a synchronized and a natural bovine estrous cycle K.E. Pfeiffer, L.J. Jury, J.E. Larson* Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS 39762, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2013 Received in revised form 16 May 2013 Accepted 17 May 2013
Anti-Müllerian hormone (AMH) has been correlated with phenotypic indicators of fertility. However, the effects of exogenous hormones used during estrus synchronization on AMH have not been evaluated. Therefore, the objective of this experiment was to determine whether concentrations of AMH at estrus are similar between a synchronized compared with a natural estrous cycle. Nulliparous dairy and beef heifers (n ¼ 68) were synchronized with the Select Synch þ controlled internal drug release (CIDR) protocol (GnRH þ CIDR-7 d-CIDR removal þ PG). Heifers were observed for expression of estrus every 6 h until 84 h after the injection of PG. Visual detection of the subsequent estrus, considered natural estrus, occurred every 6 h from day 16 to 24 after synchronized estrus. At the time of standing estrus, ovarian structures in heifers were evaluated by transrectal ultrasonography. Blood samples were collected at estrus for analysis of concentrations of AMH during the synchronized and natural estrous cycles. The GLM and CORR procedures of SAS were used to analyze data. Concentrations of AMH between natural and synchronized estrus were positively correlated (r ¼ 0.67; P < 0.001). Mean concentration of AMH did not differ (P > 0.05) between the natural (0.0543 0.0076 ng/mL) or synchronized (0.0428 0.0076 ng/mL) estrous cycles. In conclusion, concentrations of AMH were similar between natural and synchronized estrous cycles. Concentrations of AMH in natural and synchronized estrous cycles were highly correlated within individual heifers and varied among heifers with beef heifers having increased (P < 0.05) concentrations of AMH compared with dairy heifers (0.0638 0.01 and 0.0402 0.01 ng/mL, respectively). Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Anti-Müllerian hormone Bovine Estrus synchronization
1. Introduction The ability of a female to produce an oocyte and successfully sustain an embryo is the primary factor that reduces the efficiency of cow–calf production. Development of a prognostic method, using phenotypic factors, to identify females with suboptimal fertility could advance production in the cattle industry. However, establishment of these factors has been difficult because of the complexity of
* Corresponding author. Department of Animal and Dairy Sciences, Mississippi State University, 4025 Wise Center Dr, Mississippi State, MS 39762, USA. Tel.: þ1 662 325 0040; fax: þ1 662 325 8873. E-mail address: [email protected] (J.E. Larson). 0739-7240/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.05.004
interactions that affect fertility and the inability to quantify the female gonad. Ovarian dynamics of cattle are becoming increasingly relevant as indicators of fertility. The concept of the ovarian reserve has been established to both quantify and qualify the female gonad [1]; it is the number of morphologically healthy follicles contained in the ovary [2] and is associated with fertility in cattle [3]. With the use of serial ultrasonography, it is possible to measure the follicular population on the ovary and to determine antral follicle count (AFC), which could be used as an indicator of ovarian reserve [4]. The AFC is the total number of follicles 3 mm in diameter per pair of ovaries [3] and is positively correlated to the ovarian reserve. Reductions in follicle counts have an effect on ovarian function through changes in the concentrations
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
of gonadotropins [3,5–7] and progesterone (P4) [3] which may ultimately affect pregnancy [8–12]. With establishment of a correlation between AFC and pregnancy in beef heifers [13] and dairy cows [5], additional research in reproductive performance and response to estrus synchronization [14] as it relates to anti-Müllerian hormone (AMH) is necessary. The ability to estimate ovarian reserve and potential fertility via a blood sample rather than ultrasonography would be more conducive in a production setting. AntiMüllerian hormone is a glycoprotein produced exclusively in the granulosa cells of developing follicles, and concentrations are positively associated with ovarian reserve [1,3]. Thus, it is an endocrine indicator of small antral follicles that are responsive to gonadotropins [15]. Anti-Müllerian hormone has been identified as a factor that influences folliculogenesis by inhibiting the recruitment of primordial follicles and decreasing the efficacy of FSH in developing follicles [16,17]. Variations in the concentration of AMH are independent of follicular dynamics occurring in the bovine estrous cycle that allow for determination through the collection of a single blood sample and are indicative of hormone production over a continuing period [15]. Changes in concentrations of AMH in plasma have not been evaluated between a natural bovine estrous cycle and an estrous cycle synchronized by using hormones. Additional research is necessary to establish the significance of gonadotropins in secretion of AMH [18] in addition to the hormones used in an estrus synchronization protocol. The objective of this experiment was to determine whether concentrations of AMH are similar in a synchronized estrous cycle compared with a natural estrous cycle. It was hypothesized that concentrations would be similar between a synchronized and a natural estrus. It is important to understand whether exogenous hormones influence concentrations of AMH to design future experiments and to accurately use AMH as a potential predictor of ovarian reserve. 2. Materials and methods Animal care, handling, and protocols used in this study were approved by the Mississippi State University Institutional Animal Care and Use Committee. 2.1. Animals This experiment was conducted before the fall breeding season (October to December) 2011 with the use of cycling, nulliparous beef and dairy heifers. Beef heifers (n ¼ 24), consisting of Angus (n ¼ 19) and Charolais (n ¼ 5) breeds, were managed at the Mississippi Agricultural and Forestry Experiment Station’s Leveck Animal Research Center in Mississippi State, MS. Beef heifers averaged 385.1 24.5 d (mean SD) with a range of 330 to 426 d of age at the initiation of estrus synchronization which were later categorized into 3 categories (350 mo) for statistical analysis. The average weight determined at the initiation of the experiment of Angus heifers was 330.4 21.0 kg, ranging from 301.2 to 388.3 kg, and the average weight of Charolais heifers was 374.5 32.4 kg, ranging
59
from 332.9 to 442.8 kg. Weights were later categorized into 3 categories (350 kg) for statistical analysis. Reproductive tract scores (RTSs; scale of 1–5; 1 ¼ immature 30 mm in diameter, good tone, erect, and >10-mm follicles, corpus luteum present [19]) were determined at the time of the initial blood sample by the veterinarian at either the beef or dairy research center. The average RTS of the beef heifers was 4.6 0.5 with a range of 4 to 5. Dairy heifers (n ¼ 44), consisting of Holstein (n ¼ 34) and Jersey (n ¼ 10) breeds, were managed at the Joe Bearden Dairy Research Center in Mississippi State, MS. Dairy heifers averaged 423.9 25.7 d with a range of 378 to 463 d of age at the initiation of estrus synchronization which were later categorized into 3 categories (14.75 mo) for statistical analysis. The average body weight determined at the initiation of the experiment of Holstein heifers was 365.1 38.4 kg, ranging from 297.6 to 447.2 kg, and the average weight of Jersey heifers was 266.8 29.1 kg, ranging from 236.8 to 324.8 kg. Weights were later categorized into 3 categories (400 kg) for statistical analysis. Reproductive tract scores (scale of 1–5 [19]) were determined at the time of the initial blood sample and averaged 1.1 0.3 with a range of 1 to 3 in dairy heifers. 2.2. Experimental protocol Selection of heifers for inclusion in this experiment was based on the establishment of cyclicity. Two blood samples were obtained in 10-mL evacuated tubes that contained the anticoagulant K2 EDTA (BD Worldwide, Franklin Lakes, NJ) on day 20 and 10, relative to the expression of synchronized estrus (day 0). When at least 1 blood sample contained a concentration of P4 1 ng/mL, the heifer was considered to be cycling at the initiation of synchronization [20]. Synchronization of estrus was accomplished by the Select Synch þ controlled internal drug release protocol. Heifers received a controlled internal drug release (Pfizer Animal Health, New York, NY) vaginal insert that contained 1.38 g of P4 and an injection of GnRH (100 mg, intramuscularly; Fertagyl; Intervet Inc, Millsboro, DE) on day 9 (day 0 is predicated day of standing estrus). Seven days later (day 2) the insert was removed, and heifers received an injection of PG (25 mg, intramuscularly; Lutalyse; Pfizer Animal Health). Heifers were observed for expression of synchronized estrus every 6 h until 84 h after the injection of PG. Concentrations of hormones and ovarian structures were evaluated on heifers detected in standing estrus or with an activated heatmount detector (Estrotect Heat Detector, Spring Valley, WI) affixed midline to the rump between the tailhead and the tuber coxae, and secondary signs of estrus. Blood samples were collected at estrus (6 h) via venipuncture of the coccygeal vein of the tail and were analyzed to determine AMH during synchronized estrus. Transrectal ultrasonography (10.0- to 5.0-MHz linear-array transducer; MicroMaxx; SonoSite, Inc, Bothell, WA) was used at estrus to measure ovarian structures. Fourteen days after the occurrence of the synchronized estrus, additional
60
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
blood samples were collected via venipuncture of the coccygeal vein of the tail and were analyzed to determine P4 during the luteal phase of the synchronized estrous cycle. Visual detection of the subsequent estrus, considered natural estrus, occurred every 6 h from day 16 to 24 after synchronized estrus. At estrus (6 h), blood samples were collected and analyzed for concentrations of AMH, and transrectal ultrasonography was used to determine ovarian structures with the use the same procedures as in the synchronized estrus stated previously. Additional blood samples were collected 14 d after the expression of natural estrus to determine the concentration of P4 Heifers that did not exhibit estrus within the estrus detection windows for both the synchronized and the natural estrous cycles were eliminated from the experiment, resulting in complete data sets for 68 heifers. 2.3. Blood collection and hormonal analysis Concentration of P4 during the luteal phase, considered day 14 of the estrous cycles, was established during the synchronized estrous and natural estrous cycle by the collection of 1 blood sample in a 10-mL evacuated tube that contained K2 EDTA (BD Worldwide). Blood was centrifuged at 3,000 g for 20 min at 4 C, and plasma was recovered and stored at 20 C until analysis. Blood plasma was analyzed for concentrations of P4 by using a commercial RIA (Coat-A-Count; Siemens Medical Solutions Diagnostics, Los Angeles, CA). The assay kit was validated for bovine serum [21] by using an assay volume of 100 mL. Assay tubes for the standard curve contained 0.01, 0.025, 0.05, 0.2, 0.5, 1, 2, and 4 ng/tube. Assay sensitivity was 0.1 ng/mL. The intraassay and interassay CVs were 2.2% and 4.8%, respectively. Concentrations of AMH were established in 1 blood sample collected in 10-mL evacuated tubes without anticoagulant (BD Worldwide) at synchronized estrus (day 0) and natural estrus (day 21). Blood was allowed to clot while on ice, centrifuged at 3,000 g for 20 min at 4 C, and serum was recovered and stored at 20 C until analysis. Blood serum was analyzed for concentrations of AMH (AMH GEN II ELISA kit; Beckman Coulter, Inc, Webster, TX). The sensitivity of the assay was 0.002 ng/mL and has been validated for use in bovine [22]. The intra-assay and interassay CVs were 2.7% and 4.4%, respectively. Concentrations of AMH at each estrus were determined and analyzed for correlation between cycles and with P4.
of breed, RTS, age category, weight category, and day between the estrous cycles (categorized as 22 d). The model that assessed differences in concentrations of AMH in the estrous cycles (which included dairy and beef heifers) included the variables of cycle (natural or synchronized), follicle diameter, and concentration of P4. Correlations were also determined between AMH, P4, and follicle diameter and the aforementioned variables.
3. Results 3.1. Concentrations of AMH Concentration of AMH at estrus did not differ (P > 0.10) between the natural (0.0543 0.01 ng/mL) or synchronized (0.0428 0.01 ng/mL) estrous cycles. Concentrations of AMH between natural and synchronized estrus were positively correlated (r ¼ 0.67; P < 0.001; Fig. 1). No correlations (P > 0.10) existed between concentrations of AMH and P4 or concentration of AMH and follicle diameter in either the natural or synchronized estrus. Beef heifers had an increased (P < 0.05) concentration of AMH compared with dairy heifers (0.0638 0.01 ng/mL and 0.0402 0.01 ng/mL, respectively). When analyzing beef heifers only, concentrations of AMH did not differ (P > 0.10) between Angus and Charolais heifers (Table 1). Heifers with a RTS of 4 tended (P ¼ 0.06) to have increased concentrations of AMH compared with heifers with a RTS of 5 (Table 1). Concentrations of AMH did not differ (P > 0.10) among age categories of heifers, with heifers 13 mo having similar concentrations of AMH (Table 1). Concentrations of AMH did not differ (P > 0.10) among weight categories of heifers (Table 1). When analyzing dairy heifers only, concentrations of AMH did not differ (P > 0.10) between Holstein and Jersey heifers (Table 2). A significant (P < 0.05), positive correlation (r ¼ 0.31) was established between RTS and concentration of AMH in dairy heifers but not beef heifers (P > 0.10). When RTS increased, a tendency (P ¼ 0.07) was found
2.4. Statistical analyses The GLM and CORR procedures of SAS (SAS Institute Inc, Cary, NC) were used to analyze data. Least squares means were analyzed and separated when a protected F test of P 0.10 was detected. Differences were determined to be significant when P 0.05 and tendencies were reported at values of P > 0.05 and P 0.10. Variables were removed from models in a step-wise fashion when differences were not significant (P > 0.25). Results are presented as least squares means SEM. Because of significant differences in concentration of AMH between beef and dairy heifers, they were separated for analysis of concentrations of AMH and P4 and follicle diameter. That model included the variables
Fig. 1. Correlation between serum concentrations of AMH at a natural and a synchronized estrus. AMH, anti-Müllerian hormone.
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64 Table 1 Concentrations (ng/mL) of AMH at natural or synchronized estrus among characteristics of beef heifers. Parameter Breed Angus Charolais RTS 4 5 Age, mo 13 Weight, kg 350
Natural
Synchronized
Meana
0.0830 0.02 0.0600 0.03
0.0576 0.02 0.0211 0.02
0.0703 0.01 0.0406 0.02
0.1086 0.02b 0.0545 0.02c
0.0650 0.02 0.0378 0.01
0.0868 0.01b 0.0462 0.01c
0.0994 0.03 0.0887 0.02 0.0245 0.03
0.0360 0.03 0.0713 0.02 0.0211 0.03
0.0677 0.02 0.0800 0.02 0.0228 0.02
0.0555 0.04 0.0792 0.02 0.0905 0.03
0.0069 0.03 0.0644 0.02 0.0463 0.03
0.0312 0.03 0.0718 0.01 0.0684 0.02
Abbreviations: AMH, anti-Müllerian hormone; RTS, reproductive tract score. Data are presented as least squares means SEM. a Mean concentration of AMH between the natural and synchronized estruses. b,c Within column and parameter, means without a common superscript letter differ (P 0.10).
for concentration of AMH to be increased (Table 2). Concentrations of AMH did not differ (P > 0.10) among the age categories of heifers at the initiation of synchronized estrus, with heifers 14.75 mo having similar concentrations of AMH (Table 2). Concentrations of AMH did not differ (P > 0.10) among heifers in differing weight categories (Table 2). 3.2. Follicle diameter Follicle diameter did not differ (P > 0.10) between the natural (11.97 0.38 mm) or synchronized (12.72 0.38 mm) estrous cycles. Follicle diameters at estrus were smaller Table 2 Concentrations (ng/mL) of AMH at natural or synchronized estrus among characteristics of dairy heifers. Parameter Breed Holstein Jersey RTSc 1 1.5 2 2.5 3 Age, mo 14.75 Weight, kg 400
Natural
Synchronized
Meana
0.0411 0.01 0.0418 0.02
0.0354 0.01 0.0510 0.02
0.0382 0.01 0.0464 0.01
0.0020 0.0363 0.0378 0.0530 0.0730
0.03 0.02 0.01 0.03 0.02
0.0070 0.0382 0.0324 0.0353 0.0787
0.03 0.01 0.01 0.03 0.02
0.0045 0.0372 0.0351 0.0441 0.0758
61
(P < 0.001) in beef (10.99 0.43 mm) than in dairy (13.08 0.32 mm) heifers. In beef heifers, follicle diameter did not differ (P > 0.10) among the variables of breed, RTS, age classification, weight classification, or days between estrus. In addition, no correlation (P > 0.10) between the aforementioned variables was established for follicle diameter in the beef heifers. Follicle diameter of dairy heifers differed (P < 0.05) among weight classification (12.41 0.56, 13.78 0.33, 14.49 0.62 mm for 400 kg, respectively). In addition, a correlation (r ¼ 0.38; P < 0.05) was established between follicle diameter and weight of dairy heifers. Dairy heifers with >22 d between the synchronized estrus and the natural estrus had greater (P < 0.05) follicle diameter (14.41 0.66 mm) than heifers with 18 to 22 d between each estrus (12.65 0.28 mm) with heifers 0.10) between the natural (5.74 0.34 ng/mL) and synchronized (5.84 0.34 ng/mL) estrous cycles. Concentrations of P4 were similar (P > 0.10) between beef and dairy heifers (5.56 0.40 and 5.92 0.30 ng/mL, respectively). In beef heifers, concentrations of P4 did not differ (P > 0.10) among the variables of breed, RTS, age classification, weight classification, or days between estrus. In addition, no correlation (P > 0.10) between the aforementioned variables was established with concentrations of P4 in the beef heifers. Concentrations of P4 differed (P < 0.05) between Holstein and Jersey heifers, with Holstein heifers having an increased concentration of P4 compared with Jersey heifers (6.36 0.42 and 4.53 0.76 ng/mL, respectively). No correlations (P > 0.10) existed between concentrations of AMH and P4 or concentration of P4 and follicle diameter in either the natural or synchronized estrus. 4. Discussion Ovarian dynamics of cattle are becoming the focus of research to phenotypically identify females with
0.02b 0.01b,c 0.01b,c 0.02b,c 0.01b
0.0543 0.02 0.0313 0.01 0.0570 0.02
0.0512 0.02 0.0399 0.01 0.0281 0.02
0.0531 0.01 0.0356 0.01 0.0426 0.01
0.0284 0.02 0.0410 0.01 0.0553 0.02
0.0464 0.02 0.0422 0.01 0.0193 0.02
0.0374 0.01 0.0416 0.01 0.0373 0.01
Abbreviations: AMH, anti-Müllerian hormone; RTS, reproductive tract score. Data are presented as least squares means SEM. a Mean concentration of AMH between the natural and synchronized estruses. b,c Within column and parameter, means without a common superscript letter differ (P 0.10).
Fig. 2. Correlation between serum concentrations of P4 at a natural and a synchronized estrus. P4, progesterone.
62
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
suboptimal fertility. A decrease in the ovarian reserve leads to a reduction in reproductive efficiency through pregnancy failure [2]. Changes in concentrations of AMH in plasma have recently been evaluated during the natural bovine estrous cycle in cows but have not been evaluated during an estrous cycle synchronized with exogenous hormones in either cows or heifers. Estrus synchronization is frequently used to increase the feasibility of conducting large fertility trials. Its use also decreases variation in an experiment that may come from sires or timing of insemination, for example. The objective of this experiment was to determine whether concentrations of AMH are similar in a synchronized estrous cycle compared with a natural estrous cycle. Concentrations of AMH did not differ between the natural or synchronized estrus in this experiment and were highly correlated. In previous work, changes in AMH were evaluated during a natural estrous cycle in cows. In that study, concentrations of AMH were similar between estrus and the subsequent estrus in Holstein cows classified as high AFC and low AFC [18]. No change was found in concentrations of AMH from 6 to 9 d proceeding ovulation in Hereford, Charolais, or Angus heifers [4]. In addition, a high correlation (r2 ¼ 0.97) has been reported between the concentration of AMH from a single blood sample obtained regardless of day of the estrous cycle and both the overall mean of 4 to 8 blood samples from beef heifers and also 3 blood samples from dairy heifers [1]. The culmination of this previous work indicates concentration of AMH in a female may remain fairly constant within and across estrous cycles. The practical application is that a single blood sample collected at a random stage of the estrous cycle could give an accurate concentration of AMH. Although concentrations remain fairly constant within a female, the variation from female to female is problematic. Our results indicated a great degree of variation, particularly between dairy and beef breeds, but also within those categories. If the assessment of a concentration of a hormone in the blood is to be a valid predictor of a characteristic such as fertility, the variation must be within a manageable range. The extremely fertile or extremely infertile animals may be predicted with greater ease, but, to be truly effective, the fertility of all animals would need to be predictable. The variation in this present study was greater than in some previously published studies that used the first-generation ELISA to assess AMH. Variation in concentrations of AMH among Holstein cows, which were superovulated, was decreased, ranging from 0.025 to 0.228 ng/mL [2], compared with heifers in this present experiment, which ranged from 0.002 to 0.218 ng/mL. However, concentrations of AMH in the present experiment were decreased compared with an experiment that used Holstein heifers, which ranged from 0.006 to 0.433 ng/mL [1]. Although concentrations of AMH have been fairly consistent in females, the use of supraphysiological doses of FSH for superovulation may affect AMH and decrease this consistency. In dairy cows, a significant increase in concentrations of AMH occurred between the administration of the superovulatory treatment and estrus with a significant decrease in the concentrations of AMH occurring between estrus and the subsequent luteal phase 7 d later [2]. Although concentration of AMH changed over
the course of the estrous cycle, there remained a high correlation between concentrations of AMH at estrus and during the luteal phase (r ¼ 0.87 and r ¼ 0.93, respectively). Therefore, although supraphysiological doses of FSH may alter concentrations of AMH, the results of the present study indicate that the natural change of FSH which results from administration of exogenous GnRH and other hormones used in estrus synchronization does not similarly alter concentrations of AMH. Although the mechanism for how exogenous FSH used for superovulation might alter concentrations of AMH are not known, it may be a result of the increased recruitment of 3- to 7-mm follicles. Concentrations of AMH at administration of a superovulatory treatment were highly correlated with numbers of follicles at estrus and corpora lutea at day 7 of the cycle (r ¼ 0.83 and r ¼ 0.64, respectively [15]). Regardless, concentration of AMH of heifers in the present experiment did not affect follicle diameter at estrus or concentration of P4 on day 14. In addition, no correlations between AMH and follicle diameter or AMH and concentration of P4 on day 14 were present. Differences between these results and those that involved superovulated Holstein cows in the aforementioned experiment could simply be a result of the amount of FSH in circulation and the numbers of follicles being recruited. To further support this hypothesis, changes in the concentrations of AMH have been reported to occur independently of follicular waves during the estrous cycle [15]. Initial development of follicles that are 3 to 4 mm in size corresponds to the maximum production of AMH [23]. As the follicle reaches this size, FSH is necessary for the continued growth and development. As follicles become dependent on gonadotropins, a reduction in AMH occurs [23]. Production of AMH decreases during terminal follicular growth and atresia, independent of the follicular wave during the estrous cycle [18]. When superovulation treatments are used, this process can be exaggerated which does result in changes in circulating concentrations of AMH which are not otherwise detected. Although concentrations of AMH are fairly consistent and highly correlated [1,4,18] during a normal estrous cycle, concentrations of AMH decrease after estrus, with minimal concentrations between day 4 and 8 of the estrous cycle [18]. In the present study, blood samples were collected within 6 h after the initiation of standing estrus, but this timing may explain, in part, the decreased concentrations and increased variability that was detected compared with others [1,2]. It has been hypothesized that the decreased concentration of AMH after estrus results from inhibition by FSH and is not associated with a decreased population of follicles [18]. A low follicle count corresponds with a 15% to 50% greater circulating concentration of FSH when compared with a high AFC [5–7], although the number of receptors for FSH and LH does not differ [24,25]. Concentrations of AMH have been inversely correlated with concentrations of FSH in heifers [4]. This inverse relationship is also expressed in a 2-fold increase in basal concentration of LH [3]. On the basis of these findings, low AFC corresponds to a deleterious increased secretion of gonadotropins. This relationship among gonadotropins, AFC, and AMH is not clear. The capacity of granulosa cells to produce AMH
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
depends on the concentration of FSH. Increased and decreased concentrations of FSH produce variation in the production of AMH by granulosa cells. Increased concentrations of FSH have been correlated with decreased production of AMH, as previously stated. The decline in production of AMH could possibly be attributed to luteinization of granulosa cells [2,4]. The effects of gonadotropins on regulation of AMH require further elucidation [18], although the results of this present study indicate the use of exogenous hormones for estrus synchronization does not alter this delicate balance, at least enough to alter circulating concentrations of AMH. Decreased concentrations of FSH have been correlated with an increased production of AMH concomitant with an increased production of estradiol [6,7]. In contrast, an inverse relationship between AMH, during the first and last follicular wave of the estrous cycle, and estradiol has also been reported. Anti-Müllerian hormone has been negatively (r ¼ 0.94) then positively (r ¼ 0.94) correlated with concentrations of estradiol during the first and last wave of the estrous cycle. The dominant follicle during both waves was associated with an increased concentration of estradiol [18]. Follicle diameter in this experiment did not differ between the natural or synchronized estrous cycles, and no correlation between diameter and concentration of AMH was established. The variation in concentrations of AMH specifically during the emergence and regression of follicular waves requires further establishment [18]. 5. Conclusions In conclusion, concentrations of AMH at estrus were similar between a natural and a synchronized estrous cycle but differed between beef and dairy heifers. Concentrations of AMH at estrus in a natural and a synchronized estrous cycle were highly correlated within individual heifers and variable among heifers in this experiment. The results indicate that the use of this estrus synchronization protocol did not have an effect on concentration of AMH. This allows for further applicability of using AMH in future fertility trials in which estrus synchronization can be used, allowing for further assessment of females with differing follicular populations. Acknowledgments We thank Janet Ireland at Michigan State University for her assistance in analysis of concentrations of AMH. Approved for publication as Journal Article No.54354 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. References [1] Ireland JJ, Smith GW, Scheetz D, Jimenez-Krassel F, Folger JK, Ireland JL, Mossa F, Lonergan P, Evans AC. Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of antiMüllerian hormone as a diagnostic marker for fertility and causes of variation in the ovarian reserve in cattle. Reprod Fertil Dev 2011;23: 1–14.
63
[2] Ireland JJ, Zielak-Steciwko AE, Jimenez-Krassel F, Folger J, Bettegowda A, Scheetz D, Walsh S, Mossa F, Knight PG, Smith GW, Lonergan P, Evans AC. Variation in the ovarian reserve is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle. Biol Reprod 2009;80:954–64. [3] Jimenez-Krassel F, Folger J, Ireland JH, Smith GW, Hou X, Davis JS, Lonergan P, Evans AC, Ireland JJ. Evidence that high variation in ovarian reserves of healthy young adults has a negative impact on the corpus luteum and endometrium during reproductive cycles of single-ovulating species. Biol Reprod 2009;80:1272–81. [4] Ireland JL, Scheetz D, Jimenez-Krassel F, Themmen AP, Ward F, Lonergan P, Smith GW, Perez GI, Evans AC, Ireland JJ. Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle. Biol Reprod 2008;79: 1219–25. [5] Mossa F, Jimenez-Krassel F, Walsh S, Berry DP, Butler ST, Folger J, Smith GW, Ireland JL, Lonergan P, Ireland JJ, Evans AC. Inherent capacity of the pituitary gland to produce gonadotropins is not influenced by the number of ovarian follicles > or ¼ 3 mm in diameter in cattle. Reprod Fertil Dev 2010;22:550–7. [6] Burns DS, Jimenez-Krassel F, Ireland JL, Knight PG, Ireland JJ. Numbers of antral follicles during follicular waves in cattle: evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations. Biol Reprod 2005; 73:54–62. [7] Ireland JJ, Ward F, Jimenez-Krassel F, Ireland JL, Smith GW, Lonergan P, Evans AC. Follicle numbers are highly repeatable within individual animals but are inversely correlated with FSH concentrations and the proportion of good-quality embryos after ovarian stimulation in cattle. Hum Reprod 2007;22:1687–95. [8] Mann GE, Lamming GE. The influence of progesterone during early pregnancy in cattle. Reprod Domest Anim 1999;34:269–74. [9] Inskeep EK. Preovulatory, postovulatory, and postmaternal recognition effects of concentrations of progesterone on embryonic survival in the cow. J Anim Sci 2004;82:E24–39. [10] Stronge AJ, Sreenan JM, Diskin MG, Mee JF, Kenny DA, Morris DG. Post-insemination milk progesterone concentration and embryo survival in dairy cows. Theriogenology 2005;64:1212–24. [11] McNeill RE, Diskin MG, Sreenan JM, Morris DG. Associations between milk progesterone concentration on different days and with embryo survival during the early luteal phase in dairy cows. Theriogenology 2006;65:1435–41. [12] Diskin MG, Morris DG. Embryonic and early foetal losses in cattle and other ruminants. Reprod Domest Anim 2008;43:260–7. [13] Cushman RA, Allan MF, Kuehn LA, Snelling WM, Cupp AS, Freetly HC. Evaluation of antral follicle count and ovarian morphology in crossbred beef cows: investigation of influence of stage of the estrous cycle, age, and birth weight. J Anim Sci 2009;87:1971–80. [14] Perry GA. Physiology and endocrinology symposium: harnessing basic knowledge of factors controlling puberty to improve synchronization of estrus and fertility in heifers. J Anim Sci 2012;90: 1172–82. [15] Rico C, Fabre S, Medigue C, di Clemente N, Clement F, Bontoux M, Touze JL, Dupont M, Briant E, Remy B, Beckers JF, Monniaux D. Antimullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow. Biol Reprod 2009;80:50–9. [16] di Clemente N, Goxe B, Remy JJ, Cate RL, Josso N, Vigier B, Salesse R. Inhibitory effect of amh upon the expression of aromatase and LH receptors by cultured cytochrome p450 aromatase activity in human granulosa lutein cell culture. Fertil Steril 1994; 89:1364–70. [17] Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM, Rose UM, de Jong FH, Uilenbroek JT, Grootegoed JA, Themmen AP. Anti-Müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 2001; 142:4891–9. [18] Rico C, Medigue C, Fabre S, Jarrier P, Bontoux M, Clement F, Monniaux D. Regulation of anti-Müllerian hormone production in the cow: a multiscale study at endocrine, ovarian, follicular, and granulosa cell levels. Biol Reprod 2011;84:560–71. [19] Anderson KJ, LeFever DG, Brinks JS, Odde KG. The use of reproductive tract scoring in beef heifers. Agri-Practice 1991;12:19–26. [20] Perry RC, Corah LR, Cochran RC, Beal WE, Stevenson JS, Minton JE, Simms DD, Brethour JR. Influence of dietary energy on follicular development, serum gonadotropins, and first postpartum ovulation in suckled beef cows. J Anim Sci 1991;69:3762–73.
64
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
[21] Kirby CJ, Smith MF, Keisler DH, Lucy MC. Follicular function in lactating dairy cows with sustained-release bovine somatotropin. J Dairy Sci 1997;80:273–85. [22] Kumar A, Kalra B, Patel A, McDavid L, Roudebush WE. Development of a second generation anti-Müllerian hormone (AMH) ELISA. J Immunol Methods 2010;362:51–9. [23] Monniaux D, Clemente N, Touze JL, Belville C, Rico C, Bontoux M, Picard JY, Fabre S. Intrafollicular steroids and anti-mullerian
hormone during normal and cystic ovarian follicular development in the cow. Biol Reprod 2008;79:387–96. [24] Conti M, Harwood JP, Hsueh AJ, Dufau ML, Catt KJ. Gonadotropininduced loss of hormone receptors and desensitization of adenylate cyclase in the ovary. J Biol Chem 1976;251:7729–31. [25] Amsterdam A, Hanoch T, Dantes A, Tajima K, Strauss JF, Seger R. Mechanisms of gonadotropin desensitization. Mol Cell Endocrinol 2002;187:69–74.
Contents lists available at ScienceDirect
Domestic Animal Endocrinology journal homepage: www.domesticanimalendo.com
Determination of anti-Müllerian hormone at estrus during a synchronized and a natural bovine estrous cycle K.E. Pfeiffer, L.J. Jury, J.E. Larson* Department of Animal and Dairy Sciences, Mississippi State University, Mississippi State, MS 39762, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 January 2013 Received in revised form 16 May 2013 Accepted 17 May 2013
Anti-Müllerian hormone (AMH) has been correlated with phenotypic indicators of fertility. However, the effects of exogenous hormones used during estrus synchronization on AMH have not been evaluated. Therefore, the objective of this experiment was to determine whether concentrations of AMH at estrus are similar between a synchronized compared with a natural estrous cycle. Nulliparous dairy and beef heifers (n ¼ 68) were synchronized with the Select Synch þ controlled internal drug release (CIDR) protocol (GnRH þ CIDR-7 d-CIDR removal þ PG). Heifers were observed for expression of estrus every 6 h until 84 h after the injection of PG. Visual detection of the subsequent estrus, considered natural estrus, occurred every 6 h from day 16 to 24 after synchronized estrus. At the time of standing estrus, ovarian structures in heifers were evaluated by transrectal ultrasonography. Blood samples were collected at estrus for analysis of concentrations of AMH during the synchronized and natural estrous cycles. The GLM and CORR procedures of SAS were used to analyze data. Concentrations of AMH between natural and synchronized estrus were positively correlated (r ¼ 0.67; P < 0.001). Mean concentration of AMH did not differ (P > 0.05) between the natural (0.0543 0.0076 ng/mL) or synchronized (0.0428 0.0076 ng/mL) estrous cycles. In conclusion, concentrations of AMH were similar between natural and synchronized estrous cycles. Concentrations of AMH in natural and synchronized estrous cycles were highly correlated within individual heifers and varied among heifers with beef heifers having increased (P < 0.05) concentrations of AMH compared with dairy heifers (0.0638 0.01 and 0.0402 0.01 ng/mL, respectively). Ó 2014 Elsevier Inc. All rights reserved.
Keywords: Anti-Müllerian hormone Bovine Estrus synchronization
1. Introduction The ability of a female to produce an oocyte and successfully sustain an embryo is the primary factor that reduces the efficiency of cow–calf production. Development of a prognostic method, using phenotypic factors, to identify females with suboptimal fertility could advance production in the cattle industry. However, establishment of these factors has been difficult because of the complexity of
* Corresponding author. Department of Animal and Dairy Sciences, Mississippi State University, 4025 Wise Center Dr, Mississippi State, MS 39762, USA. Tel.: þ1 662 325 0040; fax: þ1 662 325 8873. E-mail address: [email protected] (J.E. Larson). 0739-7240/$ – see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.domaniend.2013.05.004
interactions that affect fertility and the inability to quantify the female gonad. Ovarian dynamics of cattle are becoming increasingly relevant as indicators of fertility. The concept of the ovarian reserve has been established to both quantify and qualify the female gonad [1]; it is the number of morphologically healthy follicles contained in the ovary [2] and is associated with fertility in cattle [3]. With the use of serial ultrasonography, it is possible to measure the follicular population on the ovary and to determine antral follicle count (AFC), which could be used as an indicator of ovarian reserve [4]. The AFC is the total number of follicles 3 mm in diameter per pair of ovaries [3] and is positively correlated to the ovarian reserve. Reductions in follicle counts have an effect on ovarian function through changes in the concentrations
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
of gonadotropins [3,5–7] and progesterone (P4) [3] which may ultimately affect pregnancy [8–12]. With establishment of a correlation between AFC and pregnancy in beef heifers [13] and dairy cows [5], additional research in reproductive performance and response to estrus synchronization [14] as it relates to anti-Müllerian hormone (AMH) is necessary. The ability to estimate ovarian reserve and potential fertility via a blood sample rather than ultrasonography would be more conducive in a production setting. AntiMüllerian hormone is a glycoprotein produced exclusively in the granulosa cells of developing follicles, and concentrations are positively associated with ovarian reserve [1,3]. Thus, it is an endocrine indicator of small antral follicles that are responsive to gonadotropins [15]. Anti-Müllerian hormone has been identified as a factor that influences folliculogenesis by inhibiting the recruitment of primordial follicles and decreasing the efficacy of FSH in developing follicles [16,17]. Variations in the concentration of AMH are independent of follicular dynamics occurring in the bovine estrous cycle that allow for determination through the collection of a single blood sample and are indicative of hormone production over a continuing period [15]. Changes in concentrations of AMH in plasma have not been evaluated between a natural bovine estrous cycle and an estrous cycle synchronized by using hormones. Additional research is necessary to establish the significance of gonadotropins in secretion of AMH [18] in addition to the hormones used in an estrus synchronization protocol. The objective of this experiment was to determine whether concentrations of AMH are similar in a synchronized estrous cycle compared with a natural estrous cycle. It was hypothesized that concentrations would be similar between a synchronized and a natural estrus. It is important to understand whether exogenous hormones influence concentrations of AMH to design future experiments and to accurately use AMH as a potential predictor of ovarian reserve. 2. Materials and methods Animal care, handling, and protocols used in this study were approved by the Mississippi State University Institutional Animal Care and Use Committee. 2.1. Animals This experiment was conducted before the fall breeding season (October to December) 2011 with the use of cycling, nulliparous beef and dairy heifers. Beef heifers (n ¼ 24), consisting of Angus (n ¼ 19) and Charolais (n ¼ 5) breeds, were managed at the Mississippi Agricultural and Forestry Experiment Station’s Leveck Animal Research Center in Mississippi State, MS. Beef heifers averaged 385.1 24.5 d (mean SD) with a range of 330 to 426 d of age at the initiation of estrus synchronization which were later categorized into 3 categories (350 mo) for statistical analysis. The average weight determined at the initiation of the experiment of Angus heifers was 330.4 21.0 kg, ranging from 301.2 to 388.3 kg, and the average weight of Charolais heifers was 374.5 32.4 kg, ranging
59
from 332.9 to 442.8 kg. Weights were later categorized into 3 categories (350 kg) for statistical analysis. Reproductive tract scores (RTSs; scale of 1–5; 1 ¼ immature 30 mm in diameter, good tone, erect, and >10-mm follicles, corpus luteum present [19]) were determined at the time of the initial blood sample by the veterinarian at either the beef or dairy research center. The average RTS of the beef heifers was 4.6 0.5 with a range of 4 to 5. Dairy heifers (n ¼ 44), consisting of Holstein (n ¼ 34) and Jersey (n ¼ 10) breeds, were managed at the Joe Bearden Dairy Research Center in Mississippi State, MS. Dairy heifers averaged 423.9 25.7 d with a range of 378 to 463 d of age at the initiation of estrus synchronization which were later categorized into 3 categories (14.75 mo) for statistical analysis. The average body weight determined at the initiation of the experiment of Holstein heifers was 365.1 38.4 kg, ranging from 297.6 to 447.2 kg, and the average weight of Jersey heifers was 266.8 29.1 kg, ranging from 236.8 to 324.8 kg. Weights were later categorized into 3 categories (400 kg) for statistical analysis. Reproductive tract scores (scale of 1–5 [19]) were determined at the time of the initial blood sample and averaged 1.1 0.3 with a range of 1 to 3 in dairy heifers. 2.2. Experimental protocol Selection of heifers for inclusion in this experiment was based on the establishment of cyclicity. Two blood samples were obtained in 10-mL evacuated tubes that contained the anticoagulant K2 EDTA (BD Worldwide, Franklin Lakes, NJ) on day 20 and 10, relative to the expression of synchronized estrus (day 0). When at least 1 blood sample contained a concentration of P4 1 ng/mL, the heifer was considered to be cycling at the initiation of synchronization [20]. Synchronization of estrus was accomplished by the Select Synch þ controlled internal drug release protocol. Heifers received a controlled internal drug release (Pfizer Animal Health, New York, NY) vaginal insert that contained 1.38 g of P4 and an injection of GnRH (100 mg, intramuscularly; Fertagyl; Intervet Inc, Millsboro, DE) on day 9 (day 0 is predicated day of standing estrus). Seven days later (day 2) the insert was removed, and heifers received an injection of PG (25 mg, intramuscularly; Lutalyse; Pfizer Animal Health). Heifers were observed for expression of synchronized estrus every 6 h until 84 h after the injection of PG. Concentrations of hormones and ovarian structures were evaluated on heifers detected in standing estrus or with an activated heatmount detector (Estrotect Heat Detector, Spring Valley, WI) affixed midline to the rump between the tailhead and the tuber coxae, and secondary signs of estrus. Blood samples were collected at estrus (6 h) via venipuncture of the coccygeal vein of the tail and were analyzed to determine AMH during synchronized estrus. Transrectal ultrasonography (10.0- to 5.0-MHz linear-array transducer; MicroMaxx; SonoSite, Inc, Bothell, WA) was used at estrus to measure ovarian structures. Fourteen days after the occurrence of the synchronized estrus, additional
60
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
blood samples were collected via venipuncture of the coccygeal vein of the tail and were analyzed to determine P4 during the luteal phase of the synchronized estrous cycle. Visual detection of the subsequent estrus, considered natural estrus, occurred every 6 h from day 16 to 24 after synchronized estrus. At estrus (6 h), blood samples were collected and analyzed for concentrations of AMH, and transrectal ultrasonography was used to determine ovarian structures with the use the same procedures as in the synchronized estrus stated previously. Additional blood samples were collected 14 d after the expression of natural estrus to determine the concentration of P4 Heifers that did not exhibit estrus within the estrus detection windows for both the synchronized and the natural estrous cycles were eliminated from the experiment, resulting in complete data sets for 68 heifers. 2.3. Blood collection and hormonal analysis Concentration of P4 during the luteal phase, considered day 14 of the estrous cycles, was established during the synchronized estrous and natural estrous cycle by the collection of 1 blood sample in a 10-mL evacuated tube that contained K2 EDTA (BD Worldwide). Blood was centrifuged at 3,000 g for 20 min at 4 C, and plasma was recovered and stored at 20 C until analysis. Blood plasma was analyzed for concentrations of P4 by using a commercial RIA (Coat-A-Count; Siemens Medical Solutions Diagnostics, Los Angeles, CA). The assay kit was validated for bovine serum [21] by using an assay volume of 100 mL. Assay tubes for the standard curve contained 0.01, 0.025, 0.05, 0.2, 0.5, 1, 2, and 4 ng/tube. Assay sensitivity was 0.1 ng/mL. The intraassay and interassay CVs were 2.2% and 4.8%, respectively. Concentrations of AMH were established in 1 blood sample collected in 10-mL evacuated tubes without anticoagulant (BD Worldwide) at synchronized estrus (day 0) and natural estrus (day 21). Blood was allowed to clot while on ice, centrifuged at 3,000 g for 20 min at 4 C, and serum was recovered and stored at 20 C until analysis. Blood serum was analyzed for concentrations of AMH (AMH GEN II ELISA kit; Beckman Coulter, Inc, Webster, TX). The sensitivity of the assay was 0.002 ng/mL and has been validated for use in bovine [22]. The intra-assay and interassay CVs were 2.7% and 4.4%, respectively. Concentrations of AMH at each estrus were determined and analyzed for correlation between cycles and with P4.
of breed, RTS, age category, weight category, and day between the estrous cycles (categorized as 22 d). The model that assessed differences in concentrations of AMH in the estrous cycles (which included dairy and beef heifers) included the variables of cycle (natural or synchronized), follicle diameter, and concentration of P4. Correlations were also determined between AMH, P4, and follicle diameter and the aforementioned variables.
3. Results 3.1. Concentrations of AMH Concentration of AMH at estrus did not differ (P > 0.10) between the natural (0.0543 0.01 ng/mL) or synchronized (0.0428 0.01 ng/mL) estrous cycles. Concentrations of AMH between natural and synchronized estrus were positively correlated (r ¼ 0.67; P < 0.001; Fig. 1). No correlations (P > 0.10) existed between concentrations of AMH and P4 or concentration of AMH and follicle diameter in either the natural or synchronized estrus. Beef heifers had an increased (P < 0.05) concentration of AMH compared with dairy heifers (0.0638 0.01 ng/mL and 0.0402 0.01 ng/mL, respectively). When analyzing beef heifers only, concentrations of AMH did not differ (P > 0.10) between Angus and Charolais heifers (Table 1). Heifers with a RTS of 4 tended (P ¼ 0.06) to have increased concentrations of AMH compared with heifers with a RTS of 5 (Table 1). Concentrations of AMH did not differ (P > 0.10) among age categories of heifers, with heifers 13 mo having similar concentrations of AMH (Table 1). Concentrations of AMH did not differ (P > 0.10) among weight categories of heifers (Table 1). When analyzing dairy heifers only, concentrations of AMH did not differ (P > 0.10) between Holstein and Jersey heifers (Table 2). A significant (P < 0.05), positive correlation (r ¼ 0.31) was established between RTS and concentration of AMH in dairy heifers but not beef heifers (P > 0.10). When RTS increased, a tendency (P ¼ 0.07) was found
2.4. Statistical analyses The GLM and CORR procedures of SAS (SAS Institute Inc, Cary, NC) were used to analyze data. Least squares means were analyzed and separated when a protected F test of P 0.10 was detected. Differences were determined to be significant when P 0.05 and tendencies were reported at values of P > 0.05 and P 0.10. Variables were removed from models in a step-wise fashion when differences were not significant (P > 0.25). Results are presented as least squares means SEM. Because of significant differences in concentration of AMH between beef and dairy heifers, they were separated for analysis of concentrations of AMH and P4 and follicle diameter. That model included the variables
Fig. 1. Correlation between serum concentrations of AMH at a natural and a synchronized estrus. AMH, anti-Müllerian hormone.
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64 Table 1 Concentrations (ng/mL) of AMH at natural or synchronized estrus among characteristics of beef heifers. Parameter Breed Angus Charolais RTS 4 5 Age, mo 13 Weight, kg 350
Natural
Synchronized
Meana
0.0830 0.02 0.0600 0.03
0.0576 0.02 0.0211 0.02
0.0703 0.01 0.0406 0.02
0.1086 0.02b 0.0545 0.02c
0.0650 0.02 0.0378 0.01
0.0868 0.01b 0.0462 0.01c
0.0994 0.03 0.0887 0.02 0.0245 0.03
0.0360 0.03 0.0713 0.02 0.0211 0.03
0.0677 0.02 0.0800 0.02 0.0228 0.02
0.0555 0.04 0.0792 0.02 0.0905 0.03
0.0069 0.03 0.0644 0.02 0.0463 0.03
0.0312 0.03 0.0718 0.01 0.0684 0.02
Abbreviations: AMH, anti-Müllerian hormone; RTS, reproductive tract score. Data are presented as least squares means SEM. a Mean concentration of AMH between the natural and synchronized estruses. b,c Within column and parameter, means without a common superscript letter differ (P 0.10).
for concentration of AMH to be increased (Table 2). Concentrations of AMH did not differ (P > 0.10) among the age categories of heifers at the initiation of synchronized estrus, with heifers 14.75 mo having similar concentrations of AMH (Table 2). Concentrations of AMH did not differ (P > 0.10) among heifers in differing weight categories (Table 2). 3.2. Follicle diameter Follicle diameter did not differ (P > 0.10) between the natural (11.97 0.38 mm) or synchronized (12.72 0.38 mm) estrous cycles. Follicle diameters at estrus were smaller Table 2 Concentrations (ng/mL) of AMH at natural or synchronized estrus among characteristics of dairy heifers. Parameter Breed Holstein Jersey RTSc 1 1.5 2 2.5 3 Age, mo 14.75 Weight, kg 400
Natural
Synchronized
Meana
0.0411 0.01 0.0418 0.02
0.0354 0.01 0.0510 0.02
0.0382 0.01 0.0464 0.01
0.0020 0.0363 0.0378 0.0530 0.0730
0.03 0.02 0.01 0.03 0.02
0.0070 0.0382 0.0324 0.0353 0.0787
0.03 0.01 0.01 0.03 0.02
0.0045 0.0372 0.0351 0.0441 0.0758
61
(P < 0.001) in beef (10.99 0.43 mm) than in dairy (13.08 0.32 mm) heifers. In beef heifers, follicle diameter did not differ (P > 0.10) among the variables of breed, RTS, age classification, weight classification, or days between estrus. In addition, no correlation (P > 0.10) between the aforementioned variables was established for follicle diameter in the beef heifers. Follicle diameter of dairy heifers differed (P < 0.05) among weight classification (12.41 0.56, 13.78 0.33, 14.49 0.62 mm for 400 kg, respectively). In addition, a correlation (r ¼ 0.38; P < 0.05) was established between follicle diameter and weight of dairy heifers. Dairy heifers with >22 d between the synchronized estrus and the natural estrus had greater (P < 0.05) follicle diameter (14.41 0.66 mm) than heifers with 18 to 22 d between each estrus (12.65 0.28 mm) with heifers 0.10) between the natural (5.74 0.34 ng/mL) and synchronized (5.84 0.34 ng/mL) estrous cycles. Concentrations of P4 were similar (P > 0.10) between beef and dairy heifers (5.56 0.40 and 5.92 0.30 ng/mL, respectively). In beef heifers, concentrations of P4 did not differ (P > 0.10) among the variables of breed, RTS, age classification, weight classification, or days between estrus. In addition, no correlation (P > 0.10) between the aforementioned variables was established with concentrations of P4 in the beef heifers. Concentrations of P4 differed (P < 0.05) between Holstein and Jersey heifers, with Holstein heifers having an increased concentration of P4 compared with Jersey heifers (6.36 0.42 and 4.53 0.76 ng/mL, respectively). No correlations (P > 0.10) existed between concentrations of AMH and P4 or concentration of P4 and follicle diameter in either the natural or synchronized estrus. 4. Discussion Ovarian dynamics of cattle are becoming the focus of research to phenotypically identify females with
0.02b 0.01b,c 0.01b,c 0.02b,c 0.01b
0.0543 0.02 0.0313 0.01 0.0570 0.02
0.0512 0.02 0.0399 0.01 0.0281 0.02
0.0531 0.01 0.0356 0.01 0.0426 0.01
0.0284 0.02 0.0410 0.01 0.0553 0.02
0.0464 0.02 0.0422 0.01 0.0193 0.02
0.0374 0.01 0.0416 0.01 0.0373 0.01
Abbreviations: AMH, anti-Müllerian hormone; RTS, reproductive tract score. Data are presented as least squares means SEM. a Mean concentration of AMH between the natural and synchronized estruses. b,c Within column and parameter, means without a common superscript letter differ (P 0.10).
Fig. 2. Correlation between serum concentrations of P4 at a natural and a synchronized estrus. P4, progesterone.
62
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
suboptimal fertility. A decrease in the ovarian reserve leads to a reduction in reproductive efficiency through pregnancy failure [2]. Changes in concentrations of AMH in plasma have recently been evaluated during the natural bovine estrous cycle in cows but have not been evaluated during an estrous cycle synchronized with exogenous hormones in either cows or heifers. Estrus synchronization is frequently used to increase the feasibility of conducting large fertility trials. Its use also decreases variation in an experiment that may come from sires or timing of insemination, for example. The objective of this experiment was to determine whether concentrations of AMH are similar in a synchronized estrous cycle compared with a natural estrous cycle. Concentrations of AMH did not differ between the natural or synchronized estrus in this experiment and were highly correlated. In previous work, changes in AMH were evaluated during a natural estrous cycle in cows. In that study, concentrations of AMH were similar between estrus and the subsequent estrus in Holstein cows classified as high AFC and low AFC [18]. No change was found in concentrations of AMH from 6 to 9 d proceeding ovulation in Hereford, Charolais, or Angus heifers [4]. In addition, a high correlation (r2 ¼ 0.97) has been reported between the concentration of AMH from a single blood sample obtained regardless of day of the estrous cycle and both the overall mean of 4 to 8 blood samples from beef heifers and also 3 blood samples from dairy heifers [1]. The culmination of this previous work indicates concentration of AMH in a female may remain fairly constant within and across estrous cycles. The practical application is that a single blood sample collected at a random stage of the estrous cycle could give an accurate concentration of AMH. Although concentrations remain fairly constant within a female, the variation from female to female is problematic. Our results indicated a great degree of variation, particularly between dairy and beef breeds, but also within those categories. If the assessment of a concentration of a hormone in the blood is to be a valid predictor of a characteristic such as fertility, the variation must be within a manageable range. The extremely fertile or extremely infertile animals may be predicted with greater ease, but, to be truly effective, the fertility of all animals would need to be predictable. The variation in this present study was greater than in some previously published studies that used the first-generation ELISA to assess AMH. Variation in concentrations of AMH among Holstein cows, which were superovulated, was decreased, ranging from 0.025 to 0.228 ng/mL [2], compared with heifers in this present experiment, which ranged from 0.002 to 0.218 ng/mL. However, concentrations of AMH in the present experiment were decreased compared with an experiment that used Holstein heifers, which ranged from 0.006 to 0.433 ng/mL [1]. Although concentrations of AMH have been fairly consistent in females, the use of supraphysiological doses of FSH for superovulation may affect AMH and decrease this consistency. In dairy cows, a significant increase in concentrations of AMH occurred between the administration of the superovulatory treatment and estrus with a significant decrease in the concentrations of AMH occurring between estrus and the subsequent luteal phase 7 d later [2]. Although concentration of AMH changed over
the course of the estrous cycle, there remained a high correlation between concentrations of AMH at estrus and during the luteal phase (r ¼ 0.87 and r ¼ 0.93, respectively). Therefore, although supraphysiological doses of FSH may alter concentrations of AMH, the results of the present study indicate that the natural change of FSH which results from administration of exogenous GnRH and other hormones used in estrus synchronization does not similarly alter concentrations of AMH. Although the mechanism for how exogenous FSH used for superovulation might alter concentrations of AMH are not known, it may be a result of the increased recruitment of 3- to 7-mm follicles. Concentrations of AMH at administration of a superovulatory treatment were highly correlated with numbers of follicles at estrus and corpora lutea at day 7 of the cycle (r ¼ 0.83 and r ¼ 0.64, respectively [15]). Regardless, concentration of AMH of heifers in the present experiment did not affect follicle diameter at estrus or concentration of P4 on day 14. In addition, no correlations between AMH and follicle diameter or AMH and concentration of P4 on day 14 were present. Differences between these results and those that involved superovulated Holstein cows in the aforementioned experiment could simply be a result of the amount of FSH in circulation and the numbers of follicles being recruited. To further support this hypothesis, changes in the concentrations of AMH have been reported to occur independently of follicular waves during the estrous cycle [15]. Initial development of follicles that are 3 to 4 mm in size corresponds to the maximum production of AMH [23]. As the follicle reaches this size, FSH is necessary for the continued growth and development. As follicles become dependent on gonadotropins, a reduction in AMH occurs [23]. Production of AMH decreases during terminal follicular growth and atresia, independent of the follicular wave during the estrous cycle [18]. When superovulation treatments are used, this process can be exaggerated which does result in changes in circulating concentrations of AMH which are not otherwise detected. Although concentrations of AMH are fairly consistent and highly correlated [1,4,18] during a normal estrous cycle, concentrations of AMH decrease after estrus, with minimal concentrations between day 4 and 8 of the estrous cycle [18]. In the present study, blood samples were collected within 6 h after the initiation of standing estrus, but this timing may explain, in part, the decreased concentrations and increased variability that was detected compared with others [1,2]. It has been hypothesized that the decreased concentration of AMH after estrus results from inhibition by FSH and is not associated with a decreased population of follicles [18]. A low follicle count corresponds with a 15% to 50% greater circulating concentration of FSH when compared with a high AFC [5–7], although the number of receptors for FSH and LH does not differ [24,25]. Concentrations of AMH have been inversely correlated with concentrations of FSH in heifers [4]. This inverse relationship is also expressed in a 2-fold increase in basal concentration of LH [3]. On the basis of these findings, low AFC corresponds to a deleterious increased secretion of gonadotropins. This relationship among gonadotropins, AFC, and AMH is not clear. The capacity of granulosa cells to produce AMH
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
depends on the concentration of FSH. Increased and decreased concentrations of FSH produce variation in the production of AMH by granulosa cells. Increased concentrations of FSH have been correlated with decreased production of AMH, as previously stated. The decline in production of AMH could possibly be attributed to luteinization of granulosa cells [2,4]. The effects of gonadotropins on regulation of AMH require further elucidation [18], although the results of this present study indicate the use of exogenous hormones for estrus synchronization does not alter this delicate balance, at least enough to alter circulating concentrations of AMH. Decreased concentrations of FSH have been correlated with an increased production of AMH concomitant with an increased production of estradiol [6,7]. In contrast, an inverse relationship between AMH, during the first and last follicular wave of the estrous cycle, and estradiol has also been reported. Anti-Müllerian hormone has been negatively (r ¼ 0.94) then positively (r ¼ 0.94) correlated with concentrations of estradiol during the first and last wave of the estrous cycle. The dominant follicle during both waves was associated with an increased concentration of estradiol [18]. Follicle diameter in this experiment did not differ between the natural or synchronized estrous cycles, and no correlation between diameter and concentration of AMH was established. The variation in concentrations of AMH specifically during the emergence and regression of follicular waves requires further establishment [18]. 5. Conclusions In conclusion, concentrations of AMH at estrus were similar between a natural and a synchronized estrous cycle but differed between beef and dairy heifers. Concentrations of AMH at estrus in a natural and a synchronized estrous cycle were highly correlated within individual heifers and variable among heifers in this experiment. The results indicate that the use of this estrus synchronization protocol did not have an effect on concentration of AMH. This allows for further applicability of using AMH in future fertility trials in which estrus synchronization can be used, allowing for further assessment of females with differing follicular populations. Acknowledgments We thank Janet Ireland at Michigan State University for her assistance in analysis of concentrations of AMH. Approved for publication as Journal Article No.54354 of the Mississippi Agricultural and Forestry Experiment Station, Mississippi State University. References [1] Ireland JJ, Smith GW, Scheetz D, Jimenez-Krassel F, Folger JK, Ireland JL, Mossa F, Lonergan P, Evans AC. Does size matter in females? An overview of the impact of the high variation in the ovarian reserve on ovarian function and fertility, utility of antiMüllerian hormone as a diagnostic marker for fertility and causes of variation in the ovarian reserve in cattle. Reprod Fertil Dev 2011;23: 1–14.
63
[2] Ireland JJ, Zielak-Steciwko AE, Jimenez-Krassel F, Folger J, Bettegowda A, Scheetz D, Walsh S, Mossa F, Knight PG, Smith GW, Lonergan P, Evans AC. Variation in the ovarian reserve is linked to alterations in intrafollicular estradiol production and ovarian biomarkers of follicular differentiation and oocyte quality in cattle. Biol Reprod 2009;80:954–64. [3] Jimenez-Krassel F, Folger J, Ireland JH, Smith GW, Hou X, Davis JS, Lonergan P, Evans AC, Ireland JJ. Evidence that high variation in ovarian reserves of healthy young adults has a negative impact on the corpus luteum and endometrium during reproductive cycles of single-ovulating species. Biol Reprod 2009;80:1272–81. [4] Ireland JL, Scheetz D, Jimenez-Krassel F, Themmen AP, Ward F, Lonergan P, Smith GW, Perez GI, Evans AC, Ireland JJ. Antral follicle count reliably predicts number of morphologically healthy oocytes and follicles in ovaries of young adult cattle. Biol Reprod 2008;79: 1219–25. [5] Mossa F, Jimenez-Krassel F, Walsh S, Berry DP, Butler ST, Folger J, Smith GW, Ireland JL, Lonergan P, Ireland JJ, Evans AC. Inherent capacity of the pituitary gland to produce gonadotropins is not influenced by the number of ovarian follicles > or ¼ 3 mm in diameter in cattle. Reprod Fertil Dev 2010;22:550–7. [6] Burns DS, Jimenez-Krassel F, Ireland JL, Knight PG, Ireland JJ. Numbers of antral follicles during follicular waves in cattle: evidence for high variation among animals, very high repeatability in individuals, and an inverse association with serum follicle-stimulating hormone concentrations. Biol Reprod 2005; 73:54–62. [7] Ireland JJ, Ward F, Jimenez-Krassel F, Ireland JL, Smith GW, Lonergan P, Evans AC. Follicle numbers are highly repeatable within individual animals but are inversely correlated with FSH concentrations and the proportion of good-quality embryos after ovarian stimulation in cattle. Hum Reprod 2007;22:1687–95. [8] Mann GE, Lamming GE. The influence of progesterone during early pregnancy in cattle. Reprod Domest Anim 1999;34:269–74. [9] Inskeep EK. Preovulatory, postovulatory, and postmaternal recognition effects of concentrations of progesterone on embryonic survival in the cow. J Anim Sci 2004;82:E24–39. [10] Stronge AJ, Sreenan JM, Diskin MG, Mee JF, Kenny DA, Morris DG. Post-insemination milk progesterone concentration and embryo survival in dairy cows. Theriogenology 2005;64:1212–24. [11] McNeill RE, Diskin MG, Sreenan JM, Morris DG. Associations between milk progesterone concentration on different days and with embryo survival during the early luteal phase in dairy cows. Theriogenology 2006;65:1435–41. [12] Diskin MG, Morris DG. Embryonic and early foetal losses in cattle and other ruminants. Reprod Domest Anim 2008;43:260–7. [13] Cushman RA, Allan MF, Kuehn LA, Snelling WM, Cupp AS, Freetly HC. Evaluation of antral follicle count and ovarian morphology in crossbred beef cows: investigation of influence of stage of the estrous cycle, age, and birth weight. J Anim Sci 2009;87:1971–80. [14] Perry GA. Physiology and endocrinology symposium: harnessing basic knowledge of factors controlling puberty to improve synchronization of estrus and fertility in heifers. J Anim Sci 2012;90: 1172–82. [15] Rico C, Fabre S, Medigue C, di Clemente N, Clement F, Bontoux M, Touze JL, Dupont M, Briant E, Remy B, Beckers JF, Monniaux D. Antimullerian hormone is an endocrine marker of ovarian gonadotropin-responsive follicles and can help to predict superovulatory responses in the cow. Biol Reprod 2009;80:50–9. [16] di Clemente N, Goxe B, Remy JJ, Cate RL, Josso N, Vigier B, Salesse R. Inhibitory effect of amh upon the expression of aromatase and LH receptors by cultured cytochrome p450 aromatase activity in human granulosa lutein cell culture. Fertil Steril 1994; 89:1364–70. [17] Durlinger AL, Gruijters MJ, Kramer P, Karels B, Kumar TR, Matzuk MM, Rose UM, de Jong FH, Uilenbroek JT, Grootegoed JA, Themmen AP. Anti-Müllerian hormone attenuates the effects of FSH on follicle development in the mouse ovary. Endocrinology 2001; 142:4891–9. [18] Rico C, Medigue C, Fabre S, Jarrier P, Bontoux M, Clement F, Monniaux D. Regulation of anti-Müllerian hormone production in the cow: a multiscale study at endocrine, ovarian, follicular, and granulosa cell levels. Biol Reprod 2011;84:560–71. [19] Anderson KJ, LeFever DG, Brinks JS, Odde KG. The use of reproductive tract scoring in beef heifers. Agri-Practice 1991;12:19–26. [20] Perry RC, Corah LR, Cochran RC, Beal WE, Stevenson JS, Minton JE, Simms DD, Brethour JR. Influence of dietary energy on follicular development, serum gonadotropins, and first postpartum ovulation in suckled beef cows. J Anim Sci 1991;69:3762–73.
64
K.E. Pfeiffer et al. / Domestic Animal Endocrinology 46 (2014) 58–64
[21] Kirby CJ, Smith MF, Keisler DH, Lucy MC. Follicular function in lactating dairy cows with sustained-release bovine somatotropin. J Dairy Sci 1997;80:273–85. [22] Kumar A, Kalra B, Patel A, McDavid L, Roudebush WE. Development of a second generation anti-Müllerian hormone (AMH) ELISA. J Immunol Methods 2010;362:51–9. [23] Monniaux D, Clemente N, Touze JL, Belville C, Rico C, Bontoux M, Picard JY, Fabre S. Intrafollicular steroids and anti-mullerian
hormone during normal and cystic ovarian follicular development in the cow. Biol Reprod 2008;79:387–96. [24] Conti M, Harwood JP, Hsueh AJ, Dufau ML, Catt KJ. Gonadotropininduced loss of hormone receptors and desensitization of adenylate cyclase in the ovary. J Biol Chem 1976;251:7729–31. [25] Amsterdam A, Hanoch T, Dantes A, Tajima K, Strauss JF, Seger R. Mechanisms of gonadotropin desensitization. Mol Cell Endocrinol 2002;187:69–74.
