This study evaluated Anti-Mullerian hormone (AMH) and/or follicle counts as predictors of subsequent superovulatory response and embryo production in 79 beef cows. Before superovulation, 3 to 5 mm follicles presented on the ovaries of donor cows were counted, and blood was collected for measure of serum AMH. Across cows, serum AMH ranged from 0.013 to 0.898 ng/mL, with a mean of 0.293 ng/mL. The distribution of both AMH concentrations and follicle counts were divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest) for analysis. Donor cows in AMH Q4 had a greater (P < 0.001) number of 3 to 5 mm follicles at the start of superovulation than did donors in either Q1 or Q2. At embryo collection, cows in AMH Q3 and 4 had more (P < 0.001) palpable corpora lutea (CL) than cows in AMH Q1. The mean number of embryos recovered from donor cows in AMH Q4 was greater (P < 0.001) than those recovered from cows in either AMH Q1 or 2, but similar to that of AMH Q3. Analysis indicated that AMH was positively correlated (P < 0.001) with mean follicles (r = 0.458), CL (r = 0.452) and embryos recovered (r = 0.430). Donor cows with higher follicle counts (F Q3 and 4) at the start of superovulation had more (P < 0.001) palpable CL at embryo collection than donor cows in F Q1 or 2. More (P < 0.001) embryos were recovered from cows with the highest follicle counts (F Q4) as compared with cows having lower (F Q1 and 2) follicle counts. Results confirm that relative AMH concentration was positively correlated with number of small antral follicles in the ovaries of cows; both AMH measure and antral follicle counts might be used to predict subsequent superovulatory response in beef cows.
Since the development of cattle superovulation and non-surgical embryo recovery in the 1970s, the unpredictability and variability of the superovulatory response has remained a major obstacle. Hasler [
There is an inherent variability in the number of follicles present in the ovaries of heifers. Erickson [
Ultrasonography can be used to quantify the number of antral follicles present in the ovaries at the initiation of superovulation, but requires experienced technicians, expensive equipment and careful evaluation of ultrasound images. Anti-Mullerian hormone (AMH) is a glycoprotein that is expressed in granulosa cells of growing follicles [
A total of 79 cows, including 31 Angus, 2 Chianina, 10 Polled Hereford, 1 Maine-Anjou, 15 Shorthorn, 3 Simmental, and 17 crossbred of various breeds were housed at the Food Animal Veterinary Services donor care facility located in Rensselaer, Indiana. Animal care was under the supervision of the veterinary staff at the donor care facility, following standard veterinary practice and animal welfare guidelines. The embryo donors consisted of 14 yearling to 2 year old heifers, 24 head of 3 to 5 year old cows, 27 head of 6 to 8 year old cows, and 14 head of 9 to 13 year old cows. Donor body condition score (BCS; 1 to 9 scale) [
Depending upon scheduling, client preference and donor history, superovulatory treatment was initiated either during the luteal phase of cow's natural estrous cycle, or after insertion of a 1.38 g progesterone insert (CIDR; EAZI-Breed CIDR; Zoetis, Florham Park, NJ). Cows superovulated based on their natural cycle had superovulatory treatment initiated between Day 8 and 12 of the estrous cycle. Cows received twice daily (10 to 12 hours apart), decreasing doses of porcine follicle stimulating hormone (FSH; Follitropin-V, Bioniche Animal Health, Belleville Ontario CA) over a 4-day period. The cows were injected (i.m) with 50 mg (NIH-FSH-P1 units) of FSH twice daily the first 2 days, then 40 and 30 mg twice daily on days 3 and 4, respectively. On the morning of the fourth day of FSH treatment, cows received an injection of 2.5 cc (~625 µg) of Cloprostenol (Estrumate, Merck Animal Health, Summit, NJ) to induce luteal regression. After Cloprostenol treatment, cows were observed until onset of estrus.
Cows superovulated regardless of the day of the estrous cycle, received an EAZI-Breed CIDR on Day 0, in conjunction with an injection (i.m.) of 2.5 mg estradiol and 50 mg of progesterone to suppress the dominant follicle and initiate a new follicular wave [
Timing of insemination after detected estrus depended on the number of straws and type of frozen-thawed semen used. When a single straw of semen was used, insemination occurred 12 to 16 hours after detected estrus. When multiple straws of semen were used, insemination occurred within 12 hours of detected estrus, and again 6 to 8 hours later. When X- or Y-sorted semen was used, insemination occurred 16 and again at 24 hours after detected estrus, with 3 to 6 straws of semen used in total, depending on sperm concentration per straw.
Non-surgical embryo recoveries were performed ~Day 7 of the subsequent estrous cycle, using a Foley catheter and ViGRO complete flush medium (ViGRO™, Bioniche Animal Health, Pullman, WA). At the time of embryo collection, the ovaries of donor cows were palpated to determine the number of corpora lutea (CL) present on each ovary. Embryos recovered from the flush medium were evaluated for stage of development (morula, early blastocyst or blastocyst) and morphological quality (grade 1, 2, degenerate or unfertilized), using evaluation criteria established by the International Embryo Transfer Society [
Serum samples were shipped on dry ice over night to the Texas A&M Veterinary Medical Diagnostic Laboratory at College Station, TX. The diagnostic laboratory used a bovine AMH enzyme-linked immunosorbent assay kit (Bovine AMH ELISA AL-114; Ansh Labs, Webster, TX) to determine AMH concentration (ng/mL) in duplicate samples, following the assay kit manufacturer’s recommended procedures. The AMH assay had an analytical sensitivity of 0.011 ng/mL.
Analysis was performed, using JMP Pro 12.0 statistical software (SAS Institute, Inc.). Variables considered in the analysis were embryo donor breed, superovulation protocol, AMH concentration, follicle and CL number, total, transferable (grade 1 or 2), degenerate embryos, and unfertilized ova. Superovulation protocol had no effect (P ³ 0.293) on number of corpora lutea, or the number of total, transferrable, degenerate embryos or unfertilized ova, so was removed from the analysis. There were no breed differences (P = 0.321) for serum AMH concentration.
Frequency distribution was used to assign AMH concentration measured in serum samples to quartiles. Analysis of variance was then used to make comparisons between AMH quartiles for number of 3 to 5 mm follicles, number of corpora lutea at embryo collection, number of embryos recovered, and the percentages of transferrable and degenerate embryos, and unfertilized ova. Percent transferrable embryos were defined as the portion of total embryos recovered that were of grade 1 or 2 morphological quality. Percent degenerate embryos were defined as the portion of total embryos recovered that exhibited limited cleavage and/or were of poor morphological quality. Percent unfertilized ova were the portion of all recovered embryos/structures that exhibited no cleavage.
Frequency distribution was also used to assign follicle counts to quartiles. Analysis of variance was then used to make comparisons between follicle quartiles and number of corpora lutea at embryo collection, number of embryos recovered, and the percentages of transferrable and degenerate embryos, and unfertilized ova. Multivariate analysis was used to determine any correlations between AMH concentrations and number of 3 to 5 mm follicles, number of corpora lutea at embryo collection, number of embryos recovered, and the percentages of transferrable and degenerate embryos, and unfertilized ova. All values are expressed as the mean ± SEM. Statistical differences were considered significant where P < 0.05.
Anti-Mullerian hormone measured in serum samples ranged from 0.013 to 0.898 ng/mL, with a mean of 0.293 ng/mL. The distribution of AMH concentrations was divided into quartiles (AMH Q1 through Q4, with Q1 the lowest and Q4 the highest ng/mL) for analysis. The range of AMH concentrations within each quartile are presented in
The number of 3 to 5 mm follicles counted on the ovaries of donor cows ranged from 5 to over 30, with a mean of 16. In order to determine if follicle counts at the start of superovulation might be predictive of subsequent superovulatory
Item | Quartile of AMH concentration | P-value | |||
---|---|---|---|---|---|
AMH, ng/mL | Q1 0.013 - 0.168 | Q2 0.169 - 0.263 | Q3 0.264 - 0.363 | Q4 0.364 - 0.898 | |
No. of donors/collections* | 26 | 23 | 24 | 24 | |
No. of follicles | 13.46 ± 0.91b | 14.95 ± 0.98b | 16.79 ± 0.94ab | 19.33 ± 0.94a | 0.001 |
No. of CL | 11.62 ± 1.54c | 13.68 ± 1.67bc | 17.58 ± 1.60ab | 20.54 ± 1.60a | 0.001 |
No. of embryos | 9.77 ± 1.76b | 9.36 ± 1.91b | 15.50 ± 1.83ab | 20.13 ± 1.83a | 0.001 |
Transferable % | 69.32 ± 6.62 | 57.06 ± 7.08 | 58.75 ± 6.62 | 51.05 ± 6.62 | 0.275 |
Degenerate % | 5.52 ± 2.52 | 7.89 ± 2.69 | 9.40 ± 2.52 | 9.87 ± 2.52 | 0.614 |
Unfertilized % | 25.16 ± 6.73 | 35.06 ± 7.19 | 31.85 ± 6.73 | 39.09 ± 6.73 | 0.519 |
*Included 77 of 79 donors, with 20 donors collected twice; a,b,cMeans ± S.E.M within rows with different superscripts are significantly different (P < 0.05).
Variable 1 | Variable 2 | Correlation | P-value |
---|---|---|---|
Anti-Mullerian hormone | Mean number of follicles | 0.458 | 0.001 |
Mean number of corpora lutea | 0.452 | 0.001 | |
Mean number of embryos | 0.430 | 0.001 | |
Percent transferrable embryos | -0.126 | 0.231 | |
Percent degenerate embryos | 0.195 | 0.061 | |
Percent unfertilized ova | 0.052 | 0.621 | |
Mean number of follicles | Mean number of corpora lutea | 0.556 | 0.001 |
Mean number of embryos | 0.423 | 0.001 | |
Percent transferrable embryos | -0.037 | 0.724 | |
Percent degenerate embryos | 0.147 | 0.153 | |
Percent unfertilized ova | -0.018 | 0.857 | |
Mean number of corpora lutea | Mean number of embryos | 0.887 | 0.001 |
Percent transferrable embryos | -0.008 | 0.942 | |
Percent degenerate embryos | 0.191 | 0.063 | |
Percent unfertilized ova | -0.064 | 0.539 | |
Mean number of embryos | Percent transferrable embryos | 0.011 | 0.919 |
Percent degenerate | 0.236 | 0.021 | |
Percent unfertilized | -0.098 | 0.341 | |
Percent transferrable embryos | Percent degenerate embryos | -0.184 | 0.073 |
Percent unfertilized ova | -0.931 | 0.001 | |
Percent unfertilized ova | Percent degenerate | -0.188 | 0.067 |
response, the distribution of follicle counts were also divided into quartiles (F Q1 through Q4, with Q1 the lowest and Q4 the highest) for analysis (
As was noted for AMH, mean follicles at the start of superovulation was positively correlated (P < 0.001;
The objective of the present study was to further investigate the use of AMH and/or follicle counts as a predictor of subsequent superovulatory response and embryo production in beef cows. The goal of superovulation is to stimulate a number of small antral follicles to grow and mature, resulting in multiple ovulations. Therefore, the pool of small antral follicles available for stimulation should dictate the superovulatory response. Ireland et al. [
Item | Quartile of follicle counts | P-value | |||
---|---|---|---|---|---|
Q1 | Q2 | Q3 | Q4 | ||
No. of donors | 26 | 35 | 20 | 18 | |
Follicle range | 5 - 12 | 13 - 17 | 18 - 20 | 21 - 30 | |
No. of CL | 10.65 ± 1.40b | 13.51 ± 1.20b | 19.15 ± 1.59a | 23.33 ± 1.68a | 0.001 |
No. of embryos | 9.62 ± 1.79b | 11.57 ± 1.55b | 16.50 ± 2.04ab | 20.00 ± 2.16a | 0.001 |
Transferable % | 58.20 ± 6.44 | 64.77 ± 5.63 | 54.51 ± 7.53 | 58.46 ± 7.97 | 0.716 |
Degenerate % | 4.82 ± 2.23b | 6.71 ± 1.95b | 17.09 ± 2.61a | 5.38 ± 2.76b | 0.002 |
Unfertilized % | 36.99 ± 6.44 | 28.53 ± 5.63 | 28.39 ± 7.54 | 36.16 ± 7.97 | 0.688 |
a,b Means ± S.E.M within rows with different superscripts are significantly different (P < 0.05).
were highly variable between individual animals, but highly repeatable (r = 0.89) within individual heifers for average number of follicles per follicular wave. Likewise, AMH in circulation, which is a reflection of the number of growing follicles within the ovaries, is repeatable within individuals. Ireland et al. [
Assay of AMH in the serum of 77 embryo donor beef cows in the present study found a range of 0.013 to 0.898 ng/mL. This range of serum AMH is somewhat broader than that reported by others. Ireland et al. [
Different AMH assays, blood sample types, sample storage conditions, and criteria for measuring superovulatory response can alter recommended AMH cut-off values for selecting potential donor cows. Whereas other studies utilized animals of similar age and breeding, embryo donors in the present study ranged from breeding age heifers to 13-year-old cows, and were of various beef breeds. Therefore, embryo donors were classified into quartiles of circulating AMH concentration rather than by specific concentrations in blood. Embryo donor cows in the lowest AMH quartile (0.013 to 0.168 ng/mL) had fewer follicles at the start of superovulation, fewer palpable CL at embryo collection, and produced fewer embryos than donors in the highest AMH quartile. Donors in AMH quartiles 2 and 3 were intermediate for follicle, CL and embryo counts. Souza et al. [
In this study, AMH in circulation was positively correlated (P = 0.001) with follicles present at start of superovulation (r = 0.458), and number of CL (r = 0.452) and embryos recovered at collection (r = 0.430). Rico et al. [
A difference between the present study and others [
Assay for AMH is relatively expensive and limited in availability to most embryo transfer practitioners. However, many practitioners have access to ultrasonography and often evaluate ovarian structures before superovulation. As an alternate to AMH, the number of 3 to 5 mm follicles present in the ovaries at initiation of superovulatory treatment was evaluated as a predictor of superovulatory response. As with serum AMH, donors were assigned to quartiles for comparison. Results indicate that follicle counts are of value for predicting subsequent superovulatory response. Donor cows in the highest two quartiles for 3 to 5 mm follicles had more ovulations, based on CL present at embryo collection than cows in the lowest two follicle quartiles. Donors in the highest follicle quartile also produced more embryos that cows in the lowest two quartiles. As with circulating AMH, mean number of follicles at start of superovulation was positively correlated (P = 0.001) with ovulations (CL) after superovulation (r = 0.556) and number of embryos recovered (r = 0.423). In a previous study using ultrasound to determine antral follicle count, Ireland et al. [
Currently, embryo donor information such as breed, age, parity and past superovulatory response are used to adjust the superovulation regimen in an effort to improve embryo production. While improvements have been made in hormonal treatment and synchronization protocols [
The authors acknowledge Dr. Scott Jacques at the Texas A&M Veterinary Diagnostic Laboratory for performing Anti-Mullerian hormone assays, and the University of Arkansas Agricultural Experiment Station and the Department of Animal Science for their financial support.
Center, K., Dixon, D., Looney, C. and Rorie, R. (2018) Anti-Mullerian Hormone and Follicle Counts as Predictors of Superovulatory Response and Embryo Production in Beef Cattle. Advances in Reproductive Sciences, 6, 22-33. http://dx.doi.org/10.4236/arsci.2018.61003