The effect of anti-müllerian hormone and progesterone
concentrations on superovulation response and embryo yield in goats
Kubra Karakas Alkan
a,*, Hasan Alkan
b, Mustafa Kaymaz
aaAnkara University Faculty of Veterinary Medicine Department of Obstetrics and Gynecology, Ankara, 06110, Turkey bSelcuk University Faculty of Veterinary Medicine Department of Obstetrics and Gynecology, Konya, 42250, Turkey
a r t i c l e i n f o
Article history: Received 26 June 2019 Received in revised form 29 November 2019 Accepted 30 November 2019 Available online 3 December 2019 Keywords: AMH Goat MOET Progesterone
a b s t r a c t
We aimed to evaluate the relationship of anti-Müllerian hormone (AMH) and progesterone concentra-tions with superovulation response in goats and to determine donors exhibiting better superovulation response by measuring AMH concentrations. For this, blood samples were collected from multiparous Angora goats (n¼ 24) for measuring the progesterone and AMH concentrations on the day the syn-chronization protocol was initiated (Day 0), on the day of thefirst FSH administration (Day 9), on the day the progesterone source was removed (Day 11), and on the day of uterineflushing. Descriptive statistics (mean, standard deviation, median, minimum value, maximum value, and percentile) were given for superovulation response and embryo yield. To compare the differences between the two groups, the Student’s t-test was used. The relationship between two continuous variables was assessed by the Pearson Correlation Coefficient. The AMH cutoff values in superovulation responses were evaluated by ROC analysis on the day the synchronization protocol was initiated. A strong positive correlation was found between the AMH concentrations measured on the day the synchronization protocol was initiated (Day 0), on the day of thefirst FSH administration (Day 9), and on the day of removal of the progesterone source (Day 11) and the count of total corpus luteum (CL), total oocyte/embryo, transferable embryo, and Code I quality embryo (P< 0.05). Furthermore, AMH concentration increased on the day the synchro-nization protocol was initiated, the donor’s superovulation response increased as well. The cutoff value was 4.74 ng/ml, as assessed by the ROC curve analysis conducted for selecting donors exhibiting better superovulation responses. The sensitivity and specificity of the selected cutoff value were found to be quite high (P< 0.01). However, a positive correlation was noted between the progesterone concentra-tions measured on the day of uterineflushing and total CL count, total oocyte/embryo count, transferable embryo count, and Code I quality embryo count (P< 0.01). In conclusion, it was determined that an increase in AMH concentrations in goats led to an increase in the total CL count, embryo count, and embryo quality and that AMH measurement could be used to identify donors that responded better to superovulation. Additionally, a positive correlation was found between the progesterone concentration measured on the day of uterineflushing and the total CL count, transferable embryo count, and embryo quality.
© 2019 Elsevier Inc. All rights reserved.
1. Introduction
Assisted reproductive techniques such as artificial insemination or multiple ovulation and embryo transfer (MOET) are commonly used to improve reproductive performance in livestock [1]. MOET involves the transfer of multiple embryos from the donor animals with a genetically superior capacity to recipients [2]. This tech-nology enables the rapid spread of genotypes with high yield
properties or of new breeds, reduces the risk of spreading diseases, allows generations to easily adapt to different production and administrative systems, and allows the generation interval to be quite short [3].
MOET is extensively used worldwide, mostly in cattle, sheep, and goats [2,4e6]. However, the difference in the number of su-perovulation responses and transferable embryos obtained is a problem encountered in MOET programs. Furthermore, the possi-bility of obtaining variable and low transferable embryo counts between applications and between individuals in the same appli-cation group limits the practical use of MOET [7]. Despite im-provements in superovulation and MOET protocols, different
* Corresponding author.
E-mail address:kkarakas@ankara.edu.tr(K. Karakas Alkan).
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results are obtained depending on the condition of ovarian follicles at the beginning of gonadotropin administration [1,8]. Therefore, donor selection at an early stage is becoming increasingly common for obtaining transferable embryos, and there is a requirement for a technique that can predict embryo productivity at an early stage [9].
Studies in the last decade have focused on the role of anti-Müllerian hormone (AMH) in predicting animal responses to gonadotropin treatments [ [4,10e12]]. In different animal species, higher AMH concentrations have been associated with a better performance with respect to superovulation response and a higher potential for embryo production [13e17]. In assisted reproductive technologies, AMH is the best endocrine biomarker for predicting follicle count and superovulation response [10]. Because AMH is expressed in the granulosa cells of developing follicles and highest AMH concentrations are found in small antral follicles, Measure-ment of AMH concentration can be used to determine the follicle pool of donors [12]. Some studies have reported a positive corre-lation between the small antral follicle count and serum AMH concentrations [12,16,28]. Moreover, a significant relationship was found between AMH concentrations and total corpus luteum (CL) and transferable embryo counts in cattle [8] and goat [13].
Progesterone (P4) concentration can be used for identifying donors exhibiting a poor superovulation response and poor oocyte/ embryo quality. Although the correlation between the progester-one and superovulation response remains controversial, studies have reported that abnormal progesterone concentrations nega-tively affect superovulation response, embryo quality, and embryo yield [18e20]. Callasen et al. [19], reported that abnormal proges-terone concentrations in cattle lead to low oocyte competence and decreased embryo yield and quality. Goto et al. [36] found that progesterone concentrations on the day of superovulation treat-ment in cattle were closely related to embryo yield and quality. The total CL and embryo counts were higher in cows with high pro-gesterone concentrations (>3 ng/ml) on the day of uterine flushing than in those with low progesterone concentrations. Silva et al. [21] previously determined that progesterone concentrations during diestrus (either before or after superovulation) had a positive effect on embryo quality and yield in dairy cows and heifers. In addition, the total oocyte/embryo and transferable embryo counts were increased in donors exhibiting high progesterone concentrations after superovulation [21e23]. Therefore, it is thought that proges-terone concentrations affect embryo yield in goats.
We aimed to evaluate the relationship of AMH and progesterone concentrations with superovulation response, total oocyte/embryo count, transferable embryo count, and embryo quality in MOET applications on goats. In addition, we aimed to determine the cutoff value of AMH concentrations by a ROC curve analysis to identify the donors (>14 CL) that respond better to superovulation treatments. 2. Materials and methods
The study was approved by the Ankara University Local Ethics Committee of Animal Experiments. The study was conducted in the Training, Research and Application Farm of the Ankara University Faculty of Veterinary Medicine (þ40 60 8.499600 K,þ32 37’ 18.6492” D, Turkey).
2.1. Animals
The study was conducted between September and December, which is the breeding season in the northern hemisphere. In this study, 24 Angora Goats with a weight of 35e45 (39.41 ± 0.40) kg and aged 3e7 (3.91 ± 0.18) years who had previously given birth at
least once were used as animal material. Clinical or
ultrasonographic examination revealed no reproductive problems in the animals. In this study, 12 fertile bucks with a weight of 45e55 (51.67± 2.39) kg and aged 2e5 (3.78 ± 0.35) years were used. Before the study, semen samples were obtained from bucks, and sperm color, amount, mass movement, motility and viable and abnormal spermatozoa rates were evaluated; fertility was also determined. Corn silage, alfalfa, straw, and concentrated feed were used in the rations of animals. Water and mineral salt were pro-vided ad libitum.
2.2. Synchronization and superovulation of donors
A preparation containing 0.33 g progesterone (CIDR®, Eazi-Breed, Zoetis, USA) was intravaginally placed in the donor goats (n¼ 24) on Day 0. On Day 9, the goats admininstered with 150
m
g prostaglandin intramuscularly (Cloprostenol, Dalmazin®, Vetas, Turkey). For superovulation, donors were injected with FSH (Foll-tropin-V®, Bioniche Animal Health, Canada) for three days starting from the morning of Day 9, while the synchronization protocol was in progress. In total, 200 mg FSH was administered intramuscularly with six decreasing doses (50, 50, 30, 30, 20, and 20 mg) at 12-h intervals. On Day 11, the progesterone source was removed from the vagina, and after 24 h (Day 12), the donors were mated with twelve fertile bucks.2.3. Surgical recovery of embryos
Embryos were surgically recovered 156 h after mating. Feed and water were withheld from the donors for 12 h before the operation. General anesthesia was induced to the animals prior to the oper-ation. Subcutaneous atropine sulfate at a dose of 0.1e1 mg/kg (Atropin, Vetas, Turkey) was administered 15 min prior to the anesthesia as premedication. Following this, 0.5 mg/kg diazepam (Diazepam, Deva, Turkey) and 2 mg/kg ketamine HCl (Alfamine 10%, Egevet, Turkey) were intravenously applied to induce anes-thesia. After the mid-lateral incision, the ovary and uterus were exteriorized through the incision line. The ovaries were then examined, and the CL counts were recorded. Each uterine horn was
thenflushed with a solution (20 mL mD-PBS þ 3 mg/mL bovine
serum albumin) using a catheter (1.3 130 mm) emplaced near the uterotubal junction. Theflush containing embryos was collected in a 90-mm Petri dish by using a two-way Foley catheter (No. 10, Rüsch) emplaced at the base of the uterine horns.
2.4. Evaluation and classification of goat embryos
The uterineflushes were observed under a stereo microscope (Leica M205C, Germany). The obtained embryos were evaluated according to their developmental stages and quality [24,41]. Non-fertilized ovum was considered as stage 1; embryos with 2e12 cells as stage 2; the early morula as stage 3; the compact morula as stage 4; the early blastocyst as stage 5; the blastocyst as stage 6; the expanded blastocyst as stage 7; the hatched blastocyst as stage 8; and the expanded hatched blastocyst as stage 9.
Embryo quality was determined according to its morphological integrity. In Code I embryos (excellent or good), the level of irreg-ularity between cells is very low, proportion of living embryonic cells is 85%, and the zona pellucida is round and unfolded. Code II embryos (fair) are characterized by moderate irregularities, and the proportion of living cells is 50%. In Code III embryos (poor), there are irregularities in the form, and the percentage of living cells is 25%. In Code IV embryos (dead or degenerating), there are oocytes or dead cells with unfinished division. According to these criteria, Code I and Code II embryos were considered to be of transferable quality.
2.5. Blood sampling
While the synchronization protocol was in progress, blood samples were collected to measure AMH and progesterone con-centrations on the day the synchronization protocol was initiated (Day 0; AMH/P4), on the day of thefirst FSH administration (Day 9; only AMH), on the day the progesterone source was removed from vagina (Day 11; AMH/P4), and on the day of uterineflushing (Day 19; AMH/P4).
Immediately after blood samples were obtained, blood was centrifuged at 3000g for 15 min. The collected sera were then stored at20C until AMH and P4 were measured.
2.6. Hormonal assays 2.6.1. AMH
The serum concentrations of AMH were determined using the active MIS/AMH enzyme-linked immunosorbent assay (ELISA) goat kit (Sunred Biological Technology, China). The kit used a double-antibody sandwich ELISA to assay the concentration of Goat Mullerian Inhibiting Substance/Anti-Mullerian Hormone (MIS/ AMH) in samples. The pre-coated antibody was goat MIS/AMH monoclonal antibody and the detecting antibody was polyclonal antibody with biotin labeled. Before the assay, reagents, samples and standards were prepared. First, 40
m
l sample and then both 10m
l MIS/AMH antibody and 50m
l Streptavidin-HRP were added to each well. After that, the plate was sealed with a membrane and incubated 60 min at 37C. Plate was washedfive times to remove the uncombined enzyme after incubation. Thereafter, 50m
l Chro-mogen Solution A and 50m
l Chromogen Solution B were added to each well and the plate incubated again 10 min at 37C away from light. Finally, 50m
l stop solution was added to wells to stop reaction (the blue changed into yellow immediately). 15 min after adding the stop solution, OD value was measured under 450 nm wave-length. The standard curve linear regression equation was calcu-lated according to standards’ concentration and the corresponding OD values. The test sensitivity was 0.038 ng/mL. The intra- and inter-assay coefficients of variation (CV(%) ¼ SD/meanX100) were <10 and < 12%, respectively.2.6.2. Progesterone
The serum concentrations of progesterone were determined using the active Goat Progesterone kit (Novus Biological, USA). This assay employed the competitive enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with goat-anti-rabbit IgG antibody. Reagents and samples were pre-pared. First, 50
m
l of standard or sample and then 50m
l of HRP-conjugate and 50m
l antibody were added to each well. After that, wells were mixed and incubated for 60 min at 37C. Following incubation, each well was aspirated and washed with 200m
l Wash Buffer for a total of three washes. After the last wash, any remaining Wash Buffer was removed by aspirating or decanting. Thereafter, 50m
l of Substrate A and 50m
l of Substrate B were added to each well and incubated for 15 min at 37C in the dark. Finally, 50m
l of Stop Solution was added to each well to stop reaction (the blue turned into yellow immediately). 10 min after adding the stop so-lution, optical density (OD) value was measured using a microplate reader set to 450 nm. The professional soft“Curve Expert” was used to make a standard curve. The minimum detectable dose of goat progesterone is typically less than 0.2 ng/ml. The intra- and inter-assay CV were<15 and < 15%, respectively.2.6.3. Division of donors according to AMH and progesterone concentrations
The donors were subdivided according to AMH concentrations
at the beginning of the synchronization protocol and according to progesterone concentrations on the day of uterineflushing. Mean AMH (6.9 ng/ml) and progesterone (approximately 40 ng/ml) concentrations were considered while forming the groups. The obtained superovulatory response findings were increased with increasing AMH and progesterone concentrations. Accordingly, the AMH concentrations were<3 ng/ml in the first group, 3e6 ng/ml in the second group, 6e10 ng/ml in the third group and >10 ng/ml in the fourth group [15,16,27]. In addition, the P4 concentrations were <30 ng/ml in the first group, 30e50 ng/ml in the second group, >50 ng/ml in the third group [23,34,36].
2.7. Statistical analysis
For discrete and continuous variables such as the counts of total CL, total oocyte/embryo, transferable embryos, Code I and Code II quality embryos, degenerating embryos, and unfertilized oocytes obtained on the day of uterineflushing, descriptive statistics (mean, standard deviation, median, minimum value, maximum value, and percentile) were provided. Moreover, the homogeneity of the vari-ances, one of the prerequisites of parametric tests, was checked using the Levene’s test. The assumption of normality was tested using the Shapiro-Wilk test. To compare the differences between the two groups (i.e., when comparing AMH concentrations<6.9 and >6.9 ng/ ml), the Student’s t-test was used when the parametric test pre-requisites were fulfilled, and the Mann WhitneyeU test was used when such prerequisites were not fulfilled. To compare the differ-ences among3 groups (i.e., when comparing the donors according to AMH and progesterone concentrations), one-way analysis of variance was used when the parametric test prerequisites were ful-filled, and the Kruskal Wallis test was used when such prerequisites were not fulfilled. The Bonferroni correction method, which is a multiple comparison test (i.e, when comparing the donors according with AMH and progesterone concentrations), was used to evaluate the significant results of 3 groups. Repeated measures of analysis of variance (i.e., when comparing AMH and progesterone concentra-tions on different days) was analysed by Mauchly’s sphericity test and Box’s test of equality of covariance matrices. For comparisons of means of repeated measures, repeated measures analysis of variance was used. If parametric tests (factorial design for repeated measures analysis) did not provide the preconditions, Greenhouse-Geisser [25] correction or Huynh-Feldt [26] correction was used for corrections to the degrees of freedom or Friedman Test. The corrected Bonferroni test was used for multiple comparisons. In this study, univariate parametric and nonparametric regression methods were used to estimate AMH and progesterone concentrations variables with different parameters in goats. The relationship between two contin-uous variables (i.e., when comparing correlation between AMH concentrations and the counts of total CL, total oocyte/embryos, transferable embryos, Code I and Code II quality embryos, degener-ating embryos, and unfertilized oocytes) was assessed by the Pearson correlation coefficient, and by the Spearman correlation coefficient when the parametric test prerequisites were not met. The AMH cutoff values (AMH concentration on the day the synchronization protocol was initiated) in superovulation responses were evaluated by ROC analysis. Moreover, the AUC value, sensitivity, selectivity values were calculated. For the significance level of the tests, P < 0.05 and P < 0.01 were accepted.
3. Results
3.1. Embryo yield and superovulation response relationship with AMH concentrations
superovulation. The ROC curve analysis was performed to deter-mine the circulating AMH concentration that could be used to select goats that produced>14 CL (Fig. 2). The selected cutoff point (threshold) was 4.74 ng/ml (ROC curve area¼ 0.86, P < 0.01). Ac-cording to these results, the selection of better-responding donors (>14 CL) would be easily accomplished when the AMH cutoff value was taken to be 4.74 ng/ml during selection of donors. Because the sensitivity (probability of a negative test outcome in a low-responding individual) and specificity (probability of a positive test outcome in a high-responding individual) of this value were found to be 83% (P< 0.01).
The mean AMH concentrations of the donor goats were 6.857± 1.372 ng/ml on the day the synchronization protocol was initiated (Day 0), 7.070± 0.965 ng/ml on the day of the first FSH administration (Day 9), 5.532 ± 1.102 ng/ml on the day the pro-gesterone source was removed (Day 11), and 4.443± 0.913 ng/ml on the day of uterineflushing (Day 19). The counts of total CL, total oocyte/embryo, transferable embryos, Code I and Code II quality embryos, degenerating embryos, and unfertilized oocytes obtained on the day of uterineflushing are presented inTable 1. Donors were classified according to the mean AMH concentration (<6.9 and >6.9 ng/ml) on the day the synchronization protocol was initiated. Accordingly, the counts of total CL, total oocyte/embryos, transfer-able embryos and Code I quality embryos were found to be higher in goats with an AMH concentration of>6.9 ng/ml than in those with an AMH concentration of<6.9 ng/ml on Day 0 (P < 0.05).
Table 2shows the correlation between AMH concentrations and the counts of total CL, total oocyte/embryos, transferable embryos, Code I and Code II quality embryos, degenerating embryos, and unfertilized oocytes on different days of synchronization. A strong positive correlation was found between the AMH concentrations measured on the day 0, 9, 11 and the superovulation response and embryo yield (Fig. 1, P< 0.05). In addition, donors were divided into four groups according to AMH concentrations on the day the syn-chronization protocol was initiated (Day 0). The obtained super-ovulatory responsefindings were increased with increasing AMH concentrations (Table 3). An increase in the AMH concentrations on the day the synchronization protocol (Day 0) was initiated was associated with an increase in the counts of transferable embryos and Code I quality embryos (P< 0.05). However, the counts of Code II quality embryos, degenerating embryos, and oocytes were not affected by AMH concentrations (P> 0.05).
3.2. Embryo yield and superovulation response relationship with P4 concentrations
The mean P4 concentrations of the donor goats was 5.473 ± 2.54 ng/ml on the day the synchronization protocol was initiated (Day 0), 2.951± 0.32 ng/ml on the day of removal of the
progesterone source (Day 11), and 39.577± 4.54 ng/ml on the day of uterineflushing (Day 19). The counts of total CL, total oocyte/ embryos, transferable embryos, Code I and Code II quality embryos, degenerating embryos, and unfertilized oocytes on the day of uterine flushing according to progesterone concentrations are presented inTable 4.Table 5presents the correlation between P4 concentrations and the superovulation response and embryo yield on different days of synchronization. A positive correlation was found between the P4 concentration and the counts of total CL, total oocyte/embryos, transferable embryos, and Code I quality embryos on the day of uterineflushing (Fig. 3, P< 0.05). However, although the superovulation response had a positive correlation with the progesterone concentration on the day the synchroniza-tion protocol was initiated and the source of progesterone was removed, these results were not statistically significant (P > 0.05). The donors were divided into three groups according to P4
concentrations on the day of uterine flushing. The obtained
superovulatory responses increased with increased progesterone concentrations.Table 6 shows the superovulation response and embryo yield according to this classification. Based on the findings on the day of uterineflushing, it was determined that an increase in the P4 concentration was associated with an increase in the counts of total CL, total oocyte/embryos, transferable embryos, and Code I quality embryos (P < 0.05). Moreover, as the P4 concentration increased, the Code II quality embryo count decreased (P< 0.05).
In addition, the correlation between the AMH concentration on the day the synchronization protocol was initiated (Day 0) and the P4 concentration and CL count on the day of uterineflushing (Day 19) were also evaluated (Table 7). There was a positive correlation between the AMH concentration and P4 concentration and CL count on the day the synchronization protocol was initiated (Fig. 4, P< 0.05).
4. Discussion
The most important factor affecting MOET programs is the inability to estimate the transferable embryo count and the su-perovulation response [8]. The aim of superovulation is to promote the growth and development of a large number of small antral follicles and to stimulate multiple ovulations. Therefore, to deter-mine the superovulation response, it is necessary to identify the existing pool of small antral follicles [27]. Studies have also re-ported that AMH is the most reliable endocrine marker in deter-mining ovarian reserve [4,16]. This is because AMH is produced by granulosa cells of preantral and early antral follicles [28]. Studies performed on cattle [29], sheep [14], goats [13], and pigs [30] have found a positive correlation between small antral follicle count and serum AMH concentrations. In addition, Rico et al. [8] reported a strong correlation between the plasma AMH concentration in
Table 1
The counts of total CL, total oocyte/embryos, transferable embryos, Code I and II embryos, degenerating embryos and unfertilized oocytes obtained on uterineflushing day according to AMH concentration on day 0.
Total AMH concentrations on the day
synchronization initiated (Day 0)
P <6.9 ng/ml >6.9 ng/ml Number of donors n 24 15 9 Total CL x±SD 14.75± 1.29 12.06± 5.63 19.22± 4.94 0.005 Total oocyte/embryos x±SD 10.12± 1.00 8.53± 5.06 12.77± 3.45 0.038 Transferable embryos x±SD 8.16± 0.86 6.80± 4.10 10.44± 3.64 0.039 Code I embryos x±SD 4.75± 0.78 3.46± 3.13 6.88± 4.07 0.030 Code II embryos x±SD 3.41± 0.49 3.33± 2.69 3.55± 2.12 0.835 Degenerating embryos x±SD 1.62± 0.40 1.53± 1.59 1.77± 2.63 0.779 Unfertilized oocyte x±SD 0.33± 0.22 0.20± 0.56 0.55± 1.66 0.551
Holstein cows and number of large follicles in estrus (r¼ 0.83) and high CL count obtained after superovulation (r¼ 0.64). Monniaux et al. [13] also found a high correlation between the AMH con-centrations and total CL count and the total cell and transferable
embryo counts before FSH administration in goats. The present study found a positive correlation between the AMH concentra-tions measured on the day the synchronization protocol was initiated and the counts of total CL (r¼ 0.67, P < 0.01), total oocyte/
Table 2
Correlation between the AMH concentrations and the counts of total CL, total oocyte/embryos, transferable embryos, Code I and II embryos, degenerating embryos, unfertilized oocytes on different days of synchronization.
AMH relationships on different days of synchronization
Day 0 Day 9 Day 11 Day 19
Number of donors n 24 24 24 24
Mean AMH concentration (ng/ml) x±SD 6.857± 1.372 7.070± 0.965 5.532± 1.102 4.443± 0.913
Total CL r 0.670a 0.594a 0.547a 0.341 Total oocyte/embryos r 0.451b 0.456b 0.428b 0.238 Transferable embryos r 0.584a 0.655a 0.561a 0.255 Code I embryos r 0.599a 0.700a 0.561a 0.176 Code II embryos r 0.079 0.046 0.101 0.169 Degenerating embryos r 0.100 0.228 0.177 0.087 Unfertilized oocyte r 0.061 0.083 0.077 0.080
a Correlation is significant at the0.01 level. b Correlation is significant at the0.05 level.
Fig. 1. The relationships between the AMH concentration on the day the synchronization protocol was initiated (Day 0) and (A) the counts of total corpus luteum, (B) transferable embryos, (C) Code I embryos.
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 AMH 0 days (ng/ml)
Cut-off point for superovula on response
14 CL
Sensi vity Specificity
4.74 ng/ml
Fig. 2. ROC analysis of serum AMH (ng/mL) on the day the synchronization protocol was initiated (Day 0). Determination of the AMH cutoff point for goats that had>14 corpora lutea (CL) in response to the superovulation. AMH concentrations were selected as a decision threshold (cutoff value) to define positive and negative test outcomes. The diagnostic specificity was defined as the probability of a positive test outcome in an individual with >14 CL in response to the superovulation. The diagnostic sensitivity was defined as the probability of a negative test outcome in an individual with>14 CL in response to the superovulation.
embryo (r¼ 0.45, P < 0.05), transferable embryo (r ¼ 0.58, P < 0.01), and Code I quality embryo (r¼ 0.59, P < 0.01).
In the present study, the mean AMH concentration was 6.857± 1.372 on the day the synchronization protocol was initiated. When the donors were divided into two groups depending on whether their AMH concentration on that day was<6.9 or >6.9 ng/ ml, it was found that the counts of total CL, total oocyte/embryos, transferable embryos, and Code I quality embryos were higher in those with an AMH concentration>6.9 ng/ml than in those with an AMH concentration of<6.9 ng/ml. When the donors are divided into four groups (<3, 3e6, 6e10, and >10 ng/ml) according to their mean AMH concentration on the day the synchronization protocol was initiated, the best outcomes regarding the counts of total CL, total oocyte/embryos, transferable embryos, and Code I quality embryos were observed in goats with an AMH concentration >10 ng/ml. Moreover, compared with goats with an AMH concen-tration<3 ng/ml, the total CL count and total oocyte/embryo count increased by 2.5-fold, the transferable embryo counts increased by 2.9-fold, and the Code I quality embryo counts increased by 5-fold
in goats with an AMH concentrations of>10 ng/ml. Abdel Aziz et al. [16] divided cows into four groups according to the AMH concen-tration determined prior to superovulation. The researchers re-ported that there was a 2.5-fold difference in the transferable embryo count and a 6-fold difference in the total number of Code I quality embryos between donors with the lowest and highest AMH concentrations. Souza et al. [15] reported that donors with the highest AMH concentration (243.1 pg/ml) had more than two times the total CL count and three times the total number of cells compared with those with the lowest AMH concentrations (44.9 pg/ml). Center et al. [27] reported that animals with higher AMH concentrations had more follicle and total CL counts than those with lower AMH concentrations. Ghanem et al. [31] observed more cumulus-oocyte complexes (COC) per donor and a higher ratio of oocyte to blastocyst transformation in cows with high (0.25 ng/ml) and moderate (0.1 to <0.25 ng/ml) pre-ovum pick-up AMH concentrations than those with low (<0.1 ng/mL) pre-ovum pick-up concentrations. Therefore, it is believed that it is useful to identify animals with high AMH concentrations to select
Table 3
Uterineflushing findings according to AMH concentrations on the day of synchronization was initiated (Day 0).
AMH concentrations on the day synchronization initiated (Day 0) P
<3 3e6 6e10 >10
Number of donors n 8 6 6 4
Mean AMH concentrations (ng/ml) x±SD 2.05± 0.83 4.17± 1.02 7.47± 1.89 19.60± 3.27 0.001
Total CL x±SD 9.00± 1.92 15.50± 1.58 16.50± 3.01 22.50± 4.93 0.001 Total oocyte/embryos x±SD 5.37± 4.27 11.50± 2.16 12.83± 2.85 13.50± 5.25 0.002 Transferable embryos x±SD 3.87± 2.09 9.50± 2.73 10.50± .1.87 11.25± 5.71 0.001 Code I embryos x±SD 1.37± .1.24 5.50± 2.58 6.13± 2.75 7.01± 3.65 0.009 Code II embryos x±SD 2.50± 1.77 5.12± 3.03 2.67± 2.12 4.25± 1.89 >0.05 Degenerating embryos x±SD 1.37± 1.20 2.00± 1.89 1.16± 1.47 2.25± .2.86 >0.05 Unfertilized oocyte x±SD 0.12± 0.35 0.33± 0.81 0.83± 2.04 0 >0.05 Table 4
The counts of total CL, total oocyte/embryos, transferable embryos, Code I and II embryos, degenerating embryos and unfertilized oocytes obtained on uterineflushing day according to P4 concentration on day 19.
Totals P4 concentration on the day of uterine
flushing (Day 19) P <40 ng/ml >40 ng/ml Number of donors n 24 13 11 e Mean P4 concentration (ng/ml) x±SD 39.577± 4.54 22.81± 5.13 59.38± 7.45 0.001 Total CL x±SD 14.75± 1.29 11.84± 5.59 18.18± 5.60 0.011 Total oocyte/embryos x±SD 10.12± 1.00 7.84± 4.93 12.81± 3.42 0.010 Transferable embryos x±SD 8.16± 0.86 6.46± 4.38 10.18± 3.21 0.030 Code I embryos x±SD 4.75± 0.78 2.30± 2.21 7.63± 3.29 0.001 Code II embryos x±SD 3.41± 0.49 4.15± 2.67 2.54± 1.91 >0.05 Degenerating embryos x±SD 1.62± 0.40 1.30± 1.18 2.00± 2.68 >0.05 Unfertilized oocyte x±SD 0.33± 0.22 0.07± 0.27 0.63± 1.56 >0.05 Table 5
Correlation between the P4 concentrations and the counts of total CL, total oocyte/embryos, transferable embryos, Code I and II embryos, degenerating embryos, unfertilized oocytes on different days of synchronization.
Day 0 Day 11 Day 19
Number of donors n 24 24 24 Mean P4 concentrations (ng/ml) x±SD 5.473± 2.54 2.951± 0.32 39.577± 4.54 Total CL r 0.194 0.287 0.742a Total oocyte/embryos r 0.038 0.264 0.633a Transferable embryos r 0.113 0.101 0.669a Code I embryos r 0.144 0.125 0.891a Code II embryos r 0.027 0.019 0.229 Degenerating embryos r 0.111 0.352 0.049 Unfertilized oocyte r 0.070 0.153 0.154
*Correlation is significant at the 0.05 level.
donors exhibiting better superovulation responses. This is because considering the housing, feeding, FSH, and labor costs, the cost per embryo will be less than half than usual for the embryos produced from donors with higher AMH concentrations [15]. Therefore, AMH measurements can help embryo producers in identifying high su-perovulation response donors and reduce embryo production costs. In MOET programs, it is desirable to know the CL count or transferable embryo count following superovulation before initi-ating the program. Therefore, in this study, a ROC curve analysis was performed according to the AMH concentration on the day the synchronization protocol was initiated to select>14 CL-responding goats per donor; and it was determined that superovulation response was high in donors with an AMH concentration of >4.74 ng/ml. The specificity and sensitivity values of this cutoff concentration were also quite high. Therefore, it was believed that AMH concentration can be assessed in goats to select donors that will respond well after superovulation. In their study in cows, Souza et al. [15] also reported that donors that respond more with>15 CL can be selected through a ROC curve analysis performed according to AMH concentrations. Rico et al. [32] reported that in MOET programs, the number of large follicles was<15 in cows with an AMH concentration<87 pg/ml in estrus, and <10 embryos were obtained when the AMH concentration was<74 pg/ml. In a study conducted on pre-pubertal sheep, the cutoff value of AMH
concentration was considered to be 97 pg/mL for determining fertility at thefirst mating. In sheep with an AMH concentration of 97 pg/mL, the fertility rate at the first mating was 34.8% higher than that in sheep with low AMH concentrations [33]. In another study on sheep, pre-laparoscopic ovum pick-up AMH concentra-tions were measured. On average, 5.1 extra follicles and 2.7 extra
Fig. 3. The relationships between the P4 concentration on the day of uterineflushing and (A) the counts of total corpus luteum, (B) transferable embryos, (C) Code I embryos.
Table 6
Uterineflushing findings according to P4 concentrations on the day of uterine flushing.
P4 concentrations in uterineflushing P
<30 30e50 >50 Number of donors n 8 8 8 Mean P4 concentrations (ng/ml) x±SD 15.890± 3.23 38.038± 5.79 64.803± 7.09 0.001 Total CL x±SD 8.87± 1.83 15.62± 1.41 19.75± 1.86 0.001 Total oocyte/embryos x±SD 5.75± 1.61 10.37± 1.32 12.25± 1.22 0.004 Transferable embryos x±SD 4.50± 1.37 8.62± 0.94 10.37± 1.32 0.005 Code I embryos x±SD 1.00± 0.50 4.62± 0.56 8.62± 1.13 0.001 Code II embryos x±SD 4.00± 1.05 3.50± 0.65 1.75± 0.51 0.021 Degenerating embryos x±SD 1.12± 0.42 2.75± 0.81 1.62± 0.40 >0.05 Unfertilized oocyte x±SD 0.12± 0.1 0 0.27± 0.63 >0.05 Table 7
Correlation between the AMH concentrations on the Day 0 and the counts of total CL and the P4 concentrations on Day 19.
Total CL P4 (Day 19)
AMH (Day 0) 0.670a 0.541a
aCorrelation is significant at the 0.01 level.
Fig. 4. The relationships between the AMH concentration on the day the synchroni-zation protocol was initiated (Day 0) and the P4 concentration on the day of uterine flushing.
COCs were obtained for each 100 pg/mL increase in AMH concen-tration [14]. Although the ROC curve analysis is used for appro-priate donor selection in cattle and a cutoff value is used for the evaluation of fertility in sheep, the cutoff value has so far not been determined to identify appropriate superovulation response in goats during donor selection. Therefore, this study will help in determining donors exhibiting the best superovulation response by assessing AMH concentrations in goats before MOET. However, it should be remembered that the cutoff value may be affected by factors such as breed, age, individual, the day of AMH concentration measurement, a large number of antral follicles on the day of measurement, different AMH analysis methods, and different methods used for sampling (EDTA vs. heparin) [1,14,15,27].
Reportedly, the progesterone concentration after ovulation in superovulated cows increases early and rapidly depending on the CL count. In addition, there is a strong positive correlation between the CL count and progesterone concentration [34,35]. In this study, a positive correlation was found between the P4 concentration on the day of uterineflushing and the counts of total CL, total oocyte/ embryos, transferable embryos, and Code I quality embryos, whereas a negative correlation was found with Code II quality embryo count. In addition, the mean P4 concentration on the day of uterine flushing was 39.57 ± 4.54. Accordingly, when the goats were classified according to the <40 and >40 ng/ml progesterone concentrations; total oocyte/embryo and transferable embryo counts were found to be higher in goats with a progesterone con-centration >40 ng/ml. Furthermore, the counts of total CL, total oocyte/embryos, and transferable embryos increased by more than 2-fold, that of Code I quality embryos increased by more than 8-fold, and that of Code II quality embryos increased by> 2-fold in goats with a progesterone concentrations >50 ng/ml compared with those with concentrations of<30 ng/ml. In other words, it was found that progesterone concentrations were positively correlated with Code I quality embryo counts and negatively correlated with the Code II quality embryo counts. Goto et al. [36] found results similar to those of the present study that the counts of CL, total cells, and transferable embryos were higher in animals with high P4 concentrations on the day of uterineflushing. Silva et al. [21] reported that plasma P4 concentrations were significantly and positively correlated with viable embryo, fertilized ova, and total ova counts in cattle. In a study on sheep, a positive correlation was noted between plasma progesterone concentration and the counts of total CL, total oocyte/embryos, and transferable embryos [23]. Sharma et al. [22] reported that progesterone concentrations are crucial after superovulation and that a deviation from the proges-terone concentration negatively affects embryo quality and yield. The environment of the developing embryo in the post-fertilization period significantly affects access to quality blastocysts [37]. Pro-gesterone concentrations during this period are extremely useful in embryo development and quality. This is because progesterone leads to many molecular, biochemical, and physiological in-teractions that affect the growth, development, and viability of the embryo in the uterus. Consequently, higher than normal proges-terone concentrations accelerate embryo development to a greater extent [38,39]. Contrary to these statements, Saumande et al. [34] and Lonergan et al. [40] found that P4 concentrations were posi-tively correlated with the total CL count on the day of uterine flushing, but it had no effect on transferable embryo counts. It is thought that this is owing to the different oocyte and semen quality of the animal species, to the ovulation of the animals, or to the low rate of infection or fertilization of the uterus.
5. Conclusion
Because there is a significant relationship between AMH
concentrations and superovulation response and embryo yield, AMH concentration measurement can be done to select donors for MOET in goats. Because the AMH concentrations have increased, the total counts of CL, total oocyte/embryos, and transferable em-bryos also increased. In addition, the cutoff value of AMH concen-tration was found to be > 4.74 ng/ml for determining better-responding (>14 CL) goats after gonadotropin administration. Furthermore, it was concluded that the CL count, transferable embryo count, and embryo quality were related to the P4 con-centration on the day of uterineflushing.
Author contributions statements
Kubra Karakas Alkan: Conceptualization, Methodology, Investi-gation, Writing - Original Draft, Writing - Review& Editing. Hasan Alkan: Conceptualization, Methodology, Formal analysis, Investi-gation, Writing - Original Draft. Mustafa Kaymaz: Methodology, Investigation, Writing - Original Draft.
Declaration of competing interest
The authors declare no conflicts of interest. Acknowledgement
This work was supported by the Scientific Research Projects Chieftaincy of Ankara University (Grant Number: 18H0239002). References
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