Clinical Theriogenology 2022; 14: 91 Serum anti-Müllerian hormone concentrations in female alpacas: variations during the reproductive cycle and correlation with ovarian superstimulation response Cristian Patino,a Eduardo Arroyo,a Michela Ciccarelli,a,b Jacobo Rodriguez,c Alan Conley,d Ahmed Tibarya,b aDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine and bCenter for Reproductive Biology, Washington State University, Pullman, WA cVista Equine Colorado Clinic, Fort Collins, CO dDepartment of Population Health and Reproduction, School of Veterinary Medicine University of California, Davis, CA Abstract Earlier, we validated an anti-Müllerian hormone (AMH) enzyme-linked immunosorbent assay kit for male alpacas. First, we compared the validation data with another kit. There was a high correlation (R2 = 0.94) between these 2 kits. Second, we used the latter kit to determine serum AMH concentrations during follicular and luteal phases of the reproductive cycle in female alpacas. There were no differences (p = 0.39) in serum AMH concentrations in alpacas (n = 11) between peak follicular and luteal phases (mean ± SEM, 1.33 ± 0.35 versus 1.18 ± 0.34 ng/ml, respectively). Third, we treated female alpacas (n = 13; 5 - 11 years) after 14- day treatment with decreasing doses of porcine follicle-stimulating hormone. There was no effect (p > 0.05) of day of treatment on serum AMH concentrations. Number of follicles (7 - 10 mm; mean ± SD [as determined via transrectal ultrasonography]) at end of treatment (12.69 ± 5.25; range: 6 - 24) was positively correlated (R2 = 0.7; p < 0.01) with serum AMH concentrations. To conclude, the kit tested is usable for female alpacas; serum AMH concentrations were not affected by the cycle stage nor by ovarian superstimulation treatment. Furthermore, a significant correlation between serum AMH serum concentrations and response to su- perstimulation suggested that estimation of serum AMH concentrations may be valuable in determining ovarian follicular reserve. Keywords: Camelid, anti-Müllerian hormone, enzyme-linked immunosorbent assay, follicular reserve Introduction Anti-Müllerian hormone (AMH), also known as Müllerian in- hibiting substance, is a homodimeric disulfide-linked glycopro- tein member of the transforming growth factor ß superfamily.1 In male mammals, AMH is produced by Sertoli cells during fetal development.2,3 In contrast, in females, AMH is expressed for the first time in the neonatal ovary exclusively by healthy granulosa cells of small primary growing follicles after recruitment from the primordial follicle pool.4,5 Anti-Müllerian hormone binds to specific receptors to activate intracellular signal transduction mechanisms.6-8 In females, AMH has 2 functions: it inhibits pri- mordial follicular growth (to prevent premature exhaustion of ovarian follicular reserve) and reduces FSH responses of preant- ral and small antral follicles (to suppress the development of nondominant follicles).9 Serum concentrations of AMH were determined in cows,10,11 ewes,12,13 mares,14 and women.9,15,16 Se- rum concentrations increased a few months after birth, declined until the onset of puberty, then increased again and remained relatively stable throughout the cycle until ovarian senescence/ menopause.11,17,18 In cattle,19-22 women,7,9,23 mice,17 and small ruminants,10,24 se- rum AMH concentrations remained constant during cycles and were positively correlated with the number of antral follicles in women.9 Serum AMH serum concentrations are considered an indirect measure of the number of morphologically healthy ovarian follicles. It is speculated that a single measurement of AMH can predict follicular reserve. There was a good correlation between AMH serum concentrations and quality of embryos produced after ovarian superstimulation in cattle,19,25-27 goats,28 and sheep.11,12,29 Additionally, in cattle, serum AMH concentra- tions were highly correlated with pregnancy rate and early em- bryo survival.22,30 There was a positive correlation between AMH concentrations (measured just before FSH treatment) and the number of gonadotropin-responsive follicles that developed to the preovulatory stage in several ruminant species (cattle,20,25-27 goats,28 and sheep 11,12,29). Although assisted reproductive technologies in camels31 and south American camelids32,33 have increased since the 1990s, studies on AMH serum concentrations in female camelids are lacking. Female camels present overlapping follicular waves in anovulation that makes ovarian superstimulation more difficult (reviewed by34). Ovarian superstimulation protocol for came- Clinical Theriogenology 2022; 14: 92 lids has been extrapolated from other species, despite many differences in reproductive physiology. In camelids, however, embryo yield after superstimulation is highly variable and often poor due to lack of response, overstimulation, or poor fertiliza- tion.31-34 Studies on AMH in female camelids may shed some light on reasons for this variability in response to superstimu- lation and ovarian follicular dynamic abnormalities. Further- more, like several mammalian species, estimation of serum AMH concentrations in camelids may be a good indicator of ovarian follicular reserve, fertility, and ovarian response to su- perstimulation. Most studies used either human or species-specific en- zyme-linked immunosorbent assay (ELISA) commercial kits for AMH serum concentration determination. Validated ELISA kits are available for cattle, dogs, horses, humans, mice, and sheep.35 Earlier, we validated an AMH enzyme-linked immunosorbent assay kit for male alpacas36 and determined that serum AMH concentrations remained relatively stable in prepubertal and post pubertal males, but declined in adult males after castra- tion.36 Our aims were to validate a commercial ELISA kit for female alpacas, characterize serum concentrations of AMH in cycling alpacas, and to determine whether there was a correlation be- tween AMH serum concentrations and number of antral folli- cles after FSH ovarian superstimulation. Materials and methods Animals and sample collection Healthy, multiparous, and reproductively sound Huacaya fe- male alpacas (5 - 11 years) were used. Animals were housed and evaluated at the Washington State University Veterinary Teach- ing Hospital. Animals were fed alfalfa and grass hay at a mainte- nance level to sustain a body condition score between 2.5 and 3 out of 5 (1 = emaciated, 5 = obese). Water and mineral salt were provided ad libitum. Animals were on regular internal and ex- ternal parasite control and vaccination program. Animals were not subjected to prior ovarian superstimulation. Procedures were approved by the institutional animal care and use commit- tee of Washington State University. Validation of anti-Müllerian hormone enzyme-linked immu- nosorbent assay commercial kit Unavailability of a validated36 commercial AMH assay (AMH ELISA test Kit Cat # KAMH-01; Preventia Diagnositcs, Siascon- set, MA) prompted us to validate an alternate commercial kit (AMH ELISA AL-113 kit [Ansh Labs, Webster, TX AL-113]). Sera collected from 5 male alpacas (before and after castration)36 were used. Serum anti-Müllerian hormone concentrations during cycle Female alpacas (n = 11) were evaluated via transrectal ultraso- nography (Exago®, Universal Imaging, New York) using a 7.5 MHz linear array transducer mounted on a polyvinyl chloride extension handle. Alpacas were monitored every other day until they developed a mature follicle (7 - 10 mm in diameter) and had uterine edema. Ovulation was induced with intramuscular GnRH (50 μg/animal, [Cystorelin®, Merial, Duluth, GA]) and verified via ultrasonography (corpus luteum) and serum proges- terone concentrations. Blood samples were collected via jugu- lar venipuncture at ovulation induction (day 0) and during the luteal phase (7 days after ovulation induction). Blood samples were allowed to clot, centrifuged (1,500 g for 10 minutes), and harvested sera were stored at – 20°C until analysis. Ovarian superstimulation and serum anti-Müllerian hormone concentrations Females (n = 13) were synchronized with intramuscular GnRH (50 µg/animal [Cystorelin®) and progesterone vaginal implants (controlled internal drug release [CIDR, Easy breed®, 0.3 mg progesterone, Zoetis, Kalamazoo, MI]), were inserted. Ovaries were superstimulated with intramuscular porcine FSH (Foll- tropin®, Vetoquinol Inc, Fort Worth, TX), twice daily at 12-hour intervals (day 12 - 16) in decreasing doses (30, 25, 20, 15, and 10 mg). Vaginal implant was removed on day 14, and animals were given intramuscular cloprostenol (250 µg). Transrectal ultrasonography was performed on days 0, 7, 12, 17, and 24; ovarian follicular status was determined on days of examination and mature follicles (7 - 10 mm) were counted at the end of porcine follicle-stimulating hormone (pFSH) treatment. Blood samples were collected via jugular venipuncture for AMH and progesterone assays on days 0, 3, 7, 12, 17, and 24 (Figure 1). Ovulation was induced with GnRH when most follicles reached 8 mm diameter. Blood samples were allowed to clot, centrifuged (1,500 g for 10 minutes), and harvested sera were stored at – 20°C until essayed. Hormone analyses Serum AMH concentrations were determined from 50 µl dupli- cate aliquots. Monoclonal antibody pairs bind to the noncova- lent AMH complex that do not detect other related members of the TGF-ß superfamily. Samples from each female were included in the same analytical run. Intra-assay coefficients of variation for serum pools with high (13.5 ng/ml, n = 14), medium (6.2 ng/ml, n = 14), and low concentrations (2.3 ng/ml, n = 14) were 2.6, 5.5, and 4.1%, respectively. Lowest calibrator was 0.41 ng/ ml and values below were estimated by extrapolation. Figure 1. Synchronization, ovarian superstimulation, and sampling protocol; porcine follicle- stimulating hormone (pFSH), controlled internal drug release (CIDR), transrectal ultrasonography (US), progesterone (P4) anti-Müllerian hormone (AMH), and cloprostenol (Clp) Progesterone concentrations were determined using a competitive enzyme immunoassay.37 Briefly, 100 µl of serum was extracted with 2 ml of petroleum ether, the solvent was decanted and dried. Extracts were reconstituted with 150 µl of horseradish peroxidase-conjugated progesterone in buffer, and 50 µl aliquots (in duplicate) were transferred to 96-well plates precoated with antisera raised against 11α-hemi-succinate-conjugated progesterone. Progesterone analysis was completed in 2 analytical runs. The average intra-assay coefficient of variation of low (0.5 ng/ml) and high (4.9 ng/ml) pools of samples included in each run was 11.4%, with an assay sensitivity of 5 pg/well.37 Data analysis Serum AMH concentrations obtained using Preventia® kit were compared to those obtained with Ansh (AL-113) using Pearson’s correlation coefficient. Serum AMH concentration at ovulation induction (day 0) and luteal phase (day 7) was compared by paired Student’s t-test. Effect of superstimulation on AMH serum concentrations was evaluated using repeated measurement analysis. Correlation between the number of antral follicles in response to FSH treatment and serum AMH concentrations were determined by linear regression analysis. Statistical analysis was performed using commercial software (Statistix 10®, Analytical Software, Tallahassee, FL). Significance was set at p < 0.05. Figure 1. Synchronization, ovarian superstimulation, and sampling protocol; porcine follicle-stimulating hormone (pFSH), con- trolled internal drug release (CIDR), transrectal ultrasonography (US), progesterone (P4) anti-Müllerian hormone (AMH), and cloprostenol (Clp) Clinical Theriogenology 2022; 14: 93 Progesterone concentrations were determined using a com- petitive enzyme immunoassay.37 Briefly, 100 µl of serum was extracted with 2 ml of petroleum ether, the solvent was de- canted and dried. Extracts were reconstituted with 150 µl of horseradish peroxidase-conjugated progesterone in buffer, and 50 µl aliquots (in duplicate) were transferred to 96-well plates precoated with antisera raised against 11α-hemi-suc- cinate-conjugated progesterone. Progesterone analysis was completed in 2 analytical runs. The average intra-assay coef- ficient of variation of low (0.5 ng/ml) and high (4.9 ng/ml) pools of samples included in each run was 11.4%, with an assay sensitivity of 5 pg/well.37 Data analysis Serum AMH concentrations obtained using Preventia® kit were compared to those obtained with Ansh (AL-113) using Pearson’s correlation coefficient. Serum AMH concentration at ovulation induction (day 0) and luteal phase (day 7) was compared by paired Student’s t-test. Effect of superstimulation on AMH serum concentrations was evaluated using repeated measurement analysis. Correlation between the number of antral follicles in response to FSH treatment and serum AMH concentrations were determined by linear regression analysis. Statistical analysis was performed using commercial software (Statistix 10®, Analytical Software, Tallahassee, FL). Signifi- cance was set at p < 0.05. Results Validation of anti-Müllerian hormone enzyme-linked immu- nosorbent assay commercial kit Serum concentrations of AMH from 5 male alpacas, before and after castration, as measured by the Ansh assay, were closely associated with the previously published results using the Preventia kit (r = 0.94, n = 10, p < 0.01) (Figure 2). Results Validation of anti-Müllerian hormone enzyme-linked immunosorbent assay commercial kit Serum concentrations of AMH from 5 male alpacas, before and after castration, as measured by the Ansh assay, were closely associated with the previously published results using the Preventia kit (r = 0.94, n = 10, p < 0.01) (Figure 2). Figure 2. Correlation between serum anti-Müllerian hormone concentrations measured via 2 kits in samples from male (n = 5) alpacas (before and after castration) Serum anti-Müllerian hormone concentrations during cycle Serum AMH concentrations (mean ± SEM) on the day of ovulation induction (day 0) and during luteal phase (day 7) were 1.33 ± 0.35 ng/ml and 1.14 ± 0.34 ng/ml, respectively (Figure 3). Luteal phase (occurrence of ovulation in response to GnRH treatment) was verified on day 7 by visible corpus luteum via transrectal ultrasonography and by serum progesterone concentrations (samples had > 1.5 ng/ml; 3.26 ± 1.26 ng/ml [mean ± SEM]). There was no difference (p = 0.39) in serum AMH concentrations between estrus and diestrus. However, there was a significant variation in serum AMH concentrations among females. Minimum and maximum serum AMH concentrations were 0.027 and 4.28 ng/ml on day 0 and 0.01 and 3.72 ng/ml on day 7, respectively. Figure 2. Correlation between serum anti-Müllerian hormone concentrations measured via 2 kits in samples from male (n = 5) alpacas (before and after castration) Serum anti-Müllerian hormone concentrations during cycle Serum AMH concentrations (mean ± SEM) on the day of ovu- lation induction (day 0) and during luteal phase (day 7) were 1.33 ± 0.35 ng/ml and 1.14 ± 0.34 ng/ml, respectively (Figure 3). Luteal phase (occurrence of ovulation in response to GnRH treatment) was verified on day 7 by visible corpus luteum via transrectal ultrasonography and by serum progesterone concen- trations (samples had > 1.5 ng/ml; 3.26 ± 1.26 ng/ml [mean ± SEM]). There was no difference (p = 0.39) in serum AMH con- centrations between estrus and diestrus. However, there was a significant variation in serum AMH concentrations among fe- males. Minimum and maximum serum AMH concentrations were 0.027 and 4.28 ng/ml on day 0 and 0.01 and 3.72 ng/ml on day 7, respectively. Figure 3. Serum anti-Müllerian hormone concentrations on day 0 (estrus) and day 7 after ovulation induction in alpacas (n = 11) Ovarian superstimulation and serum anti-Müllerian hormone concentrations Number of antral follicles (> 7 mm) after FSH treatment (day 17) was variable (12.69 ± 5.25 [mean ± SD]; range 6 - 24). There were no differences (p > 0.005) in serum AMH concentrations within animals across sampling dates (Table). However, there was a difference (p > 0.005) among female alpacas. Correlation between mean AMH serum concentration for each female alpaca and the number of follicles at the end of superstimulation treatment (day 17) was determined. There was a correlation (r = 0.46, p < 0.01) between the number of follicles after FSH treatment and serum AMH concentrations (Figure 4). Further analysis after excluding 1 outlier had higher positive correlation (r = 0.7, p < 0.001; [Figure 5]). Correlation between AMH serum concentrations and the number of corpora lutea could not be assessed as there was ovulation failure in several animals. Serum progesterone concentrations increased in animals immediately after CIDR insertion and then dropped; however, they remained above 1.5 ng/ml until CIDR removal (Figure 6). Table. Serum anti-Müllerian hormone concentration (ng/ml) on various days of ovarian superstimulation Female Day 3 Day 7 Day 12 Day 17 Day 24 Mean (SEM) 1 0.525 0.612 0.564 0.564 0.653 0.58 (0.022) 2 1.337 1.298 0.791 0.736 0.887 1.01 (0.12) 3 3.193 3.123 3.698 2.497 3.917 3.29 (0.24) 4 3.193 3.123 3.698 2.497 3.917 3.29 (0.25) 5 3.213 2.529 2.771 2.04 2.404 2.59 (0.19) 6 4.282 3.719 4.314 7.719 4.582 4.92 (0.71) 7 0.741 0.972 0.802 0.828 0.775 0.82 (0.04) 8 2.961 2.785 2.498 2.131 3.429 2.76 (0.22) 9 0.491 0.491 0.491 1.769 2.286 1.11 (0.39) 10 1.627 4.785 2.454 1.576 2.506 2.59 (0.58) 11 1.292 1.535 1.439 2.052 1.708 1.61 (0.13) 12 0.027 0.01 0.01 0.01 0.061 0.02 (0.01) 13 0.541 0.32 0.313 0.357 0.515 0.41 (0.05) Figure 3. Serum anti-Müllerian hormone concentrations on day 0 (estrus) and day 7 after ovulation induction in alpacas (n = 11) Clinical Theriogenology 2022; 14: 94 Ovarian superstimulation and serum anti-Müllerian hor- mone concentrations Number of antral follicles (> 7 mm) after FSH treatment (day 17) was variable (12.69 ± 5.25 [mean ± SD]; range 6 - 24). There were no differences (p > 0.005) in serum AMH concen- trations within animals across sampling dates (Table). How- ever, there was a difference (p > 0.005) among female alpac- as. Correlation between mean AMH serum concentration for each female alpaca and the number of follicles at the end of superstimulation treatment (day 17) was determined. There was a correlation (r = 0.46, p < 0.01) between the number of follicles after FSH treatment and serum AMH concentrations (Figure 4). Further analysis after excluding 1 outlier had high- er positive correlation (r = 0.7, p < 0.001; [Figure 5]). Correla- tion between AMH serum concentrations and the number of corpora lutea could not be assessed as there was ovulation failure in several animals. Serum progesterone concentrations increased in animals immediately after CIDR insertion and then dropped; however, they remained above 1.5 ng/ml until CIDR removal (Figure 6). Table. Serum anti-Müllerian hormone concentration (ng/ml) on various days of ovarian superstimulation Female Day 3 Day 7 Day 12 Day 17 Day 24 Mean (SEM) 1 0.525 0.612 0.564 0.564 0.653 0.58 (0.022) 2 1.337 1.298 0.791 0.736 0.887 1.01 (0.12) 3 3.193 3.123 3.698 2.497 3.917 3.29 (0.24) 4 3.193 3.123 3.698 2.497 3.917 3.29 (0.25) 5 3.213 2.529 2.771 2.04 2.404 2.59 (0.19) 6 4.282 3.719 4.314 7.719 4.582 4.92 (0.71) 7 0.741 0.972 0.802 0.828 0.775 0.82 (0.04) 8 2.961 2.785 2.498 2.131 3.429 2.76 (0.22) 9 0.491 0.491 0.491 1.769 2.286 1.11 (0.39) 10 1.627 4.785 2.454 1.576 2.506 2.59 (0.58) 11 1.292 1.535 1.439 2.052 1.708 1.61 (0.13) 12 0.027 0.01 0.01 0.01 0.061 0.02 (0.01) 13 0.541 0.32 0.313 0.357 0.515 0.41 (0.05) Figure 4. Correlation between serum anti-Müllerian hormone concentrations and number of follicles after FSH treatment in alpacas (n = 13) Figure 5. Correlation between serum anti-Müllerian hormone concentrations and number of follicles after FSH treatment in alpacas (n = 12) after removal of an outlier Figure 4. Correlation between serum anti-Müllerian hormone concentrations and number of follicles after FSH treatment in alpacas (n = 13) Clinical Theriogenology 2022; 14: 95 Figure 4. Correlation between serum anti-Müllerian hormone concentrations and number of follicles after FSH treatment in alpacas (n = 13) Figure 5. Correlation between serum anti-Müllerian hormone concentrations and number of follicles after FSH treatment in alpacas (n = 12) after removal of an outlier Figure 5. Correlation between serum anti-Müllerian hormone concentrations and number of follicles after FSH treatment in alpacas (n = 12) after removal of an outlier Figure 6. Serum progesterone concentrations (mean ± SEM) on days 3, 7, and 12 after CIDR insertion (day 0) Discussion Anti-Müllerian hormone is a well-conserved glycoprotein among mammalian species. Commercial AMH assays kits are available for humans, cattle, small ruminants, horses, dogs, and rodents.35 Recently, we validated36 a commercial test kit (AMH ELISA test Kit Cat # KAMH-01; Preventia Diagnostics) for serum AMH in male alpacas. However, the unavailability of this kit necessitated validation of an alternate kit. We used male alpaca serum collected earlier36 to validate the alternate commercial kit (AMH ELISA, AL-113®, Ansh lab). There was a high correlation among data obtained with these 2 commercial assay kits. Both kits utilized monoclonal antibody pairs in a sandwich assay format. Determination of the effect of cycle stage on AMH serum concentration has diagnostic applications. If the cycle stage had no effect, a single random test could be used for prognostication of ovarian reserves. Serum AMH concentrations (with repeatability across multiple estrous cycles) were not influenced by cycle stage (estrus versus diestrus) in cows,11,21,38 goats,24 and mares.39,40 These patterns are expected since AMH is expressed only in follicles that have been recruited from primordial follicle pool and not selected for dominance. Neither dominant follicles nor corpora lutea are believed to contribute to circulating AMH concentrations in these species.7,41,42 Only in pigs is AMH is expressed in preovulatory follicles and corpora lutea.43 Serum AMH concentrations during peak follicular activity (follicle diameter > 8 with uterine edema) and luteal phase were not different in our study, similar to other species (cattle,11,44 goats,24 and humans7,42). Hence a random determination of serum AMH concentration can be used (as in other species) to evaluate ovarian follicular reserve in alpacas. Figure 6. Serum progesterone concentrations (mean ± SEM) on days 3, 7, and 12 after CIDR insertion (day 0) Discussion Anti-Müllerian hormone is a well-conserved glycoprotein among mammalian species. Commercial AMH assays kits are available for humans, cattle, small ruminants, horses, dogs, and rodents.35 Recently, we validated36 a commercial test kit (AMH ELISA test Kit Cat # KAMH-01; Preventia Diagnostics) for serum AMH in male alpacas. However, the unavailability of this kit necessitated validation of an alternate kit. We used male alpaca serum collected earlier36 to validate the alternate commercial kit (AMH ELISA, AL-113®, Ansh lab). There was a high correlation among data obtained with these 2 commer- cial assay kits. Both kits utilized monoclonal antibody pairs in a sandwich assay format. Determination of the effect of cycle stage on AMH serum con- centration has diagnostic applications. If the cycle stage had no effect, a single random test could be used for prognostica- tion of ovarian reserves. Serum AMH concentrations (with re- peatability across multiple estrous cycles) were not influenced by cycle stage (estrus versus diestrus) in cows,11,21,38 goats,24 and mares.39,40 These patterns are expected since AMH is expressed only in follicles that have been recruited from primordial fol- licle pool and not selected for dominance. Neither dominant follicles nor corpora lutea are believed to contribute to circu- lating AMH concentrations in these species.7,41,42 Only in pigs is AMH is expressed in preovulatory follicles and corpora lu- tea.43 Serum AMH concentrations during peak follicular activity (follicle diameter > 8 with uterine edema) and luteal phase were not different in our study, similar to other species (cat- tle,11,44 goats,24 and humans7,42). Hence a random determina- tion of serum AMH concentration can be used (as in other species) to evaluate ovarian follicular reserve in alpacas. The potential for AMH serum concentrations as a biomarker for fertility was established for cattle.11,21,30,44 Lower AMH se- rum concentrations reflected suboptimal fertility, lower preg- nancy rate, and higher incidence of pregnancy loss compared to higher AMH serum concentrations. Poor reproductive effi- ciency, a major problem in the camelid industry, is primarily Clinical Theriogenology 2022; 14: 96 due to ‘repeat breeding’ (75.6%) and recurrent early preg- nancy loss (18.3%).45 Determination of AMH concentrations could help to define the reproductive potential of a female alpaca, prevent excessive mating, and delay diagnosing fol- licular dynamics disorders. Multiple ovulation and embryo transfer programs’ are important tools for genetic improve- ment in camelids. Ovarian superstimulation treatments have often been extrapolated from ruminant studies. Such treat- ments (e.g. pFSH and eCG alone or in combination) were used during the luteal phase and follicular wave emergence with variable results. Initiation of treatment in relation to fol- licular dynamics, dose, protocol, and individual variation was considered for individual variability.31-34 A better understand- ing of camelids’ ovarian follicular dynamics and physiology of ovulation is needed to develop species-specific protocols. We used pFSH daily in decreasing doses over 5 days after a 14-day course of progesterone.34,46,47 Despite a good response (number of follicles), number of ovulations (as estimated by corpora lutea) was very low, a common problem in alpac- as.34,47,48 Response to hormonal ovarian stimulation treatments in buf- falo,49 cattle,19,21,22,25,26 goats,24,28 sheep,12,29 and woman41 was predicted using AMH blood concentrations. In our study, serum AMH concentrations were highly correlated with the number of follicles developed after ovarian superstimulation treatment. However, further studies using a larger number of females are warranted in order to determine whether alpacas with higher circulating AMH concentrations will consistently develop a higher number of follicles in response to FSH ovari- an superstimulation. Consistent with research in cattle,19,20,25-27 in our study, there were no differences in serum AMH concen- trations before and after FSH treatment. Conclusion We determined serum AMH concentrations in male and fe- male alpacas using a commercial assay kit. Consistent with other species, serum AMH concentrations in female alpacas remained constant during peak follicular and luteal phases. Hence a single sample to estimate female ovarian follicular re- serve and potentially predict their reproductive performance. Serum AMH concentrations in female alpacas were positively correlated with the number of dominant follicles that devel- oped following ovarian superstimulation. As demonstrated in ruminants, future experiments should investigate whether there are correlations among AMH serum concentrations, oo- cyte quality, and embryo survival in alpacas. Funding Partial funding from Northwest Camelid Research Founda- tion. Conflict of interest The authors do not have any conflict of interest to declare. References 1. Cate RL, Mattaliano RJ, Hession C, et al: Isolation of the bovine and human genes for Müllerian inhibiting substance and expression of the human gene in animal cells. Cell 1986;45:685-698. 2. Josso N, Clemente N: Transduction pathway of anti-Müllerian hormone, a sex-specific member of the TGF-beta family. Trends Endocrinol Metab 2003;14:91-97. 3. Jost A. Hormonal factors in the development of the fetus. Cold Spring Harb Symp Quant Biol 1954;19:167-181. 4. 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