Trio a novel bovine high-fecundity allele: II. Hormonal profile and follicular dynamics underlying the high ovulation rate

Trio a novel bovine high-fecundity allele: II. Hormonal profile and follicular dynamics... Abstract The newly discovered Trio high-fecundity allele produces multiple ovulations in cattle. This study evaluated (1) size and growth rates of follicles in Trio carriers during a synchronized follicular wave, induced by follicle aspiration; (2) follicle-stimulating hormone (FSH) patterns associated with the follicular wave; (3) size of corpora lutea (CL) and circulating progesterone; and (4) intrafollicular estradiol concentrations prior to normal deviation. Trio carriers had mean dominant follicles that were significantly smaller in diameter and volume than noncarriers. Onset of diameter deviation occurred at ∼3 days after the last follicle aspiration in both genotypes despite Trio carriers having much smaller individual follicles. Follicles of Trio carriers grew at a slower rate than noncarrier follicles (∼65% in mm/day or ∼30% in mm3/day) resulting in much smaller individual dominant follicles (∼25% volume). However, total dominant follicle volume, calculated as the sum of all dominant follicles in each animal, was similar in carriers and noncarriers of Trio throughout the entire follicular wave. Circulating FSH was greater in Trio carriers during the 24 h encompassing deviation. Trio carriers had significantly more ovulations than noncarriers, and individual CL volume was smaller, although total luteal tissue volume and circulating P4 were not different. Thus, increased ovulation rate in Trio carriers relates to smaller individual follicles (one-third the volume) near the time of deviation due to slower follicle growth rate, although time of deviation is similar, with increased circulating FSH near deviation leading to selection of multiple dominant follicles in Trio carriers with similar total follicle volume. Introduction The mechanisms underlying the determination of litter size, both in polytocous and monotocous species, have been the subject of extensive research. One of the major determinants of litter size is ovulation rate, which generally determines the upper limit on the number of offspring. Ovulation rate is, in turn, determined by the number of follicles that are selected for dominance and thereby have the capacity to ovulate at the time of the luteinizing hormone (LH) surge. In polytocous species, such as mice and swine, there is substantial variation in ovulation rate, with multiple loci explaining much of the variation in ovulation rate [1–3]. For example, in swine most of the variation in ovulation rate (71.1%) could be accounted for by variation in 22 quantitative trait loci (QTL) that were present on various chromosomes and included candidate genes such as estrogen receptor 1 (ESR1), growth differentiation factor 9 (GDF9), bone morphogenetic protein 7 (BMP7), bone morphogenetic protein 4 (BMP4), SMA, and SMAD family member 2 (SMAD2, previously known as MAD, mothers against decapentaplegic homolog 2), cAMP responsive element binding protein 1 (CREB1), and nhibin beta A subunit (INHBA) [4]. Nevertheless, the specific links between the genetic variation and the physiological and molecular alterations that lead to increased ovulation rate have not been completely defined. In monotocous species or species with a low ovulation rate, such as sheep, there is substantial variation in ovulation rate due to environmental and genotypic effects with clear linkages between increased ovulation rate and specific genetic variations [5,6]. Single gene mutations such as Booroola/FecB [7], Inverdale/FecXI [8], Hannah/FecXH [9], and FecGH [10] led to the discovery of the essential role of members of the transforming growth factor beta (TGFB) superfamily in folliculogenesis and ovulation rate. Two members of the TGFB superfamily, bone morphogenetic protein 15 (BMP15) and GDF9, are secreted by the oocyte and regulate proliferation and other aspects of granulosa cell function [5,11]. Eight different mutations in the BMP15 gene [10,12–15] and four different mutations in the GDF9 gene [10,16–18] have been identified in sheep. These mutations produce increased ovulation rate (35–100%) in heterozygous carriers and sometimes premature ovarian failure in homozygous carriers with failure of normal folliculogenesis past the primary stage [5]. Interestingly, homozygous carriers of certain fecundity alleles had a further increase in ovulation rate and no premature ovarian failure [19], apparently due to the mutations causing attenuation of BMP15/GDF9 activity and not loss of function [5,11]. Another mutation, termed Booroola fecundity, FecB, was found to reside in the kinase region of the bone morphogenetic protein receptor type 1B (BMPR1B), and produced increased ovulation rate in heterozygous carriers with further increases in ovulation rate in homozygotes [20]. Furthermore, another X-linked mutation (FecX2W) increased ovulation rate in both heterozygotes and homozygotes, although the causative mutation has not been identified [21]. Additive increases in ovulation rate are produced when heterozygous carriers for mutations in BMP15, BMPR1B, and the FecX2W mutation are combined, producing ovulation rates over 10 in heterozygous carriers of all 3 mutations, compared to 1-2 ovulations in the noncarrier control ewes [22]. In monovular species, such as cattle and humans, QTLs for high ovulation rate have been identified [23]. In addition, long-term selection of cattle for high ovulation rate and twinning has led to the formation of the USDA-MARC twinner herd, which exhibits a much greater frequency of multiple ovulations than wild-type control cattle [24]. Genetic analysis of this population indicated that the increased ovulation rate was related to multiple genes rather than a single gene mutation [25]. Recently, a major gene for high ovulation rate in cattle has been identified [26] with clear evidence for segregation in a 1.2-Mb region of bovine chromosome 10 [27]. The resulting high-fecundity allele, termed “Trio,” appears to increase ovulation rate through a novel mechanism that involves upregulation of MAD family member 6 (SMAD6, previously known as MAD, mothers against decapentaplegic homolog 6) [28]. Carriers of the Trio allele offer a unique opportunity to evaluate the dynamics and interactions of follicular growth and circulating follicle-stimulating hormone (FSH) in a high ovulation rate genotype of a monovular species. The bovine model has been a frequently utilized model for study of the mechanisms involved in follicular selection [29,30]. Growth of follicular waves and morphological selection of a single dominant follicle, termed follicular deviation, can be clearly distinguished by current resolution of ultrasound technology available for cattle. In addition, the similarities of cattle and humans in morphological follicular growth patterns and follicle size at deviation make cattle a particularly interesting biological model for human follicular physiology (reviewed in [31]). This study was designed to evaluate the hormonal profile and associated follicle growth dynamics in cattle that are heterozygous carriers and half-sibling noncarriers of the novel Trio allele that produces increased ovulation rate. Based on our preliminary observations and the previous model proposed for ovine high-fecundity genotypes [5], it was hypothesized that (1) carriers of the high-fecundity allele would ovulate smaller-sized follicles compared to noncarriers, although the total ovulatory follicular volume would be similar; (2) follicles in the carrier animals would grow at a slower rate than in noncarriers; (3) concentrations of FSH would be greater in carriers than in noncarriers, particularly near the time of follicular deviation; and (4) follicular deviation would occur at smaller follicle sizes in carrier animals than in noncarriers. Materials and methods All animal procedures were approved by the Animal Care and Use Committee of the College of Agriculture and Life Sciences at the University of Wisconsin–Madison. All animals used in this study were kept in outdoor paddocks with free access to a run-in shelter and water. Cattle were fed mixed hay ad libitum and had access to a standard mineral mix. Females used in the following studies were produced by mating (through artificial insemination) bulls which were carriers of the Trio allele with cows that in most cases were of predominantly Angus breeding (noncarriers). In some cases, project females were the product of superovulation and embryo transfer with both parents being Trio allele carriers. Breed composition of the Trio allele carrier parents was >50% Angus with the remainder of the breed composition being a mix of Hereford, Holstein, and Jersey. Trio allele genotype was determined by analysis of haplotypes based on three linked markers as described previously [27]. All females were thus either half or full sibs, and Trio carriers were heterozygous for the Trio allele. Experiment 1 Experiment 1 evaluated the follicle dynamics and circulating FSH of cattle carrying a high-fecundity allele (Trio) under a controlled progesterone (P4) environment. Nulliparous and multiparous nonlactating crossbred cattle, ranging in age from 18 to 57 months, carrying the high-fecundity allele (n = 13) or age-matched half-sib controls (n = 11) had their estrous cycle synchronized (Figure 1). All animals received a P4 intravaginal controlled internal drug-releasing device (CIDR, Eazi-Breed, Zoetis, Florham Park, NJ) on D−9 and this was removed 6 days later (D−3). Prostaglandin F2α (PGF, Estroplan, Parnell, Overland Park, KS) was administered twice 24 h apart on D−3 and D−2. In order to synchronize the emergence of a new follicular wave, animals had ultrasound-guided follicular ablation of all follicles ≥4 mm on D−2, D−1, and D0, as previously described with minor modifications [32]. Briefly, transvaginal ultrasound-guided follicle ablation was performed with an 8 MHz microconvex array transducer attached to a B-mode ultrasound scanner using a 2″ 18G hypodermic needle. On D0, after follicle ablation was performed, a new CIDR was inserted and remained in place until D5. Ultrasound examinations of the ovaries were performed every 12 h starting on D0 until cows ovulated, using a B-mode ultrasound scanner equipped with a 7.5 MHz transducer. A sketch was made of each ovary recording follicle number, size, and relative location on the ovary. Due to the complexity of follicle wave patterns in Trio carriers (i.e. multiple dominant follicles of smaller size), follicle growth profiles were constructed based on the nonidentity method as previously described [33]. Blood collection was performed via coccygeal venipuncture into evacuated tubes every 24 h from D3 to D1 and then every 12 h from D0 until ovulation. All animals were fitted with Kamar heat mount detectors (Kamar Products Inc., Zionsville, IN) on the day of removal of the CIDR (D5) to aid in the detection of standing estrus, and detectors were checked every 12 h. Figure 1. View largeDownload slide Treatment schedules of experiment 1 for cattle carrying the high-fecundity allele Trio and age-matched, half-sib controls. A CIDR was inserted on D−9 and left in place for 6 days. Prostaglandin F2α (PGF) was administered 24 h apart on D−3 and D−2. Transvaginal ultrasound-guided follicle (≥4 mm) ablation was performed in all animals on D−2, D−1, and D0 and a new CIDR was inserted after the last follicle ablation. The CIDR was removed on D5 and animals were examined by transrectal ultrasound (US) and blood samples collected (BS) every 12 h from D0 until ovulation (D8 approximately). The treatment schedule for experiment 2 is also shown. All animals had their estrous cycle synchronized with two doses (500 μg/each) of PGF administered 24 h apart on D0 and D1 and on D3 GnRH (100 μg) was administered. On D10, GnRH (200 μg) was administered again and finally two doses of PGF were administered on D15 and D16. The following complete estrous cycle was evaluated by daily ultrasound and blood sampling for circulating progesterone concentrations. In experiment 3, animals were synchronized by 6 days of treatment (D−8 to D−2) with a CIDR and PGF (500 μg) was given on D−2 and D−1, followed by follicular aspiration on D−1 and D0. Animals were then evaluated by ultrasound every 12 h and follicular fluid collection was done when the largest follicle reach 7 mm in diameter. Figure 1. View largeDownload slide Treatment schedules of experiment 1 for cattle carrying the high-fecundity allele Trio and age-matched, half-sib controls. A CIDR was inserted on D−9 and left in place for 6 days. Prostaglandin F2α (PGF) was administered 24 h apart on D−3 and D−2. Transvaginal ultrasound-guided follicle (≥4 mm) ablation was performed in all animals on D−2, D−1, and D0 and a new CIDR was inserted after the last follicle ablation. The CIDR was removed on D5 and animals were examined by transrectal ultrasound (US) and blood samples collected (BS) every 12 h from D0 until ovulation (D8 approximately). The treatment schedule for experiment 2 is also shown. All animals had their estrous cycle synchronized with two doses (500 μg/each) of PGF administered 24 h apart on D0 and D1 and on D3 GnRH (100 μg) was administered. On D10, GnRH (200 μg) was administered again and finally two doses of PGF were administered on D15 and D16. The following complete estrous cycle was evaluated by daily ultrasound and blood sampling for circulating progesterone concentrations. In experiment 3, animals were synchronized by 6 days of treatment (D−8 to D−2) with a CIDR and PGF (500 μg) was given on D−2 and D−1, followed by follicular aspiration on D−1 and D0. Animals were then evaluated by ultrasound every 12 h and follicular fluid collection was done when the largest follicle reach 7 mm in diameter. Experiment 2 Experiment 2 evaluated circulating P4 and volume of corpora lutea (CL) in cattle that were heterozygous carriers or noncarriers of the Trio allele during an estrous cycle. Nulliparous and multiparous nonlactating crossbred cattle, ranging in age from 18 to 57 months, carrying the Trio allele (n = 7) or age-matched, half-sib controls (n = 5) were used. All animals had their estrous cycle synchronized as follows: two doses (500 μg/each) of PGF (Estroplan, Parnell, Overland Park, KS) were administered 24 h apart on D0 and D1 and on D3 GnRH (100 μg of gonadorelin acetate) was administered (Gonabreed, Parnell, Overland Park, KS). On D10, GnRH (200 μg) was administered again and finally two doses of PGF were administered on D15 and D16. A change in synchronization protocol was done in experiment 2 to increase circulating progesterone concentrations during follicle growth in order to potentially decrease the double ovulations that occurred in experiment 1. Ultrasound examinations of the ovaries was performed every 24 h starting on the day of the last PGF until D10 after standing estrus. Examinations were done using a B-mode ultrasound scanner equipped with a 7.5 MHz transducer and were recorded in a 20-s cine loop. Images were analyzed, and size and relative location of each CL were recorded in a sketch. Blood collection was performed via coccygeal venipuncture into evacuated tubes every 24 h for the complete interovulatory interval. Experiment 3 Experiment 3 was designed to evaluate the concentrations of estradiol (E2) and P4 in the follicular fluid of follicles before selection in cattle that were heterozygous carriers or noncarriers of the Trio allele. Nulliparous and multiparous nonlactating crossbred cattle, ranging in age from 2 to 4 yr, carrying the Trio allele (n = 8) or age-matched, half-sib controls (n = 5) were used. All animals received a CIDR on D8 and this was removed 6 days later (D2). Prostaglandin F2α was administered twice, 24 h apart, on D2 and D1. In order to synchronize the emergence of a new follicular wave, ultrasound-guided transvaginal follicle aspiration was performed as described in experiment 1 on D1 and D0. All animals were monitored by transrectal ultrasonography of their ovaries every 12 h beginning on D0. The follicular fluid of the three largest follicles was collected individually at the time that the largest follicle (F1) reached a diameter of at least 7 mm, as previously described [34]. Upon collection of follicular fluid, animals continued to be monitored by ultrasonography every 12 h until the largest follicle reached 7 mm, and the process of collecting follicular fluid from the three largest follicles was repeated. All animals were fitted with Kamar heat mount detectors on the day of the second PGF (D1) to aid in the detection of standing estrus. Three criteria were used to determine if data from a collection would be used in analyses. First, if the F1 follicle diameter was >8 mm (nine collections removed, five carriers and four noncarriers). Second, if animals were exhibiting estrus at the time of collection (potential exposure to LH surge; three carriers removed). Third, if the F1 follicle had nondetectable concentrations of estradiol in the follicular fluid (three carriers removed). Thus, from 26 total collections, data from only 11 collections were available for the final analysis. Hormone assays Blood samples were centrifuged at 1300 × g and 4°C for 20 min, and serum was transferred into vials, frozen, and stored at –20°C until assayed. Assay of P4 was performed using one of two solid-phase radioimmunoassay (RIA) kits (Coat-A-Count Progesterone; Siemens Healthcare Diagnostics, Los Angeles, CA or ImmuChem Progesterone Coated Tube; MP Biomedicals, Santa Ana, CA). Mean assay sensitivity, calculated as 2 SD less than the mean counts per minute of maximum binding, was 0.018 and 0.015 ng/ml for each kit, respectively. Intra- and interassay coefficients of variation were 4.7% and 5.9% for the Coat-A-Count kit and 5.0% was the intra-assay coefficient of variation for the one assay with the ImmuChem kit. Serum FSH was measured using an RIA that was previously validated for cattle [35,36]. The FSH assay incorporated the primary antibody NIDDK-anti-oFSH-I-2 and the radiolabeled and standard antigen USDA-bFSH-I-2. Mean assay sensitivity was 0.04 ng/ml, and intra- and inter-assay coefficients of variation were 2.8% and 12.9% using the high FSH quality control sample and 4.3% and 14.1% using the low FSH quality control sample. Luteinizing hormone concentrations were determined by RIA as described in [37]. Average sensitivity of the assays was 0.11 ng/mL and intra- and inter-assay coefficients of variation were 2.0% and 8.6%, respectively. Follicular fluid samples were centrifuged at 600 × g for 10 minutes to remove granulosa cells from the samples and the supernatant was stored frozen at –20°C until assayed. Intrafollicular estradiol concentrations were determined using a commercially available competitive ELISA (Estradiol Serum EIA Kit, #KB30-H1, Arbor Assays, Ann Arbor, MI). Samples were diluted in assay buffer provided by the manufacturer at 1:5000 or 1:20,000. Samples were analyzed in a single assay, and the intra-assay coefficient of variation was 7.0%. Intrafollicular concentrations of P4 were assessed using the solid-phase RIA kit described above (Coat-A-Count Progesterone). Samples were diluted 1/20 in PBS, and all samples were evaluated in one assay. Intra-assay coefficient of variation was 7.8%. Terminology and data arrangement statistical analyses Experiment 1 Dominant follicles were defined as any follicle that ovulated at the end of the evaluation period and were numbered from largest to smallest (F1, F2, etc.) based on their maximum diameter. Subordinate follicles (SFs) were designated retrospectively as one that appeared to originate from the same pool as the dominant follicles, its first detection was within 2 days of detection of the dominant follicle, increased in diameter during the time the dominant follicle was also initially increasing, failed to ovulate, and later ceased to grow while a dominant follicle continued to grow [38]. The beginning of deviation was defined as the beginning of the greatest difference in growth rate between the largest dominant follicle (F1) and the first SF at or before the largest SF attained maximal diameter [39]. The beginning of deviation was determined for each animal individually. Follicle volume was calculated assuming the shape of a sphere with the following formula: V = 4/3πr3. Mean dominant follicle dimensions (diameter and volume) was calculated as the sum of all dominant follicles for a given animal and ultrasound examination divided by the number of dominant follicles. Total dominant follicle volume was calculated as the sum of the volume for all individual dominant follicles for a given animal and time point. Follicular growth rate was estimated by calculating the diameter/volume of a given follicle for each animal at the beginning and end of a defined period of time. The difference in diameter/volume between the time points divided by the number of days between time points was used as the growth rate. Experiment 2 Individual CL volume was calculated by using the formula shown above with the radius determined from the CL diameter, assuming that the CL had the shape of a sphere. If a cavity was present, the volume of the cavity was calculated and subtracted from the total CL volume. Volume was calculated for each individual CL and time point. Mean CL volume was calculated as the sum of all CL for a given animal and examination divided by the number of CL present. Total CL volume was calculated as the sum of each CL volume for a given animal and time point. For experiment 3, follicles were numbered F1 to F3, from largest to smallest, based on their diameter at the time of follicular fluid collection. Statistical analyses The analysis of variables measured over time (e.g. follicle diameter and volume; FSH, LH, P4 concentrations) was performed by analysis of variance using the repeated option in PROC MIXED with day as the repeated variable and cow as the subject with an autoregressive covariance structure (Statistical Analysis System, SAS Institute, Version 9.4 Cary, NC, USA). Genotype, time, and genotype by time interaction were included in the model as fixed effects. Analysis of data over time was performed in relation to the last follicle aspiration and in other analyses was normalized to the day of deviation. The main effects of treatment, day, and their interactions were determined, and preplanned comparisons between genotypes for specific time points were done by least square difference. For individual comparisons between genotypes, the two-sample t-test was used for continuous outcome (e.g. follicle diameter and volume at deviation, hormone concentrations at specific time points). For comparisons in experiment 3, each collection was considered to be independent and follicle within each collection was the experimental unit. Analysis of variables in this experiment was performed by analysis of variance using the MIXED procedure including genotype, follicle hierarchy (F1, F2, or F3), and their interaction as fixed effects. The repeated statement was utilized in order to account for clustering of follicles within animal, and cow was included as a random effect. Differences between follicles and genotypes were determined by least square means. Assumptions of normality and homogeneity of variance were evaluated for all outcomes, and transformations were attempted if assumptions were not met. For data in which transformations were not successful, the Wilcoxon rank-sum test was used. Data are presented as mean (±SEM). Values were considered statistically different when P ≤ 0.05. Results Experiment 1 In preliminary analyses of follicular data, it became apparent that noncarrier cows primarily had double ovulations, whereas noncarrier heifers primarily had single ovulation. Therefore, results in experiment 1 were analyzed separately for cows (32–57 months of age) and heifers (18–25 months of age). One heifer in the noncarrier group was removed from the analysis due to double ovulation. Thus, eight carrier and six noncarrier heifers were available for further analysis. All cows in the noncarrier group had codominant follicles and double ovulations. As a result, five carrier and four noncarrier cows were available for further analysis. Two noncarriers (one heifer and one cow) had P4 concentrations that failed to decrease after CIDR removal, resulting in a delayed onset of estrus (192 and 132 h after CIDR removal, respectively). As a result, data from these animals were considered only for outcomes that evaluated the period up to CIDR removal. Follicle growth pattern by genotype and parity Follicle growth patterns for Trio carriers and noncarrier controls are shown in Figure 2 with the top panel showing noncarrier heifers with single ovulation (left) and noncarrier cows with double ovulations (right) and the bottom panel showing Trio carrier heifers with three ovulations (left) and Trio carriers cows with five to six ovulations (right). In all genotypes and parities, there was consistent growth of the F1 from the time of first detection (0.5 days) until 5 days. Further, in all genotypes and parities a clear SF could be identified. As shown in Figure 2, the SF, on average, grew from day 0.5 to 2.5 and then growth of the SF was reduced in all of the four groups. The major difference between groups was clearly the number of codominant follicles that were present. In noncarrier heifers, only a single dominant follicle was identified. In noncarrier cows, a second dominant follicle was present as the F2 continued growth during the same time period as the F1 (0.5 to 5.5 days). In carrier heifers, the F1, F2, and F3 grew during the entire evaluation period (0.5 to 5.5 days) and grew faster than the SF after day 2.5. In carrier cows, a total of at least five dominant follicles could be identified (F1 to F5 shown with F6 [present in some cows] not shown) and clear differences in growth could be observed between the SF and all of the dominant follicles after day 2.5. Figure 2. View largeDownload slide Growth profiles (mean ± SEM) of the largest (F1), second largest (F2), third largest (F3), fourth largest (F4), and fifth largest (F5) dominant follicle and first subordinate follicle (SF) in animals carrying the Trio allele or age-matched, half-sib controls (experiment 1). The upper left panel depicts noncarrier heifers (n = 6) with a single dominant follicle, while the lower left panel depicts Trio carrier heifers (n = 3) with triple dominant follicles. The upper right panel depicts noncarrier cows (n = 4) with double dominant follicles, while the lower right panel depicts Trio carrier cows (n = 3) with quintuple or sextuple dominant follicles. Figure 2. View largeDownload slide Growth profiles (mean ± SEM) of the largest (F1), second largest (F2), third largest (F3), fourth largest (F4), and fifth largest (F5) dominant follicle and first subordinate follicle (SF) in animals carrying the Trio allele or age-matched, half-sib controls (experiment 1). The upper left panel depicts noncarrier heifers (n = 6) with a single dominant follicle, while the lower left panel depicts Trio carrier heifers (n = 3) with triple dominant follicles. The upper right panel depicts noncarrier cows (n = 4) with double dominant follicles, while the lower right panel depicts Trio carrier cows (n = 3) with quintuple or sextuple dominant follicles. Comparison of follicle growth normalized to time of follicle aspiration In heifers, the diameter of F1 (Supplemental Figure S1) indicated a main effect of genotype (P < 0.0001), day (P < 0.0001), but no interaction between genotype and day (P = 0.20). Noncarrier heifers had greater (P < 0.02) F1 diameter than Trio carriers from the first detection of F1 at 12 h after the last aspiration and throughout the evaluation period (Supplemental Figure S1). Also in heifers, the mean dominant follicle diameter (Figure 3) during the 5 days after follicle aspiration showed a main effect of genotype (P < 0.0001), day (P < 0.0001), and a genotype by day interaction (P = 0.02). Noncarrier heifers had greater (P < 0.02) mean dominant follicle diameter than Trio carrier heifers beginning at first detection on D0.5 until D5 (Figure 3). Figure 3. View largeDownload slide Growth profile of mean dominant follicle diameter, mean dominant follicle volume, and total dominant follicle volume in heifers (left) and cows (right) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration until the removal of the intravaginal P4 implant. Each point represents mean (±SEM). *Indicates significant differences between genotype for a given time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Figure 3. View largeDownload slide Growth profile of mean dominant follicle diameter, mean dominant follicle volume, and total dominant follicle volume in heifers (left) and cows (right) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration until the removal of the intravaginal P4 implant. Each point represents mean (±SEM). *Indicates significant differences between genotype for a given time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. When dominant follicle growth in heifers was considered on a volume basis (Figure 3; second panel), there was a main effect of genotype (P < 0.0001), day (P < 0.0001), but no genotype by day interaction (P = 0.29). Noncarrier heifers had a greater (P < 0.01) mean dominant follicle volume than Trio carriers from first detection at 12 h after aspiration until D5. On average, noncarrier heifers had a 3.2-fold greater mean dominant follicle volume than Trio carrier heifers. In contrast, total dominant follicle volume in heifers showed a main effect of day (P < 0.0001) but no effect of genotype (P = 0.25) or genotype by day interaction (P = 0.37). Trio carrier and noncarrier heifers had similar (P > 0.05) total dominant follicle volume at all time points with the exception of D0.5 when Trio carriers had greater (P = 0.004) total follicle volume than noncarrier heifers (Figure 3). In cows, analysis of F1 diameter (Supplemental Figure S1) showed a main effect of genotype (P = 0.0005), day (P < 0.0001), but no genotype by day interaction (P = 0.63). Noncarrier cows had greater (P < 0.01) F1 diameter than Trio carriers from D1 until D5 (Supplemental Figure S1). Also in cows, mean dominant follicle diameter showed a main effect of genotype (P = 0.0002) and day (P < 0.0001) but no genotype by day interaction (P = 0.75). Noncarrier cows consistently had a greater (P < 0.01) mean dominant follicle diameter than Trio carrier cows beginning at first detection on D0.5 until the last examination on D5 (Figure 3). On a volume basis, mean dominant follicle volume in cows showed a main effect of genotype (P = 0.0002), day (P < 0.0001), but no genotype by day interaction (P = 0.76). Noncarrier cows had a greater (P < 0.01) mean dominant follicle volume than Trio carriers starting on D0.5 and continuing throughout the observation period until D5. On average, noncarrier cows had a twofold greater mean dominant follicle volume than Trio carrier cows. In contrast, analysis of total dominant follicle volume in cows showed a main effect of day (P < 0.0001) but no effect of genotype (P = 0.41) and no genotype by day interaction (P = 0.76). Trio carrier and noncarrier cows had similar (P > 0.15) total dominant follicle volume at all time points (Figure 3). Comparison of follicle growth normalized to time of follicle deviation The time of deviation (days from last aspiration) between the F1 and the first subordinate follicle in heifers (Table 1) was not different between carriers and noncarriers (3.2 vs. 3.0 d; P = 0.66). At the time of follicle deviation in heifers, the diameter of F1, the mean dominant follicle diameter, and the diameter of the SF1 were all greater (P < 0.01) in noncarrier heifers than the corresponding follicles in Trio carriers (Table 1). Analysis of follicle volume at the time of deviation indicated that noncarrier heifers had a greater (P < 0.01) F1 (+229%) and SF1 (261%) volume than Trio carrier heifers (Table 1). Mean dominant follicle volume at time of deviation was 287% larger (P < 0.0001) in noncarrier than Trio carriers, but total dominant follicle volume was not different (P = 0.22). For cows, time of deviation and follicle characteristics at deviation are also shown in Table 1. Time from last follicle aspiration until follicle deviation in cows was not different (P = 0.49) between Trio carriers and noncarriers (2.9 vs. 3.1 d). Analysis of follicle diameter at the time of deviation showed that noncarrier cows had greater (P < 0.02) F1, F2, SF1, and mean dominant follicle diameter than carriers. Analysis of follicle volume at the time of deviation also indicated that noncarrier cows had greater (P < 0.03) F1 (+213%), F2 (+171%), and SF1 (+258%) volume than Trio carrier cows. Mean dominant follicle volume at deviation in noncarrier cows was 207% larger (P = 0.0005) than in Trio carriers. Total dominant follicle volume at the time of deviation did not differ between genotypes (P = 0.41). Follicle growth rate Analysis of overall follicle growth rate from first detection (D0.5) until ovulation is shown in Table 2. Growth rate of F1 and mean dominant follicle growth rate were greater (P < 0.02) in noncarrier heifers compared to Trio carrier heifers, both on a diameter and volume basis. Mean dominant follicle growth rate on a volume basis was 4.3-fold greater (P < 0.01) in noncarrier heifers than in Trio carriers. The total dominant follicle volume growth rate was not different (P = 0.40) between Trio carrier and noncarrier heifers. Table 1. Mean (±SEM) follicle diameter and volume, at the time of deviation in heifers and cows that are Trio carriers or noncarriers.   Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  6    5  4    Days to deviation  3.2 ± 0.4  3.0 ± 0.4  0.66  2.9 ± 0.2  3.1 ± 0.2  0.49  Follicle diameter (mm)  F1  6.5 ± 0.3  8.6 ± 0.3  0.0004  7.1 ± 0.1  8.9 ± 0.3  0.011  F2  5.9 ± 0.2      6.8 ± 0.2  8.1 ± 0.3  0.006  F3a  5.8 ± 0.3      6.7 ± 0.4      First subordinate  5.3 ± 0.1  7.2 ± 0.4  0.0001  5.6 ± 0.1  7.6 ± 0.5  0.016  Mean dominant follicle  6.0 ± 0.2  8.6 ± 0.3  <0.0001  6.6 ± 0.1  8.5 ± 0.3  0.0006  Follicle volume (mm3)  F1  149.3 ± 20  340.2 ± 35  0.0003  186.2 ± 8  369.6 ± 43  0.022  F2  116.3 ± 13      163.6 ± 16  280.5 ± 29  0.007  F3a  109.5 ± 15      162.5 ± 30      First subordinate  79.2 ± 6  207.1 ± 32  0.009  92.7 ± 7  239.2 ± 43  0.016  Mean dominant  118.5 ± 14  340.2 ± 35  <0.0001  157.0 ± 10  325.1 ± 34  0.0005  Total dominant  432.0 ± 56  340.2 ± 35  0.22  719.3 ± 78  650.1 ± 68  0.41    Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  6    5  4    Days to deviation  3.2 ± 0.4  3.0 ± 0.4  0.66  2.9 ± 0.2  3.1 ± 0.2  0.49  Follicle diameter (mm)  F1  6.5 ± 0.3  8.6 ± 0.3  0.0004  7.1 ± 0.1  8.9 ± 0.3  0.011  F2  5.9 ± 0.2      6.8 ± 0.2  8.1 ± 0.3  0.006  F3a  5.8 ± 0.3      6.7 ± 0.4      First subordinate  5.3 ± 0.1  7.2 ± 0.4  0.0001  5.6 ± 0.1  7.6 ± 0.5  0.016  Mean dominant follicle  6.0 ± 0.2  8.6 ± 0.3  <0.0001  6.6 ± 0.1  8.5 ± 0.3  0.0006  Follicle volume (mm3)  F1  149.3 ± 20  340.2 ± 35  0.0003  186.2 ± 8  369.6 ± 43  0.022  F2  116.3 ± 13      163.6 ± 16  280.5 ± 29  0.007  F3a  109.5 ± 15      162.5 ± 30      First subordinate  79.2 ± 6  207.1 ± 32  0.009  92.7 ± 7  239.2 ± 43  0.016  Mean dominant  118.5 ± 14  340.2 ± 35  <0.0001  157.0 ± 10  325.1 ± 34  0.0005  Total dominant  432.0 ± 56  340.2 ± 35  0.22  719.3 ± 78  650.1 ± 68  0.41  aFor carrier animals, dimensions for up to the third largest dominant follicle are shown as all animals had at least three dominant follicles. View Large Table 2. Mean (±SEM) follicle growth rates in heifers and cows that are Trio carriers or noncarrier controls.   Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Overall growth rate (mm/day)  F1  0.82 ± 0.1  1.22 ± 0.2  0.017  0.93 ± 0.1  1.39 ± 0.1  0.018  F2  0.66 ± 0.1      0.96 ± 0.1  1.20 ± 0.1  0.029  F3a  0.63 ± 0.1      0.94 ± 0.1      Mean dominant follicle  0.70 ± 0.1  1.22 ± 0.2  0.003  0.93 ± 0.1  1.30 ± 0.1  0.013  Overall volume growth rate (mm3/day)  F1  54.9 ± 7  175.2 ± 40  0.0006  73.6 ± 10  191.7 ± 30  0.006  F2  40.0 ± 6      71.9 ± 7  134.9 ± 11  0.003  F3a  32.2 ± 5      65.1 ± 8      Mean dominant follicle  40.9 ± 6  175.2 ± 40  0.0001  67.0 ± 9  163.3 ± 18  0.002  Total dominant follicle  142.7 ± 17  175.2 ± 40  0.404  293.7 ± 32  326.6 ± 37  0.54    Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Overall growth rate (mm/day)  F1  0.82 ± 0.1  1.22 ± 0.2  0.017  0.93 ± 0.1  1.39 ± 0.1  0.018  F2  0.66 ± 0.1      0.96 ± 0.1  1.20 ± 0.1  0.029  F3a  0.63 ± 0.1      0.94 ± 0.1      Mean dominant follicle  0.70 ± 0.1  1.22 ± 0.2  0.003  0.93 ± 0.1  1.30 ± 0.1  0.013  Overall volume growth rate (mm3/day)  F1  54.9 ± 7  175.2 ± 40  0.0006  73.6 ± 10  191.7 ± 30  0.006  F2  40.0 ± 6      71.9 ± 7  134.9 ± 11  0.003  F3a  32.2 ± 5      65.1 ± 8      Mean dominant follicle  40.9 ± 6  175.2 ± 40  0.0001  67.0 ± 9  163.3 ± 18  0.002  Total dominant follicle  142.7 ± 17  175.2 ± 40  0.404  293.7 ± 32  326.6 ± 37  0.54  aFor carrier animals, dimensions for up to the third largest dominant follicle are shown as all animals had at least three dominant follicles. View Large Noncarrier cows had a greater (P < 0.03) growth rate of F1, F2, and mean dominant follicle compared to Trio carriers, both on a diameter and volume basis. Mean dominant follicle growth rate on a volume basis was 2.4-fold greater (P = 0.002) in noncarrier cows. Growth rate of total dominant follicles, on a volume basis, was not different (P = 0.54) between carrier and noncarrier cows (Table 2). Circulating FSH Circulating FSH concentrations in heifers tended (P = 0.06) to be different between genotypes (Figure 4A). There was a significant effect of time (P < 0.001) reflecting the rise in FSH concentrations after follicle aspiration and subsequent decline, but no interaction between genotype and time was identified (P = 0.95). Comparisons between genotypes for individual time points were not different (P > 0.05) with the exception of D−2 (day of the first follicle aspiration) when carrier heifers had greater FSH concentrations than noncarriers (P = 0.03). Peak FSH concentrations were not different (P = 0.38) between Trio carriers and noncarrier heifers (0.57 ± 0.04 and 0.65 ± 0.09 ng/ml, respectively). Figure 4. View largeDownload slide Mean (±SEM) serum FSH for heifers (A), cows (B), and all animals combined (C) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration. *Indicates significant differences between genotype for a given time point (P < 0.05), while ‡indicates a tendency (P < 0.10). G, genotype; D, day; G*D, genotype by day interaction. Figure 4. View largeDownload slide Mean (±SEM) serum FSH for heifers (A), cows (B), and all animals combined (C) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration. *Indicates significant differences between genotype for a given time point (P < 0.05), while ‡indicates a tendency (P < 0.10). G, genotype; D, day; G*D, genotype by day interaction. Circulating FSH concentrations were further evaluated after normalization to deviation (data not shown). Three distinct periods were evaluated: predeviation (mean FSH from –48 to –24 h); at deviation (mean FSH from –12 to 12 h); and postdeviation (mean FSH from 24 to 48 h). Predeviation FSH concentrations were not different (P = 0.83) between Trio carriers and noncarrier heifers (0.32 ± 0.02 and 0.31 ± 0.05 ng/ml, respectively). Similarly, postdeviation FSH concentrations were not different (P = 0.26) between genotypes (0.27 ± 0.02 and 0.23 ± 0.02 ng/ml, for carrier and noncarrier heifers respectively). However, there was a tendency (P = 0.09) for greater FSH concentrations at deviation in Trio carriers (0.28 ± 0.02 ng/ml) compared to noncarrier heifers (0.23 ± 0.03 ng/ml). Circulating FSH concentrations in cows showed no main effect of genotype (P = 0.12) and no genotype by day interaction (P = 0.92, Figure 4B). There was a significant effect of time (P < 0.001) reflecting the rise in FSH concentrations after follicle aspiration and the subsequent decline during follicle growth. Comparison between genotypes for each time point were not different (P > 0.05). Peak FSH concentrations were not different (P = 0.54) between Trio carriers and noncarriers cows (0.67 ± 0.06 and 0.61 ± 0.08 ng/ml, respectively). Predeviation FSH concentrations were not different (P = 0.32) between Trio carriers and noncarrier cows (0.30 ± 0.05 and 0.24 ± 0.04 ng/ml, respectively). Similarly, postdeviation FSH were not different (P = 0.39) between genotypes (0.23 ± 0.07 and 0.19 ± 0.06 ng/ml, for Trio carriers and noncarrier cows, respectively). However, there was a tendency (P = 0.06) for greater FSH concentrations at deviation in Trio carriers (0.27 ± 0.03 ng/ml) compared to noncarrier cows (0.19 ± 0.03 ng/ml). In order to further evaluate potential differences in circulating FSH due to genotype, data were combined from heifers and cows (Figure 4C). There was a main effect of genotype (P = 0.01), day (P < 0.001), but no genotype by day interaction (P = 0.93). There was a main effect of parity (P = 0.006) but no genotype by parity interaction (P = 0.57). Trio carriers tended (P < 0.09) to have greater FSH concentrations on days 2.5, 3.5, and 4.5. Mean circulating FSH concentrations were also normalized to follicular deviation and grouped into three distinct periods (data not shown). Predeviation and postdeviation mean FSH concentrations were not different between genotypes (P > 0.10). However, circulating FSH concentrations encompassing deviation were greater (P = 0.01) in Trio carriers (0.28 ± 0.01 ng/ml) than noncarriers (0.21 ± 0.02 ng/ml). Circulating LH Analysis of circulating LH concentrations in heifers (data not shown) showed no main effect of genotype (P = 0.12), day (P = 0.81), and no genotype by day interaction (P = 0.83). Predeviation LH concentrations were not different (P = 0.11) between Trio carriers and noncarrier heifers (0.84 ± 0.07 and 0.70 ± 0.03 ng/ml, respectively). Similarly, postdeviation LH was not different (P = 0.71) between genotypes (0.78 ± 0.08 and 0.82 ± 0.04 ng/ml, for carrier and noncarrier heifers). Circulating LH at deviation was also not different between Trio carriers (0.82 ± 0.04 ng/ml) and noncarrier heifers (0.76 ± 0.04 ng/ml, P = 0.35). Circulating LH concentrations in cows showed a main effect of genotype (P = 0.05) but no effect of day (P = 0.52), and no genotype by day interaction (P = 0.99). Comparisons between genotypes for each time point were not different (P > 0.05). Predeviation LH concentrations were not different (P = 0.39) between Trio carriers and noncarrier cows (0.97 ± 0.07 and 0.89 ± 0.07 ng/ml). Similarly, postdeviation LH was not different (P = 0.50) between genotypes (1.00 ± 0.09 and 0.92 ± 0.08 ng/ml, for carrier and noncarrier cows). Circulating LH at deviation was also not different between Trio carriers (0.96 ± 0.05 ng/ml) and noncarrier cows (0.88 ± 0.05 ng/ml, P = 0.29). In order to further evaluate potential differences in circulating LH, data from heifers and cows were combined for each genotype. Mean LH, normalized to follicle deviation, and grouped into three distinct periods, was analyzed (data not shown). Mean LH concentrations were not different (P > 0.10) at any of the times that were evaluated including during predeviation, near deviation, and postdeviation. Circulating P4 Serum P4 concentrations from D0 (insertion of the CIDR) until D7 are shown in Supplemental Figure S2. Concentrations of P4 in heifers, during the time the CIDR was in place, showed no main effect of genotype (P = 0.84), day (P = 0.13), and no genotype by day interaction (P = 0.80). Analysis of P4 in cows indicated no effect of genotype (P = 0.18), day (P = 0.09), and no genotype by day interaction (P = 0.53). Ovulation and preovulatory follicle characteristics Interval from CIDR removal to estrus was not different (P = 0.23) between heifers that were Trio carriers (43.5 ± 2 h) or noncarriers (48 ± 0 h). Interval from estrus to ovulation was also not different (P = 0.11) between genotypes in heifers (30 ± 2 h vs 36 ± 0 h, for carriers and noncarriers). The interval from CIDR removal to ovulation was shorter (P = 0.005) in heifers that were Trio carriers (73.5 ± 1 h) compared to noncarriers (84 ± 0 h). Trio carrier heifers had 3.8-fold greater (P = 0.0003) number of ovulations than noncarrier heifers (Table 3). Analysis of preovulatory follicle dimensions indicated a greater (P < 0.01) size of the largest, smallest, and mean dominant preovulatory follicle, both when expressed on a diameter basis or on a volume basis, in noncarrier heifers as compared to Trio carrier heifers (Table 3). Mean dominant preovulatory follicle volume was 4.4-fold greater (P < 0.0001) in noncarrier heifers than in Trio carriers. However, total preovulatory follicle volume was not different between genotypes (P = 0.33). Table 3. Preovulatory follicle diameter and volume, and number of ovulations in heifers and cows that are Trio carriers or noncarrier controls.   Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Number of ovulations  3.8 ± 0.3  1.0 ± 0.0  0.0003  4.6 ± 0.5  2.0 ± 0.0  0.016  Follicle diameter (mm)  Largest preovulatory follicle  9.1 ± 0.3  13.5 ± 0.9  0.0004  10.0 ± 0.4  13.8 ± 0.6  0.002  Smallest preovulatory follicle  7.4 ± 0.3  13.5 ± 0.9  < 0.0001  9.1 ± 0.5  12.4 ± 0.3  0.005  Mean ovulatory follicle  8.2 ± 0.3  13.5 ± 0.9  < 0.0001  9.6 ± 0.5  13.1 ± 0.4  0.002  Follicle volume (mm3)  Largest preovulatory follicle  410.7 ± 51  1358.4 ± 296  0.0003  539.1 ± 59  1394.2 ± 187  0.002  Smallest preovulatory follicle  219.7 ± 35  1358.4 ± 296  < 0.0001  413.4 ± 67  993.2 ± 67  0.001  Mean ovulatory follicle  310.2 ± 43  1358.4 ± 296  < 0.0001  479.0 ± 64  1193.7 ± 106  0.0008  Total preovulatory follicle  1084.1 ± 114  1358.4 ± 296  0.33  2095.5 ± 197  2387.4 ± 212  0.37    Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Number of ovulations  3.8 ± 0.3  1.0 ± 0.0  0.0003  4.6 ± 0.5  2.0 ± 0.0  0.016  Follicle diameter (mm)  Largest preovulatory follicle  9.1 ± 0.3  13.5 ± 0.9  0.0004  10.0 ± 0.4  13.8 ± 0.6  0.002  Smallest preovulatory follicle  7.4 ± 0.3  13.5 ± 0.9  < 0.0001  9.1 ± 0.5  12.4 ± 0.3  0.005  Mean ovulatory follicle  8.2 ± 0.3  13.5 ± 0.9  < 0.0001  9.6 ± 0.5  13.1 ± 0.4  0.002  Follicle volume (mm3)  Largest preovulatory follicle  410.7 ± 51  1358.4 ± 296  0.0003  539.1 ± 59  1394.2 ± 187  0.002  Smallest preovulatory follicle  219.7 ± 35  1358.4 ± 296  < 0.0001  413.4 ± 67  993.2 ± 67  0.001  Mean ovulatory follicle  310.2 ± 43  1358.4 ± 296  < 0.0001  479.0 ± 64  1193.7 ± 106  0.0008  Total preovulatory follicle  1084.1 ± 114  1358.4 ± 296  0.33  2095.5 ± 197  2387.4 ± 212  0.37  Data are presented as mean (±SEM). View Large In cows, interval from CIDR removal to estrus was not different (P = 0.46) between Trio carriers (34.4 ± 2 h) and noncarriers (44.0 ± 4 h). Interval from estrus to ovulation was also not different (P = 0.99) between genotypes (31.2 ± 3 h vs 28.0 ± 4 h, for Trio carrier and noncarrier cows). The interval from CIDR removal to ovulation was not different (P = 0.99) in Trio carrier (69.6 ± 2 h) compared to noncarrier (72.0 ± 0 h) cows. Trio carrier cows had a 2.3-fold greater (P = 0.02) number of ovulations than noncarrier cows (Table 3). Comparison of preovulatory follicle dimensions indicated a greater (P < 0.01) size of the largest, smallest, and mean dominant preovulatory follicle, expressed on either a diameter or volume basis, in noncarrier cows as compared to Trio carrier cows (Table 3). Mean dominant preovulatory follicle volume was 2.5-fold greater (P < 0.001) in noncarrier cows. However, total preovulatory follicle volume was not different between genotypes (P = 0.37). Experiment 2 One animal in the noncarrier group was removed from the analysis due to being the only animal with double ovulation, all other noncarriers had single ovulations. Trio carrier animals had greater (P = 0.006) number of ovulations and CL (4.3 ± 0.7) than noncarrier controls (1.0 ± 0.0). Analysis of individual mean CL volume indicated a main effect of genotype (P < 0.0001), day (P < 0.0001), but no genotype by day interaction (P = 0.17). Noncarrier animals had, on average, a 4.7-fold greater individual CL volume than Trio carriers between 6 and 10 days after estrus (Figure 5A). Total CL volume showed a main effect of day (P < 0.0001), but no effect of genotype (P = 0.60), and no genotype by day interaction (P = 0.73, Figure 5B). Circulating P4 concentrations showed a main effect of day (P < 0.0001), and a genotype by day interaction (P = 0.01), but no effect of genotype (P = 0.82, Figure 5C). Comparison of P4 concentrations between genotypes for each time point were not different (P > 0.05) with the exception of a greater P4 concentration in Trio carriers on day 3 (P = 0.02). Figure 5. View largeDownload slide Mean (±SEM) individual (A) and total (B) CL volume and serum P4 (C) normalized to the day of estrus (experiment 2) for Trio carriers (n = 7) and age-matched, half-sib noncarrier controls (n = 4). *Indicates significant differences between genotypes at a specific time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Figure 5. View largeDownload slide Mean (±SEM) individual (A) and total (B) CL volume and serum P4 (C) normalized to the day of estrus (experiment 2) for Trio carriers (n = 7) and age-matched, half-sib noncarrier controls (n = 4). *Indicates significant differences between genotypes at a specific time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Experiment 3 The interval between follicle ablation and the time of follicular fluid collection tended (P = 0.09) to be greater in carriers (2.7 ± 0.1 days) than in noncarriers (2.1 ± 0.2 days) reflecting the slower growth rate and greater time needed for the largest follicle to reach 7 mm. Follicle diameters of F1, F2, and F3 were not significantly different between genotypes (Figure 6A). Within each genotype, all three follicles were different in noncarrier animals, while in carriers the F1 was larger than F2 and F3 but no difference was found between the F2 and F3 (Figure 6A). Analysis within genotype indicated that follicular fluid estradiol concentrations were elevated in all three dominant follicles in carriers and there was no difference between F1, F2, and F3 in carriers (Figure 6B). Intrafollicular concentrations of estradiol were greater in carriers than in noncarriers in the F1, F2, and F3 (Figure 6B). In contrast, noncarriers had follicular fluid estradiol concentrations that were lower in the F2 and F3 than in F1 (Figure 6B). Intrafollicular P4 concentrations were different between genotypes only for F1, with carriers having greater concentrations of P4 than noncarriers (Figure 6C). Analysis within each genotype indicated no differences between F1, F2, and F3 for carriers or noncarriers (Figure 6C). Figure 6. View largeDownload slide Follicle diameter (A), intrafollicular estradiol (B), and intrafollicular P4 (C) concentrations in the three largest follicles of cattle carrying the Trio allele and age-matched, half-sib controls. Number in parenthesis at the base of each column indicate the number of follicles included. a,bIndicates significant differences between groups (P < 0.05). Figure 6. View largeDownload slide Follicle diameter (A), intrafollicular estradiol (B), and intrafollicular P4 (C) concentrations in the three largest follicles of cattle carrying the Trio allele and age-matched, half-sib controls. Number in parenthesis at the base of each column indicate the number of follicles included. a,bIndicates significant differences between groups (P < 0.05). Discussion This study constitutes the first characterization of the follicle growth profile, follicle volume relationships, and associated circulating gonadotropins in carriers of the high-fecundity bovine allele, Trio. The ovulation rate and associated number of dominant follicles of Trio carrier heifers and cows were significantly greater than in their noncarrier counterparts. Follicular and hormonal dynamics could be precisely defined by first temporally synchronizing the start of a follicular wave using ultrasound-guided follicular aspiration and second using detailed twice daily ultrasound evaluations and blood sampling to compare carriers of the Trio allele to age-matched, half-sib noncarriers. Accurate definition of the phenotype of a given genotype is important for proper selection of a valid control group. In this case, we incorporated the Trio allele into a crossbred beef cattle population and then directly compared half-sibling sisters that were heterozygous carriers to genetically matched and age-matched noncarriers of the Trio allele. The most distinctive feature of follicular dynamics in carriers of the Trio allele was the smaller size of the dominant follicles and the reduced growth rate during all stages of follicular development. This is clearly consistent with the elegant studies in high-fecundity ewes, in which presumptive ovulatory follicles were smaller, corresponding to fewer granulosa cells per follicle in carrier ewes [40,41]. One of the key insights in our studies was the calculation of follicular dynamics and follicular growth in terms of the volume of a sphere rather than only as a diameter. Calculation of ultrasound results on a volume basis demonstrated the impressive magnitude of the difference in follicle size between carriers and noncarriers (∼25% of the follicular volume) but also revealed that total follicular volume and total luteal volume were not different when volume of all dominant follicles or CL was combined. This corresponds to observations in the ovine high-fecundity genotypes in which total number of granulosa cells is similar to wild types, when all presumptive ovulatory follicles are added together and that, correspondingly, total luteal tissue was similar between carriers and noncarriers [40,42]. Another key insight was that, despite a reduced growth rate in Trio carriers, the time of diameter deviation between the largest follicle (F1) and the first subordinate follicle (SF1) occurred at a similar time as that observed in noncarrier controls, although at a much smaller follicle size. This corresponds to the idea that follicles in high-fecundity ewes acquire LH receptors and become dominant at an “earlier” time [5]. However, our study more accurately describes that this selection process occurs in the same temporal sequence but at a much smaller size due to the reduced growth rate of each dominant follicle in carriers of the Trio allele. Finally, FSH concentrations were, on average, greater (P = 0.01 with combined FSH data in Figure 4) in Trio carriers, but more importantly analysis of FSH in relation to key stages during follicle development revealed a subtle but significantly greater FSH concentration (Figure 5) in Trio carriers during the 24 h encompassing deviation. These exceptional results allow us to add depth and insight into current models of follicle growth, follicle selection, and, in particular, the dynamic processes that occur during selection of multiple dominant follicles in Trio, and perhaps other, high-fecundity animal models. Recently, Juengel et al [5] proposed an intriguing model for how mutations in TGFB family members result in a high ovulation rate. They proposed that ewes that are heterozygous for mutations in either GDF9 or BMP15 or their receptors have reduced granulosa cell proliferation but an earlier acquisition of LH receptivity in the granulosa cells in follicles of smaller size. The recently discovered high-fecundity bovine genotype Trio [27] has been clearly linked to overexpression of SMAD6 in granulosa cells of heterozygous Trio carriers [28]. This result clearly places this bovine high-fecundity genotype within a similar functional category as the high-fecundity ovine mutations that affect the oocyte-derived TGFB family members, BMP15 and GDF9, and their intracellular signal transduction pathways [5]. Our first hypothesis when designing this study was that carriers of the high-fecundity allele would ovulate smaller-sized follicles compared to noncarriers, although the total ovulatory follicular volume would be similar. This hypothesis was supported by the observations that Trio carriers ovulate multiple follicles of smaller size, but the total ovulatory follicle volume was similar in carriers and noncarriers. Mean dominant follicles in Trio carriers were smaller in diameter from first detection at 12 h after the last follicle aspiration, through deviation and until ovulation. The finding of smaller follicle size in carriers of the Trio allele is in agreement with previous results for ewes that were carriers of high-fecundity alleles as determined by terminal studies or by ultrasound monitoring [40,41,43]. The USDA-MARC twinner cattle population has also been shown to have smaller preovulatory follicles as ovulation rate increases, although diameters appear to be larger than those observed in this study in Trio carriers [44]. The most revealing information, however, has resulted from the consideration of follicle size from a volume perspective, where individual follicle volume in Trio carriers was significantly smaller but total dominant follicle volume, calculated as the sum of each individual dominant follicle, was similar to the volume observed in noncarrier controls. Traditionally, follicle growth has been viewed from a two-dimensional perspective and this has served well for most of the research related to follicle selection and it is extremely easy to understand since follicles are viewed in two dimensions on the ultrasound screen. However, as obvious as it may seem, the follicle is a three-dimensional structure and thus the use of the volume of a sphere may better represent the relationship between different follicle sizes and the components of the follicle such as granulosa cell numbers. The smaller individual follicle but similar total follicle volume observed in Trio carrier heifers emphasize the relationship between follicle size and ovulation rate, as Trio carriers had ∼4-fold greater number of ovulations than controls but their preovulatory follicles were individually ∼4-fold smaller in volume than in controls; thus, the total preovulatory follicle volume was not different. Assuming that the follicle volume is related to the number of granulosa cells, then total hormonal output would be similar whether it was coming from one large follicle or four smaller follicles [40–42]. The smaller size of ovulatory follicles was due to a slower growth rate, ∼65% slower follicle growth rate in carriers compared to single-ovulating, noncarrier heifers. These results support our second hypothesis that follicles in the carrier animals would grow at a slower rate than follicles of noncarriers. Previous research demonstrated reduced proliferation of granulosa cells from Booroola FecB carrier ewes, as measured by [3H] thymidine uptake under basal conditions, as compared to granulosa cells from wild-type ewes [45,46] and this is likely to be the case in carriers of Trio. These ideas are consistent with a model in which a reduction in granulosa cell proliferation underlies the smaller follicle sizes that have been reported in previous results with high-fecundity ewes [40–42,47] and the reduced follicle growth rate observed in our study. Our third hypothesis was that concentrations of FSH would be greater in carriers than in noncarriers, particularly near the time of follicular deviation. Circulating FSH concentrations have a key regulatory role in follicle growth as demonstrated by the clear FSH surge associated with the emergence of each follicular wave and the subsequent decrease in FSH concentrations associated with diameter deviation, as proposed for two way coupling between follicles and FSH [48–50]. The use of follicle aspiration in this study allowed for the synchronization of the follicular wave at a self-appointed time, and both Trio carriers and noncarrier controls had similar FSH surges in response to follicle aspiration with similar peak FSH concentrations as observed in previous reports [32]. The evidence for a relationship between circulating FSH, follicle selection, and ovulation rate has been provided by several studies [49,51–53]. For example, the administration of low doses of exogenous FSH before diameter deviation prolonged the occurrence of deviation and stimulated the growth of the presumptive future SFs [51]. Moreover, the administration of exogenous FSH preparations has become the basis for superstimulation protocols used for superovulation and multiple embryo production [54]. The natural occurrence of multiple ovulations in cattle has also been associated with changes in FSH, as shown by the greater FSH concentrations immediately before deviation in lactating dairy cows with multiple codominant follicles [52]. Conversely, circulating concentrations of FSH were found to be similar between USDA-MARC twinner cattle and controls, although in this case FSH patterns were not normalized in relation to wave emergence or deviation [55,56]. In this study, circulating FSH was, on average, greater in Trio carriers; however, detailed analysis performed in relation to observed deviation indicated that FSH was greater only in the 24 h encompassing deviation. The finding of this subtle but significant difference in FSH encompassing deviation is in agreement with one of the proposed models for the mechanisms underlying multiple ovulation proposed originally by Baird in 1987 [57] and recently reviewed and updated by Scaramuzzi et al [58]. It is also consistent with the two-way FSH-follicle growth coupling hypothesis proposed by Ginther et al [50]. Based on these models, Trio carriers in this study exhibited greater FSH concentrations during the time encompassing deviation, which is consistent with the idea of widening the gate to allow more follicles to achieve dominance. Thus, the primary mechanism that allows selection of multiple dominant follicles in carriers of the Trio allele may be that FSH is not completely suppressed until multiple follicles undergo deviation and provide the final suppression of FSH that determines the number of “selected” follicles. The role for greater FSH near deviation in selection of multiple follicles is physiologically reasonable, based on our current understanding of follicle selection, and is clearly supported by association between increased FSH near deviation and selection of multiple dominant follicles. There has been an ongoing controversy about the role for circulating FSH in increasing multiple ovulations in high-fecundity ovine genotypes. It seems possible that the role for greater FSH near deviation in selection of multiple follicles may differ in different high-fecundity genotypes. It also seems possible that changes in FSH may be subtle making it difficult to detect without precise normalization of follicular dynamics, particularly normalization of FSH to the precise time of follicle deviation in each individual animal. Our fourth and final hypothesis was that follicular deviation would occur at smaller follicle sizes in carrier animals than in noncarriers. This hypothesis was also clearly supported by our ultrasound evaluations. Of particular importance, diameter deviation in Trio carriers was observed at a similar time as observed in noncarriers and despite smaller-sized individual follicles, the total volume of all future dominant follicles was not different between genotypes at the time of deviation. The measurements of intrafollicular estradiol concentrations in experiment 3 were planned to provide information on the acquisition of dominance in follicles of Trio carriers compared to noncarriers. The use of estradiol concentrations is based on the premise that the rise in estradiol concentrations within the follicular fluid is probably one of the most consistent findings in relation to the acquisition of dominance [59–63]. The decision to collect follicular fluid from follicles when the largest follicle reached 7 mm was based on the idea that follicles in noncarriers would not yet have acquired dominance by that size [59,60]. In agreement with our ultrasound results demonstrating follicular deviation prior to 7 mm in Trio carriers, estradiol concentrations were greatly elevated in all three follicles from Trio carriers, consistent with a dominant phenotype [60], and much greater than estradiol concentrations in any of the three follicles of noncarriers. These findings, coupled with the observation that deviation occurs when the largest follicle is ∼6 mm in diameter, provide support for our hypothesis that follicles of Trio carriers acquire dominance at a smaller size than observed in noncarriers. In conclusion, Trio carriers had smaller-sized follicles, which developed at a reduced growth rate during the follicular wave, and underwent diameter deviation at a similar time but at smaller size than observed in half-sibling noncarriers. The relationship between the number of ovulations and the individual follicle volume is such that, when taken together, the total preovulatory follicle volume and subsequent total luteal tissue volume are similar to single ovulating controls, and as a result circulating P4 is not different. The smaller follicle size at which follicles acquire dominance appears to be associated with greater circulating FSH concentrations for a short time period near deviation that could allow for more follicles to acquire dominance and this may be a key part of the mechanisms that produce multiple ovulations in carriers of the Trio allele. Supplementary data Supplementary data are available at BIOLRE online. Supplemental Figure S1. Growth profile of the largest follicle (F1) diameter in heifers (left) and cows (right) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration until the removal of the intravaginal P4 implant. Each point represents mean (±SEM). *Indicates significant differences between genotype for a given time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Supplemental Figure S2. Circulating P4 in heifers (A) and cows (B) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared during the days in which the CIDR was in place and each point represents mean (±SEM). In heifers, genotype P = 0.84; day P = 0.13; genotype by day P = 0.80. In cows, genotype P = 0.18; day P = 0.10; genotype by day P = 0.53. Acknowledgments Special thanks to A. Sallam, R.V. Barletta, M. Z. Toledo, C. Gamarra, E. Trevisol, P. L. Monteiro, J. Levandowski and E. Walleser for technical assistance; and the staff of Arlington Research Station Beef Grazing unit and the Lancaster Agricultural Research Station for animal handling and production. A patent (20150007358) assigned to the Wisconsin Alumni Research Foundation has been awarded to BWK related to determining the Trio haplotype. Footnotes † Grant support: Funding was provided by WI Experiment Station as Hatch Project WIS01240 to MCW and as Hatch Project WIS01648 and WIS01932 to BWK. References 1. Spearow JL. Major genes control hormone-induced ovulation rate in mice. Reproduction  1988; 82: 181– 186. 2. Rocha JL, Eisen EJ, Siewerdt F, Van Vleck LD, Pomp D. A large-sample QTL study in mice: III. Reproduction. Mamm Genome  2004; 15: 878– 886. Google Scholar CrossRef Search ADS PubMed  3. Sugiura K, Su Y-Q, Eppig JJ. Does Bone Morphogenetic Protein 6 (BMP6) affect female fertility in the mouse? Biol Reprod  2010; 83: 997– 1004. Google Scholar CrossRef Search ADS PubMed  4. 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Trio a novel bovine high-fecundity allele: II. Hormonal profile and follicular dynamics underlying the high ovulation rate

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Abstract

Abstract The newly discovered Trio high-fecundity allele produces multiple ovulations in cattle. This study evaluated (1) size and growth rates of follicles in Trio carriers during a synchronized follicular wave, induced by follicle aspiration; (2) follicle-stimulating hormone (FSH) patterns associated with the follicular wave; (3) size of corpora lutea (CL) and circulating progesterone; and (4) intrafollicular estradiol concentrations prior to normal deviation. Trio carriers had mean dominant follicles that were significantly smaller in diameter and volume than noncarriers. Onset of diameter deviation occurred at ∼3 days after the last follicle aspiration in both genotypes despite Trio carriers having much smaller individual follicles. Follicles of Trio carriers grew at a slower rate than noncarrier follicles (∼65% in mm/day or ∼30% in mm3/day) resulting in much smaller individual dominant follicles (∼25% volume). However, total dominant follicle volume, calculated as the sum of all dominant follicles in each animal, was similar in carriers and noncarriers of Trio throughout the entire follicular wave. Circulating FSH was greater in Trio carriers during the 24 h encompassing deviation. Trio carriers had significantly more ovulations than noncarriers, and individual CL volume was smaller, although total luteal tissue volume and circulating P4 were not different. Thus, increased ovulation rate in Trio carriers relates to smaller individual follicles (one-third the volume) near the time of deviation due to slower follicle growth rate, although time of deviation is similar, with increased circulating FSH near deviation leading to selection of multiple dominant follicles in Trio carriers with similar total follicle volume. Introduction The mechanisms underlying the determination of litter size, both in polytocous and monotocous species, have been the subject of extensive research. One of the major determinants of litter size is ovulation rate, which generally determines the upper limit on the number of offspring. Ovulation rate is, in turn, determined by the number of follicles that are selected for dominance and thereby have the capacity to ovulate at the time of the luteinizing hormone (LH) surge. In polytocous species, such as mice and swine, there is substantial variation in ovulation rate, with multiple loci explaining much of the variation in ovulation rate [1–3]. For example, in swine most of the variation in ovulation rate (71.1%) could be accounted for by variation in 22 quantitative trait loci (QTL) that were present on various chromosomes and included candidate genes such as estrogen receptor 1 (ESR1), growth differentiation factor 9 (GDF9), bone morphogenetic protein 7 (BMP7), bone morphogenetic protein 4 (BMP4), SMA, and SMAD family member 2 (SMAD2, previously known as MAD, mothers against decapentaplegic homolog 2), cAMP responsive element binding protein 1 (CREB1), and nhibin beta A subunit (INHBA) [4]. Nevertheless, the specific links between the genetic variation and the physiological and molecular alterations that lead to increased ovulation rate have not been completely defined. In monotocous species or species with a low ovulation rate, such as sheep, there is substantial variation in ovulation rate due to environmental and genotypic effects with clear linkages between increased ovulation rate and specific genetic variations [5,6]. Single gene mutations such as Booroola/FecB [7], Inverdale/FecXI [8], Hannah/FecXH [9], and FecGH [10] led to the discovery of the essential role of members of the transforming growth factor beta (TGFB) superfamily in folliculogenesis and ovulation rate. Two members of the TGFB superfamily, bone morphogenetic protein 15 (BMP15) and GDF9, are secreted by the oocyte and regulate proliferation and other aspects of granulosa cell function [5,11]. Eight different mutations in the BMP15 gene [10,12–15] and four different mutations in the GDF9 gene [10,16–18] have been identified in sheep. These mutations produce increased ovulation rate (35–100%) in heterozygous carriers and sometimes premature ovarian failure in homozygous carriers with failure of normal folliculogenesis past the primary stage [5]. Interestingly, homozygous carriers of certain fecundity alleles had a further increase in ovulation rate and no premature ovarian failure [19], apparently due to the mutations causing attenuation of BMP15/GDF9 activity and not loss of function [5,11]. Another mutation, termed Booroola fecundity, FecB, was found to reside in the kinase region of the bone morphogenetic protein receptor type 1B (BMPR1B), and produced increased ovulation rate in heterozygous carriers with further increases in ovulation rate in homozygotes [20]. Furthermore, another X-linked mutation (FecX2W) increased ovulation rate in both heterozygotes and homozygotes, although the causative mutation has not been identified [21]. Additive increases in ovulation rate are produced when heterozygous carriers for mutations in BMP15, BMPR1B, and the FecX2W mutation are combined, producing ovulation rates over 10 in heterozygous carriers of all 3 mutations, compared to 1-2 ovulations in the noncarrier control ewes [22]. In monovular species, such as cattle and humans, QTLs for high ovulation rate have been identified [23]. In addition, long-term selection of cattle for high ovulation rate and twinning has led to the formation of the USDA-MARC twinner herd, which exhibits a much greater frequency of multiple ovulations than wild-type control cattle [24]. Genetic analysis of this population indicated that the increased ovulation rate was related to multiple genes rather than a single gene mutation [25]. Recently, a major gene for high ovulation rate in cattle has been identified [26] with clear evidence for segregation in a 1.2-Mb region of bovine chromosome 10 [27]. The resulting high-fecundity allele, termed “Trio,” appears to increase ovulation rate through a novel mechanism that involves upregulation of MAD family member 6 (SMAD6, previously known as MAD, mothers against decapentaplegic homolog 6) [28]. Carriers of the Trio allele offer a unique opportunity to evaluate the dynamics and interactions of follicular growth and circulating follicle-stimulating hormone (FSH) in a high ovulation rate genotype of a monovular species. The bovine model has been a frequently utilized model for study of the mechanisms involved in follicular selection [29,30]. Growth of follicular waves and morphological selection of a single dominant follicle, termed follicular deviation, can be clearly distinguished by current resolution of ultrasound technology available for cattle. In addition, the similarities of cattle and humans in morphological follicular growth patterns and follicle size at deviation make cattle a particularly interesting biological model for human follicular physiology (reviewed in [31]). This study was designed to evaluate the hormonal profile and associated follicle growth dynamics in cattle that are heterozygous carriers and half-sibling noncarriers of the novel Trio allele that produces increased ovulation rate. Based on our preliminary observations and the previous model proposed for ovine high-fecundity genotypes [5], it was hypothesized that (1) carriers of the high-fecundity allele would ovulate smaller-sized follicles compared to noncarriers, although the total ovulatory follicular volume would be similar; (2) follicles in the carrier animals would grow at a slower rate than in noncarriers; (3) concentrations of FSH would be greater in carriers than in noncarriers, particularly near the time of follicular deviation; and (4) follicular deviation would occur at smaller follicle sizes in carrier animals than in noncarriers. Materials and methods All animal procedures were approved by the Animal Care and Use Committee of the College of Agriculture and Life Sciences at the University of Wisconsin–Madison. All animals used in this study were kept in outdoor paddocks with free access to a run-in shelter and water. Cattle were fed mixed hay ad libitum and had access to a standard mineral mix. Females used in the following studies were produced by mating (through artificial insemination) bulls which were carriers of the Trio allele with cows that in most cases were of predominantly Angus breeding (noncarriers). In some cases, project females were the product of superovulation and embryo transfer with both parents being Trio allele carriers. Breed composition of the Trio allele carrier parents was >50% Angus with the remainder of the breed composition being a mix of Hereford, Holstein, and Jersey. Trio allele genotype was determined by analysis of haplotypes based on three linked markers as described previously [27]. All females were thus either half or full sibs, and Trio carriers were heterozygous for the Trio allele. Experiment 1 Experiment 1 evaluated the follicle dynamics and circulating FSH of cattle carrying a high-fecundity allele (Trio) under a controlled progesterone (P4) environment. Nulliparous and multiparous nonlactating crossbred cattle, ranging in age from 18 to 57 months, carrying the high-fecundity allele (n = 13) or age-matched half-sib controls (n = 11) had their estrous cycle synchronized (Figure 1). All animals received a P4 intravaginal controlled internal drug-releasing device (CIDR, Eazi-Breed, Zoetis, Florham Park, NJ) on D−9 and this was removed 6 days later (D−3). Prostaglandin F2α (PGF, Estroplan, Parnell, Overland Park, KS) was administered twice 24 h apart on D−3 and D−2. In order to synchronize the emergence of a new follicular wave, animals had ultrasound-guided follicular ablation of all follicles ≥4 mm on D−2, D−1, and D0, as previously described with minor modifications [32]. Briefly, transvaginal ultrasound-guided follicle ablation was performed with an 8 MHz microconvex array transducer attached to a B-mode ultrasound scanner using a 2″ 18G hypodermic needle. On D0, after follicle ablation was performed, a new CIDR was inserted and remained in place until D5. Ultrasound examinations of the ovaries were performed every 12 h starting on D0 until cows ovulated, using a B-mode ultrasound scanner equipped with a 7.5 MHz transducer. A sketch was made of each ovary recording follicle number, size, and relative location on the ovary. Due to the complexity of follicle wave patterns in Trio carriers (i.e. multiple dominant follicles of smaller size), follicle growth profiles were constructed based on the nonidentity method as previously described [33]. Blood collection was performed via coccygeal venipuncture into evacuated tubes every 24 h from D3 to D1 and then every 12 h from D0 until ovulation. All animals were fitted with Kamar heat mount detectors (Kamar Products Inc., Zionsville, IN) on the day of removal of the CIDR (D5) to aid in the detection of standing estrus, and detectors were checked every 12 h. Figure 1. View largeDownload slide Treatment schedules of experiment 1 for cattle carrying the high-fecundity allele Trio and age-matched, half-sib controls. A CIDR was inserted on D−9 and left in place for 6 days. Prostaglandin F2α (PGF) was administered 24 h apart on D−3 and D−2. Transvaginal ultrasound-guided follicle (≥4 mm) ablation was performed in all animals on D−2, D−1, and D0 and a new CIDR was inserted after the last follicle ablation. The CIDR was removed on D5 and animals were examined by transrectal ultrasound (US) and blood samples collected (BS) every 12 h from D0 until ovulation (D8 approximately). The treatment schedule for experiment 2 is also shown. All animals had their estrous cycle synchronized with two doses (500 μg/each) of PGF administered 24 h apart on D0 and D1 and on D3 GnRH (100 μg) was administered. On D10, GnRH (200 μg) was administered again and finally two doses of PGF were administered on D15 and D16. The following complete estrous cycle was evaluated by daily ultrasound and blood sampling for circulating progesterone concentrations. In experiment 3, animals were synchronized by 6 days of treatment (D−8 to D−2) with a CIDR and PGF (500 μg) was given on D−2 and D−1, followed by follicular aspiration on D−1 and D0. Animals were then evaluated by ultrasound every 12 h and follicular fluid collection was done when the largest follicle reach 7 mm in diameter. Figure 1. View largeDownload slide Treatment schedules of experiment 1 for cattle carrying the high-fecundity allele Trio and age-matched, half-sib controls. A CIDR was inserted on D−9 and left in place for 6 days. Prostaglandin F2α (PGF) was administered 24 h apart on D−3 and D−2. Transvaginal ultrasound-guided follicle (≥4 mm) ablation was performed in all animals on D−2, D−1, and D0 and a new CIDR was inserted after the last follicle ablation. The CIDR was removed on D5 and animals were examined by transrectal ultrasound (US) and blood samples collected (BS) every 12 h from D0 until ovulation (D8 approximately). The treatment schedule for experiment 2 is also shown. All animals had their estrous cycle synchronized with two doses (500 μg/each) of PGF administered 24 h apart on D0 and D1 and on D3 GnRH (100 μg) was administered. On D10, GnRH (200 μg) was administered again and finally two doses of PGF were administered on D15 and D16. The following complete estrous cycle was evaluated by daily ultrasound and blood sampling for circulating progesterone concentrations. In experiment 3, animals were synchronized by 6 days of treatment (D−8 to D−2) with a CIDR and PGF (500 μg) was given on D−2 and D−1, followed by follicular aspiration on D−1 and D0. Animals were then evaluated by ultrasound every 12 h and follicular fluid collection was done when the largest follicle reach 7 mm in diameter. Experiment 2 Experiment 2 evaluated circulating P4 and volume of corpora lutea (CL) in cattle that were heterozygous carriers or noncarriers of the Trio allele during an estrous cycle. Nulliparous and multiparous nonlactating crossbred cattle, ranging in age from 18 to 57 months, carrying the Trio allele (n = 7) or age-matched, half-sib controls (n = 5) were used. All animals had their estrous cycle synchronized as follows: two doses (500 μg/each) of PGF (Estroplan, Parnell, Overland Park, KS) were administered 24 h apart on D0 and D1 and on D3 GnRH (100 μg of gonadorelin acetate) was administered (Gonabreed, Parnell, Overland Park, KS). On D10, GnRH (200 μg) was administered again and finally two doses of PGF were administered on D15 and D16. A change in synchronization protocol was done in experiment 2 to increase circulating progesterone concentrations during follicle growth in order to potentially decrease the double ovulations that occurred in experiment 1. Ultrasound examinations of the ovaries was performed every 24 h starting on the day of the last PGF until D10 after standing estrus. Examinations were done using a B-mode ultrasound scanner equipped with a 7.5 MHz transducer and were recorded in a 20-s cine loop. Images were analyzed, and size and relative location of each CL were recorded in a sketch. Blood collection was performed via coccygeal venipuncture into evacuated tubes every 24 h for the complete interovulatory interval. Experiment 3 Experiment 3 was designed to evaluate the concentrations of estradiol (E2) and P4 in the follicular fluid of follicles before selection in cattle that were heterozygous carriers or noncarriers of the Trio allele. Nulliparous and multiparous nonlactating crossbred cattle, ranging in age from 2 to 4 yr, carrying the Trio allele (n = 8) or age-matched, half-sib controls (n = 5) were used. All animals received a CIDR on D8 and this was removed 6 days later (D2). Prostaglandin F2α was administered twice, 24 h apart, on D2 and D1. In order to synchronize the emergence of a new follicular wave, ultrasound-guided transvaginal follicle aspiration was performed as described in experiment 1 on D1 and D0. All animals were monitored by transrectal ultrasonography of their ovaries every 12 h beginning on D0. The follicular fluid of the three largest follicles was collected individually at the time that the largest follicle (F1) reached a diameter of at least 7 mm, as previously described [34]. Upon collection of follicular fluid, animals continued to be monitored by ultrasonography every 12 h until the largest follicle reached 7 mm, and the process of collecting follicular fluid from the three largest follicles was repeated. All animals were fitted with Kamar heat mount detectors on the day of the second PGF (D1) to aid in the detection of standing estrus. Three criteria were used to determine if data from a collection would be used in analyses. First, if the F1 follicle diameter was >8 mm (nine collections removed, five carriers and four noncarriers). Second, if animals were exhibiting estrus at the time of collection (potential exposure to LH surge; three carriers removed). Third, if the F1 follicle had nondetectable concentrations of estradiol in the follicular fluid (three carriers removed). Thus, from 26 total collections, data from only 11 collections were available for the final analysis. Hormone assays Blood samples were centrifuged at 1300 × g and 4°C for 20 min, and serum was transferred into vials, frozen, and stored at –20°C until assayed. Assay of P4 was performed using one of two solid-phase radioimmunoassay (RIA) kits (Coat-A-Count Progesterone; Siemens Healthcare Diagnostics, Los Angeles, CA or ImmuChem Progesterone Coated Tube; MP Biomedicals, Santa Ana, CA). Mean assay sensitivity, calculated as 2 SD less than the mean counts per minute of maximum binding, was 0.018 and 0.015 ng/ml for each kit, respectively. Intra- and interassay coefficients of variation were 4.7% and 5.9% for the Coat-A-Count kit and 5.0% was the intra-assay coefficient of variation for the one assay with the ImmuChem kit. Serum FSH was measured using an RIA that was previously validated for cattle [35,36]. The FSH assay incorporated the primary antibody NIDDK-anti-oFSH-I-2 and the radiolabeled and standard antigen USDA-bFSH-I-2. Mean assay sensitivity was 0.04 ng/ml, and intra- and inter-assay coefficients of variation were 2.8% and 12.9% using the high FSH quality control sample and 4.3% and 14.1% using the low FSH quality control sample. Luteinizing hormone concentrations were determined by RIA as described in [37]. Average sensitivity of the assays was 0.11 ng/mL and intra- and inter-assay coefficients of variation were 2.0% and 8.6%, respectively. Follicular fluid samples were centrifuged at 600 × g for 10 minutes to remove granulosa cells from the samples and the supernatant was stored frozen at –20°C until assayed. Intrafollicular estradiol concentrations were determined using a commercially available competitive ELISA (Estradiol Serum EIA Kit, #KB30-H1, Arbor Assays, Ann Arbor, MI). Samples were diluted in assay buffer provided by the manufacturer at 1:5000 or 1:20,000. Samples were analyzed in a single assay, and the intra-assay coefficient of variation was 7.0%. Intrafollicular concentrations of P4 were assessed using the solid-phase RIA kit described above (Coat-A-Count Progesterone). Samples were diluted 1/20 in PBS, and all samples were evaluated in one assay. Intra-assay coefficient of variation was 7.8%. Terminology and data arrangement statistical analyses Experiment 1 Dominant follicles were defined as any follicle that ovulated at the end of the evaluation period and were numbered from largest to smallest (F1, F2, etc.) based on their maximum diameter. Subordinate follicles (SFs) were designated retrospectively as one that appeared to originate from the same pool as the dominant follicles, its first detection was within 2 days of detection of the dominant follicle, increased in diameter during the time the dominant follicle was also initially increasing, failed to ovulate, and later ceased to grow while a dominant follicle continued to grow [38]. The beginning of deviation was defined as the beginning of the greatest difference in growth rate between the largest dominant follicle (F1) and the first SF at or before the largest SF attained maximal diameter [39]. The beginning of deviation was determined for each animal individually. Follicle volume was calculated assuming the shape of a sphere with the following formula: V = 4/3πr3. Mean dominant follicle dimensions (diameter and volume) was calculated as the sum of all dominant follicles for a given animal and ultrasound examination divided by the number of dominant follicles. Total dominant follicle volume was calculated as the sum of the volume for all individual dominant follicles for a given animal and time point. Follicular growth rate was estimated by calculating the diameter/volume of a given follicle for each animal at the beginning and end of a defined period of time. The difference in diameter/volume between the time points divided by the number of days between time points was used as the growth rate. Experiment 2 Individual CL volume was calculated by using the formula shown above with the radius determined from the CL diameter, assuming that the CL had the shape of a sphere. If a cavity was present, the volume of the cavity was calculated and subtracted from the total CL volume. Volume was calculated for each individual CL and time point. Mean CL volume was calculated as the sum of all CL for a given animal and examination divided by the number of CL present. Total CL volume was calculated as the sum of each CL volume for a given animal and time point. For experiment 3, follicles were numbered F1 to F3, from largest to smallest, based on their diameter at the time of follicular fluid collection. Statistical analyses The analysis of variables measured over time (e.g. follicle diameter and volume; FSH, LH, P4 concentrations) was performed by analysis of variance using the repeated option in PROC MIXED with day as the repeated variable and cow as the subject with an autoregressive covariance structure (Statistical Analysis System, SAS Institute, Version 9.4 Cary, NC, USA). Genotype, time, and genotype by time interaction were included in the model as fixed effects. Analysis of data over time was performed in relation to the last follicle aspiration and in other analyses was normalized to the day of deviation. The main effects of treatment, day, and their interactions were determined, and preplanned comparisons between genotypes for specific time points were done by least square difference. For individual comparisons between genotypes, the two-sample t-test was used for continuous outcome (e.g. follicle diameter and volume at deviation, hormone concentrations at specific time points). For comparisons in experiment 3, each collection was considered to be independent and follicle within each collection was the experimental unit. Analysis of variables in this experiment was performed by analysis of variance using the MIXED procedure including genotype, follicle hierarchy (F1, F2, or F3), and their interaction as fixed effects. The repeated statement was utilized in order to account for clustering of follicles within animal, and cow was included as a random effect. Differences between follicles and genotypes were determined by least square means. Assumptions of normality and homogeneity of variance were evaluated for all outcomes, and transformations were attempted if assumptions were not met. For data in which transformations were not successful, the Wilcoxon rank-sum test was used. Data are presented as mean (±SEM). Values were considered statistically different when P ≤ 0.05. Results Experiment 1 In preliminary analyses of follicular data, it became apparent that noncarrier cows primarily had double ovulations, whereas noncarrier heifers primarily had single ovulation. Therefore, results in experiment 1 were analyzed separately for cows (32–57 months of age) and heifers (18–25 months of age). One heifer in the noncarrier group was removed from the analysis due to double ovulation. Thus, eight carrier and six noncarrier heifers were available for further analysis. All cows in the noncarrier group had codominant follicles and double ovulations. As a result, five carrier and four noncarrier cows were available for further analysis. Two noncarriers (one heifer and one cow) had P4 concentrations that failed to decrease after CIDR removal, resulting in a delayed onset of estrus (192 and 132 h after CIDR removal, respectively). As a result, data from these animals were considered only for outcomes that evaluated the period up to CIDR removal. Follicle growth pattern by genotype and parity Follicle growth patterns for Trio carriers and noncarrier controls are shown in Figure 2 with the top panel showing noncarrier heifers with single ovulation (left) and noncarrier cows with double ovulations (right) and the bottom panel showing Trio carrier heifers with three ovulations (left) and Trio carriers cows with five to six ovulations (right). In all genotypes and parities, there was consistent growth of the F1 from the time of first detection (0.5 days) until 5 days. Further, in all genotypes and parities a clear SF could be identified. As shown in Figure 2, the SF, on average, grew from day 0.5 to 2.5 and then growth of the SF was reduced in all of the four groups. The major difference between groups was clearly the number of codominant follicles that were present. In noncarrier heifers, only a single dominant follicle was identified. In noncarrier cows, a second dominant follicle was present as the F2 continued growth during the same time period as the F1 (0.5 to 5.5 days). In carrier heifers, the F1, F2, and F3 grew during the entire evaluation period (0.5 to 5.5 days) and grew faster than the SF after day 2.5. In carrier cows, a total of at least five dominant follicles could be identified (F1 to F5 shown with F6 [present in some cows] not shown) and clear differences in growth could be observed between the SF and all of the dominant follicles after day 2.5. Figure 2. View largeDownload slide Growth profiles (mean ± SEM) of the largest (F1), second largest (F2), third largest (F3), fourth largest (F4), and fifth largest (F5) dominant follicle and first subordinate follicle (SF) in animals carrying the Trio allele or age-matched, half-sib controls (experiment 1). The upper left panel depicts noncarrier heifers (n = 6) with a single dominant follicle, while the lower left panel depicts Trio carrier heifers (n = 3) with triple dominant follicles. The upper right panel depicts noncarrier cows (n = 4) with double dominant follicles, while the lower right panel depicts Trio carrier cows (n = 3) with quintuple or sextuple dominant follicles. Figure 2. View largeDownload slide Growth profiles (mean ± SEM) of the largest (F1), second largest (F2), third largest (F3), fourth largest (F4), and fifth largest (F5) dominant follicle and first subordinate follicle (SF) in animals carrying the Trio allele or age-matched, half-sib controls (experiment 1). The upper left panel depicts noncarrier heifers (n = 6) with a single dominant follicle, while the lower left panel depicts Trio carrier heifers (n = 3) with triple dominant follicles. The upper right panel depicts noncarrier cows (n = 4) with double dominant follicles, while the lower right panel depicts Trio carrier cows (n = 3) with quintuple or sextuple dominant follicles. Comparison of follicle growth normalized to time of follicle aspiration In heifers, the diameter of F1 (Supplemental Figure S1) indicated a main effect of genotype (P < 0.0001), day (P < 0.0001), but no interaction between genotype and day (P = 0.20). Noncarrier heifers had greater (P < 0.02) F1 diameter than Trio carriers from the first detection of F1 at 12 h after the last aspiration and throughout the evaluation period (Supplemental Figure S1). Also in heifers, the mean dominant follicle diameter (Figure 3) during the 5 days after follicle aspiration showed a main effect of genotype (P < 0.0001), day (P < 0.0001), and a genotype by day interaction (P = 0.02). Noncarrier heifers had greater (P < 0.02) mean dominant follicle diameter than Trio carrier heifers beginning at first detection on D0.5 until D5 (Figure 3). Figure 3. View largeDownload slide Growth profile of mean dominant follicle diameter, mean dominant follicle volume, and total dominant follicle volume in heifers (left) and cows (right) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration until the removal of the intravaginal P4 implant. Each point represents mean (±SEM). *Indicates significant differences between genotype for a given time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Figure 3. View largeDownload slide Growth profile of mean dominant follicle diameter, mean dominant follicle volume, and total dominant follicle volume in heifers (left) and cows (right) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration until the removal of the intravaginal P4 implant. Each point represents mean (±SEM). *Indicates significant differences between genotype for a given time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. When dominant follicle growth in heifers was considered on a volume basis (Figure 3; second panel), there was a main effect of genotype (P < 0.0001), day (P < 0.0001), but no genotype by day interaction (P = 0.29). Noncarrier heifers had a greater (P < 0.01) mean dominant follicle volume than Trio carriers from first detection at 12 h after aspiration until D5. On average, noncarrier heifers had a 3.2-fold greater mean dominant follicle volume than Trio carrier heifers. In contrast, total dominant follicle volume in heifers showed a main effect of day (P < 0.0001) but no effect of genotype (P = 0.25) or genotype by day interaction (P = 0.37). Trio carrier and noncarrier heifers had similar (P > 0.05) total dominant follicle volume at all time points with the exception of D0.5 when Trio carriers had greater (P = 0.004) total follicle volume than noncarrier heifers (Figure 3). In cows, analysis of F1 diameter (Supplemental Figure S1) showed a main effect of genotype (P = 0.0005), day (P < 0.0001), but no genotype by day interaction (P = 0.63). Noncarrier cows had greater (P < 0.01) F1 diameter than Trio carriers from D1 until D5 (Supplemental Figure S1). Also in cows, mean dominant follicle diameter showed a main effect of genotype (P = 0.0002) and day (P < 0.0001) but no genotype by day interaction (P = 0.75). Noncarrier cows consistently had a greater (P < 0.01) mean dominant follicle diameter than Trio carrier cows beginning at first detection on D0.5 until the last examination on D5 (Figure 3). On a volume basis, mean dominant follicle volume in cows showed a main effect of genotype (P = 0.0002), day (P < 0.0001), but no genotype by day interaction (P = 0.76). Noncarrier cows had a greater (P < 0.01) mean dominant follicle volume than Trio carriers starting on D0.5 and continuing throughout the observation period until D5. On average, noncarrier cows had a twofold greater mean dominant follicle volume than Trio carrier cows. In contrast, analysis of total dominant follicle volume in cows showed a main effect of day (P < 0.0001) but no effect of genotype (P = 0.41) and no genotype by day interaction (P = 0.76). Trio carrier and noncarrier cows had similar (P > 0.15) total dominant follicle volume at all time points (Figure 3). Comparison of follicle growth normalized to time of follicle deviation The time of deviation (days from last aspiration) between the F1 and the first subordinate follicle in heifers (Table 1) was not different between carriers and noncarriers (3.2 vs. 3.0 d; P = 0.66). At the time of follicle deviation in heifers, the diameter of F1, the mean dominant follicle diameter, and the diameter of the SF1 were all greater (P < 0.01) in noncarrier heifers than the corresponding follicles in Trio carriers (Table 1). Analysis of follicle volume at the time of deviation indicated that noncarrier heifers had a greater (P < 0.01) F1 (+229%) and SF1 (261%) volume than Trio carrier heifers (Table 1). Mean dominant follicle volume at time of deviation was 287% larger (P < 0.0001) in noncarrier than Trio carriers, but total dominant follicle volume was not different (P = 0.22). For cows, time of deviation and follicle characteristics at deviation are also shown in Table 1. Time from last follicle aspiration until follicle deviation in cows was not different (P = 0.49) between Trio carriers and noncarriers (2.9 vs. 3.1 d). Analysis of follicle diameter at the time of deviation showed that noncarrier cows had greater (P < 0.02) F1, F2, SF1, and mean dominant follicle diameter than carriers. Analysis of follicle volume at the time of deviation also indicated that noncarrier cows had greater (P < 0.03) F1 (+213%), F2 (+171%), and SF1 (+258%) volume than Trio carrier cows. Mean dominant follicle volume at deviation in noncarrier cows was 207% larger (P = 0.0005) than in Trio carriers. Total dominant follicle volume at the time of deviation did not differ between genotypes (P = 0.41). Follicle growth rate Analysis of overall follicle growth rate from first detection (D0.5) until ovulation is shown in Table 2. Growth rate of F1 and mean dominant follicle growth rate were greater (P < 0.02) in noncarrier heifers compared to Trio carrier heifers, both on a diameter and volume basis. Mean dominant follicle growth rate on a volume basis was 4.3-fold greater (P < 0.01) in noncarrier heifers than in Trio carriers. The total dominant follicle volume growth rate was not different (P = 0.40) between Trio carrier and noncarrier heifers. Table 1. Mean (±SEM) follicle diameter and volume, at the time of deviation in heifers and cows that are Trio carriers or noncarriers.   Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  6    5  4    Days to deviation  3.2 ± 0.4  3.0 ± 0.4  0.66  2.9 ± 0.2  3.1 ± 0.2  0.49  Follicle diameter (mm)  F1  6.5 ± 0.3  8.6 ± 0.3  0.0004  7.1 ± 0.1  8.9 ± 0.3  0.011  F2  5.9 ± 0.2      6.8 ± 0.2  8.1 ± 0.3  0.006  F3a  5.8 ± 0.3      6.7 ± 0.4      First subordinate  5.3 ± 0.1  7.2 ± 0.4  0.0001  5.6 ± 0.1  7.6 ± 0.5  0.016  Mean dominant follicle  6.0 ± 0.2  8.6 ± 0.3  <0.0001  6.6 ± 0.1  8.5 ± 0.3  0.0006  Follicle volume (mm3)  F1  149.3 ± 20  340.2 ± 35  0.0003  186.2 ± 8  369.6 ± 43  0.022  F2  116.3 ± 13      163.6 ± 16  280.5 ± 29  0.007  F3a  109.5 ± 15      162.5 ± 30      First subordinate  79.2 ± 6  207.1 ± 32  0.009  92.7 ± 7  239.2 ± 43  0.016  Mean dominant  118.5 ± 14  340.2 ± 35  <0.0001  157.0 ± 10  325.1 ± 34  0.0005  Total dominant  432.0 ± 56  340.2 ± 35  0.22  719.3 ± 78  650.1 ± 68  0.41    Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  6    5  4    Days to deviation  3.2 ± 0.4  3.0 ± 0.4  0.66  2.9 ± 0.2  3.1 ± 0.2  0.49  Follicle diameter (mm)  F1  6.5 ± 0.3  8.6 ± 0.3  0.0004  7.1 ± 0.1  8.9 ± 0.3  0.011  F2  5.9 ± 0.2      6.8 ± 0.2  8.1 ± 0.3  0.006  F3a  5.8 ± 0.3      6.7 ± 0.4      First subordinate  5.3 ± 0.1  7.2 ± 0.4  0.0001  5.6 ± 0.1  7.6 ± 0.5  0.016  Mean dominant follicle  6.0 ± 0.2  8.6 ± 0.3  <0.0001  6.6 ± 0.1  8.5 ± 0.3  0.0006  Follicle volume (mm3)  F1  149.3 ± 20  340.2 ± 35  0.0003  186.2 ± 8  369.6 ± 43  0.022  F2  116.3 ± 13      163.6 ± 16  280.5 ± 29  0.007  F3a  109.5 ± 15      162.5 ± 30      First subordinate  79.2 ± 6  207.1 ± 32  0.009  92.7 ± 7  239.2 ± 43  0.016  Mean dominant  118.5 ± 14  340.2 ± 35  <0.0001  157.0 ± 10  325.1 ± 34  0.0005  Total dominant  432.0 ± 56  340.2 ± 35  0.22  719.3 ± 78  650.1 ± 68  0.41  aFor carrier animals, dimensions for up to the third largest dominant follicle are shown as all animals had at least three dominant follicles. View Large Table 2. Mean (±SEM) follicle growth rates in heifers and cows that are Trio carriers or noncarrier controls.   Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Overall growth rate (mm/day)  F1  0.82 ± 0.1  1.22 ± 0.2  0.017  0.93 ± 0.1  1.39 ± 0.1  0.018  F2  0.66 ± 0.1      0.96 ± 0.1  1.20 ± 0.1  0.029  F3a  0.63 ± 0.1      0.94 ± 0.1      Mean dominant follicle  0.70 ± 0.1  1.22 ± 0.2  0.003  0.93 ± 0.1  1.30 ± 0.1  0.013  Overall volume growth rate (mm3/day)  F1  54.9 ± 7  175.2 ± 40  0.0006  73.6 ± 10  191.7 ± 30  0.006  F2  40.0 ± 6      71.9 ± 7  134.9 ± 11  0.003  F3a  32.2 ± 5      65.1 ± 8      Mean dominant follicle  40.9 ± 6  175.2 ± 40  0.0001  67.0 ± 9  163.3 ± 18  0.002  Total dominant follicle  142.7 ± 17  175.2 ± 40  0.404  293.7 ± 32  326.6 ± 37  0.54    Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Overall growth rate (mm/day)  F1  0.82 ± 0.1  1.22 ± 0.2  0.017  0.93 ± 0.1  1.39 ± 0.1  0.018  F2  0.66 ± 0.1      0.96 ± 0.1  1.20 ± 0.1  0.029  F3a  0.63 ± 0.1      0.94 ± 0.1      Mean dominant follicle  0.70 ± 0.1  1.22 ± 0.2  0.003  0.93 ± 0.1  1.30 ± 0.1  0.013  Overall volume growth rate (mm3/day)  F1  54.9 ± 7  175.2 ± 40  0.0006  73.6 ± 10  191.7 ± 30  0.006  F2  40.0 ± 6      71.9 ± 7  134.9 ± 11  0.003  F3a  32.2 ± 5      65.1 ± 8      Mean dominant follicle  40.9 ± 6  175.2 ± 40  0.0001  67.0 ± 9  163.3 ± 18  0.002  Total dominant follicle  142.7 ± 17  175.2 ± 40  0.404  293.7 ± 32  326.6 ± 37  0.54  aFor carrier animals, dimensions for up to the third largest dominant follicle are shown as all animals had at least three dominant follicles. View Large Noncarrier cows had a greater (P < 0.03) growth rate of F1, F2, and mean dominant follicle compared to Trio carriers, both on a diameter and volume basis. Mean dominant follicle growth rate on a volume basis was 2.4-fold greater (P = 0.002) in noncarrier cows. Growth rate of total dominant follicles, on a volume basis, was not different (P = 0.54) between carrier and noncarrier cows (Table 2). Circulating FSH Circulating FSH concentrations in heifers tended (P = 0.06) to be different between genotypes (Figure 4A). There was a significant effect of time (P < 0.001) reflecting the rise in FSH concentrations after follicle aspiration and subsequent decline, but no interaction between genotype and time was identified (P = 0.95). Comparisons between genotypes for individual time points were not different (P > 0.05) with the exception of D−2 (day of the first follicle aspiration) when carrier heifers had greater FSH concentrations than noncarriers (P = 0.03). Peak FSH concentrations were not different (P = 0.38) between Trio carriers and noncarrier heifers (0.57 ± 0.04 and 0.65 ± 0.09 ng/ml, respectively). Figure 4. View largeDownload slide Mean (±SEM) serum FSH for heifers (A), cows (B), and all animals combined (C) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration. *Indicates significant differences between genotype for a given time point (P < 0.05), while ‡indicates a tendency (P < 0.10). G, genotype; D, day; G*D, genotype by day interaction. Figure 4. View largeDownload slide Mean (±SEM) serum FSH for heifers (A), cows (B), and all animals combined (C) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration. *Indicates significant differences between genotype for a given time point (P < 0.05), while ‡indicates a tendency (P < 0.10). G, genotype; D, day; G*D, genotype by day interaction. Circulating FSH concentrations were further evaluated after normalization to deviation (data not shown). Three distinct periods were evaluated: predeviation (mean FSH from –48 to –24 h); at deviation (mean FSH from –12 to 12 h); and postdeviation (mean FSH from 24 to 48 h). Predeviation FSH concentrations were not different (P = 0.83) between Trio carriers and noncarrier heifers (0.32 ± 0.02 and 0.31 ± 0.05 ng/ml, respectively). Similarly, postdeviation FSH concentrations were not different (P = 0.26) between genotypes (0.27 ± 0.02 and 0.23 ± 0.02 ng/ml, for carrier and noncarrier heifers respectively). However, there was a tendency (P = 0.09) for greater FSH concentrations at deviation in Trio carriers (0.28 ± 0.02 ng/ml) compared to noncarrier heifers (0.23 ± 0.03 ng/ml). Circulating FSH concentrations in cows showed no main effect of genotype (P = 0.12) and no genotype by day interaction (P = 0.92, Figure 4B). There was a significant effect of time (P < 0.001) reflecting the rise in FSH concentrations after follicle aspiration and the subsequent decline during follicle growth. Comparison between genotypes for each time point were not different (P > 0.05). Peak FSH concentrations were not different (P = 0.54) between Trio carriers and noncarriers cows (0.67 ± 0.06 and 0.61 ± 0.08 ng/ml, respectively). Predeviation FSH concentrations were not different (P = 0.32) between Trio carriers and noncarrier cows (0.30 ± 0.05 and 0.24 ± 0.04 ng/ml, respectively). Similarly, postdeviation FSH were not different (P = 0.39) between genotypes (0.23 ± 0.07 and 0.19 ± 0.06 ng/ml, for Trio carriers and noncarrier cows, respectively). However, there was a tendency (P = 0.06) for greater FSH concentrations at deviation in Trio carriers (0.27 ± 0.03 ng/ml) compared to noncarrier cows (0.19 ± 0.03 ng/ml). In order to further evaluate potential differences in circulating FSH due to genotype, data were combined from heifers and cows (Figure 4C). There was a main effect of genotype (P = 0.01), day (P < 0.001), but no genotype by day interaction (P = 0.93). There was a main effect of parity (P = 0.006) but no genotype by parity interaction (P = 0.57). Trio carriers tended (P < 0.09) to have greater FSH concentrations on days 2.5, 3.5, and 4.5. Mean circulating FSH concentrations were also normalized to follicular deviation and grouped into three distinct periods (data not shown). Predeviation and postdeviation mean FSH concentrations were not different between genotypes (P > 0.10). However, circulating FSH concentrations encompassing deviation were greater (P = 0.01) in Trio carriers (0.28 ± 0.01 ng/ml) than noncarriers (0.21 ± 0.02 ng/ml). Circulating LH Analysis of circulating LH concentrations in heifers (data not shown) showed no main effect of genotype (P = 0.12), day (P = 0.81), and no genotype by day interaction (P = 0.83). Predeviation LH concentrations were not different (P = 0.11) between Trio carriers and noncarrier heifers (0.84 ± 0.07 and 0.70 ± 0.03 ng/ml, respectively). Similarly, postdeviation LH was not different (P = 0.71) between genotypes (0.78 ± 0.08 and 0.82 ± 0.04 ng/ml, for carrier and noncarrier heifers). Circulating LH at deviation was also not different between Trio carriers (0.82 ± 0.04 ng/ml) and noncarrier heifers (0.76 ± 0.04 ng/ml, P = 0.35). Circulating LH concentrations in cows showed a main effect of genotype (P = 0.05) but no effect of day (P = 0.52), and no genotype by day interaction (P = 0.99). Comparisons between genotypes for each time point were not different (P > 0.05). Predeviation LH concentrations were not different (P = 0.39) between Trio carriers and noncarrier cows (0.97 ± 0.07 and 0.89 ± 0.07 ng/ml). Similarly, postdeviation LH was not different (P = 0.50) between genotypes (1.00 ± 0.09 and 0.92 ± 0.08 ng/ml, for carrier and noncarrier cows). Circulating LH at deviation was also not different between Trio carriers (0.96 ± 0.05 ng/ml) and noncarrier cows (0.88 ± 0.05 ng/ml, P = 0.29). In order to further evaluate potential differences in circulating LH, data from heifers and cows were combined for each genotype. Mean LH, normalized to follicle deviation, and grouped into three distinct periods, was analyzed (data not shown). Mean LH concentrations were not different (P > 0.10) at any of the times that were evaluated including during predeviation, near deviation, and postdeviation. Circulating P4 Serum P4 concentrations from D0 (insertion of the CIDR) until D7 are shown in Supplemental Figure S2. Concentrations of P4 in heifers, during the time the CIDR was in place, showed no main effect of genotype (P = 0.84), day (P = 0.13), and no genotype by day interaction (P = 0.80). Analysis of P4 in cows indicated no effect of genotype (P = 0.18), day (P = 0.09), and no genotype by day interaction (P = 0.53). Ovulation and preovulatory follicle characteristics Interval from CIDR removal to estrus was not different (P = 0.23) between heifers that were Trio carriers (43.5 ± 2 h) or noncarriers (48 ± 0 h). Interval from estrus to ovulation was also not different (P = 0.11) between genotypes in heifers (30 ± 2 h vs 36 ± 0 h, for carriers and noncarriers). The interval from CIDR removal to ovulation was shorter (P = 0.005) in heifers that were Trio carriers (73.5 ± 1 h) compared to noncarriers (84 ± 0 h). Trio carrier heifers had 3.8-fold greater (P = 0.0003) number of ovulations than noncarrier heifers (Table 3). Analysis of preovulatory follicle dimensions indicated a greater (P < 0.01) size of the largest, smallest, and mean dominant preovulatory follicle, both when expressed on a diameter basis or on a volume basis, in noncarrier heifers as compared to Trio carrier heifers (Table 3). Mean dominant preovulatory follicle volume was 4.4-fold greater (P < 0.0001) in noncarrier heifers than in Trio carriers. However, total preovulatory follicle volume was not different between genotypes (P = 0.33). Table 3. Preovulatory follicle diameter and volume, and number of ovulations in heifers and cows that are Trio carriers or noncarrier controls.   Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Number of ovulations  3.8 ± 0.3  1.0 ± 0.0  0.0003  4.6 ± 0.5  2.0 ± 0.0  0.016  Follicle diameter (mm)  Largest preovulatory follicle  9.1 ± 0.3  13.5 ± 0.9  0.0004  10.0 ± 0.4  13.8 ± 0.6  0.002  Smallest preovulatory follicle  7.4 ± 0.3  13.5 ± 0.9  < 0.0001  9.1 ± 0.5  12.4 ± 0.3  0.005  Mean ovulatory follicle  8.2 ± 0.3  13.5 ± 0.9  < 0.0001  9.6 ± 0.5  13.1 ± 0.4  0.002  Follicle volume (mm3)  Largest preovulatory follicle  410.7 ± 51  1358.4 ± 296  0.0003  539.1 ± 59  1394.2 ± 187  0.002  Smallest preovulatory follicle  219.7 ± 35  1358.4 ± 296  < 0.0001  413.4 ± 67  993.2 ± 67  0.001  Mean ovulatory follicle  310.2 ± 43  1358.4 ± 296  < 0.0001  479.0 ± 64  1193.7 ± 106  0.0008  Total preovulatory follicle  1084.1 ± 114  1358.4 ± 296  0.33  2095.5 ± 197  2387.4 ± 212  0.37    Heifers  Cows  End point  Carrier  Noncarrier  P-value  Carrier  Noncarrier  P-value  N  8  5    5  3    Number of ovulations  3.8 ± 0.3  1.0 ± 0.0  0.0003  4.6 ± 0.5  2.0 ± 0.0  0.016  Follicle diameter (mm)  Largest preovulatory follicle  9.1 ± 0.3  13.5 ± 0.9  0.0004  10.0 ± 0.4  13.8 ± 0.6  0.002  Smallest preovulatory follicle  7.4 ± 0.3  13.5 ± 0.9  < 0.0001  9.1 ± 0.5  12.4 ± 0.3  0.005  Mean ovulatory follicle  8.2 ± 0.3  13.5 ± 0.9  < 0.0001  9.6 ± 0.5  13.1 ± 0.4  0.002  Follicle volume (mm3)  Largest preovulatory follicle  410.7 ± 51  1358.4 ± 296  0.0003  539.1 ± 59  1394.2 ± 187  0.002  Smallest preovulatory follicle  219.7 ± 35  1358.4 ± 296  < 0.0001  413.4 ± 67  993.2 ± 67  0.001  Mean ovulatory follicle  310.2 ± 43  1358.4 ± 296  < 0.0001  479.0 ± 64  1193.7 ± 106  0.0008  Total preovulatory follicle  1084.1 ± 114  1358.4 ± 296  0.33  2095.5 ± 197  2387.4 ± 212  0.37  Data are presented as mean (±SEM). View Large In cows, interval from CIDR removal to estrus was not different (P = 0.46) between Trio carriers (34.4 ± 2 h) and noncarriers (44.0 ± 4 h). Interval from estrus to ovulation was also not different (P = 0.99) between genotypes (31.2 ± 3 h vs 28.0 ± 4 h, for Trio carrier and noncarrier cows). The interval from CIDR removal to ovulation was not different (P = 0.99) in Trio carrier (69.6 ± 2 h) compared to noncarrier (72.0 ± 0 h) cows. Trio carrier cows had a 2.3-fold greater (P = 0.02) number of ovulations than noncarrier cows (Table 3). Comparison of preovulatory follicle dimensions indicated a greater (P < 0.01) size of the largest, smallest, and mean dominant preovulatory follicle, expressed on either a diameter or volume basis, in noncarrier cows as compared to Trio carrier cows (Table 3). Mean dominant preovulatory follicle volume was 2.5-fold greater (P < 0.001) in noncarrier cows. However, total preovulatory follicle volume was not different between genotypes (P = 0.37). Experiment 2 One animal in the noncarrier group was removed from the analysis due to being the only animal with double ovulation, all other noncarriers had single ovulations. Trio carrier animals had greater (P = 0.006) number of ovulations and CL (4.3 ± 0.7) than noncarrier controls (1.0 ± 0.0). Analysis of individual mean CL volume indicated a main effect of genotype (P < 0.0001), day (P < 0.0001), but no genotype by day interaction (P = 0.17). Noncarrier animals had, on average, a 4.7-fold greater individual CL volume than Trio carriers between 6 and 10 days after estrus (Figure 5A). Total CL volume showed a main effect of day (P < 0.0001), but no effect of genotype (P = 0.60), and no genotype by day interaction (P = 0.73, Figure 5B). Circulating P4 concentrations showed a main effect of day (P < 0.0001), and a genotype by day interaction (P = 0.01), but no effect of genotype (P = 0.82, Figure 5C). Comparison of P4 concentrations between genotypes for each time point were not different (P > 0.05) with the exception of a greater P4 concentration in Trio carriers on day 3 (P = 0.02). Figure 5. View largeDownload slide Mean (±SEM) individual (A) and total (B) CL volume and serum P4 (C) normalized to the day of estrus (experiment 2) for Trio carriers (n = 7) and age-matched, half-sib noncarrier controls (n = 4). *Indicates significant differences between genotypes at a specific time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Figure 5. View largeDownload slide Mean (±SEM) individual (A) and total (B) CL volume and serum P4 (C) normalized to the day of estrus (experiment 2) for Trio carriers (n = 7) and age-matched, half-sib noncarrier controls (n = 4). *Indicates significant differences between genotypes at a specific time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Experiment 3 The interval between follicle ablation and the time of follicular fluid collection tended (P = 0.09) to be greater in carriers (2.7 ± 0.1 days) than in noncarriers (2.1 ± 0.2 days) reflecting the slower growth rate and greater time needed for the largest follicle to reach 7 mm. Follicle diameters of F1, F2, and F3 were not significantly different between genotypes (Figure 6A). Within each genotype, all three follicles were different in noncarrier animals, while in carriers the F1 was larger than F2 and F3 but no difference was found between the F2 and F3 (Figure 6A). Analysis within genotype indicated that follicular fluid estradiol concentrations were elevated in all three dominant follicles in carriers and there was no difference between F1, F2, and F3 in carriers (Figure 6B). Intrafollicular concentrations of estradiol were greater in carriers than in noncarriers in the F1, F2, and F3 (Figure 6B). In contrast, noncarriers had follicular fluid estradiol concentrations that were lower in the F2 and F3 than in F1 (Figure 6B). Intrafollicular P4 concentrations were different between genotypes only for F1, with carriers having greater concentrations of P4 than noncarriers (Figure 6C). Analysis within each genotype indicated no differences between F1, F2, and F3 for carriers or noncarriers (Figure 6C). Figure 6. View largeDownload slide Follicle diameter (A), intrafollicular estradiol (B), and intrafollicular P4 (C) concentrations in the three largest follicles of cattle carrying the Trio allele and age-matched, half-sib controls. Number in parenthesis at the base of each column indicate the number of follicles included. a,bIndicates significant differences between groups (P < 0.05). Figure 6. View largeDownload slide Follicle diameter (A), intrafollicular estradiol (B), and intrafollicular P4 (C) concentrations in the three largest follicles of cattle carrying the Trio allele and age-matched, half-sib controls. Number in parenthesis at the base of each column indicate the number of follicles included. a,bIndicates significant differences between groups (P < 0.05). Discussion This study constitutes the first characterization of the follicle growth profile, follicle volume relationships, and associated circulating gonadotropins in carriers of the high-fecundity bovine allele, Trio. The ovulation rate and associated number of dominant follicles of Trio carrier heifers and cows were significantly greater than in their noncarrier counterparts. Follicular and hormonal dynamics could be precisely defined by first temporally synchronizing the start of a follicular wave using ultrasound-guided follicular aspiration and second using detailed twice daily ultrasound evaluations and blood sampling to compare carriers of the Trio allele to age-matched, half-sib noncarriers. Accurate definition of the phenotype of a given genotype is important for proper selection of a valid control group. In this case, we incorporated the Trio allele into a crossbred beef cattle population and then directly compared half-sibling sisters that were heterozygous carriers to genetically matched and age-matched noncarriers of the Trio allele. The most distinctive feature of follicular dynamics in carriers of the Trio allele was the smaller size of the dominant follicles and the reduced growth rate during all stages of follicular development. This is clearly consistent with the elegant studies in high-fecundity ewes, in which presumptive ovulatory follicles were smaller, corresponding to fewer granulosa cells per follicle in carrier ewes [40,41]. One of the key insights in our studies was the calculation of follicular dynamics and follicular growth in terms of the volume of a sphere rather than only as a diameter. Calculation of ultrasound results on a volume basis demonstrated the impressive magnitude of the difference in follicle size between carriers and noncarriers (∼25% of the follicular volume) but also revealed that total follicular volume and total luteal volume were not different when volume of all dominant follicles or CL was combined. This corresponds to observations in the ovine high-fecundity genotypes in which total number of granulosa cells is similar to wild types, when all presumptive ovulatory follicles are added together and that, correspondingly, total luteal tissue was similar between carriers and noncarriers [40,42]. Another key insight was that, despite a reduced growth rate in Trio carriers, the time of diameter deviation between the largest follicle (F1) and the first subordinate follicle (SF1) occurred at a similar time as that observed in noncarrier controls, although at a much smaller follicle size. This corresponds to the idea that follicles in high-fecundity ewes acquire LH receptors and become dominant at an “earlier” time [5]. However, our study more accurately describes that this selection process occurs in the same temporal sequence but at a much smaller size due to the reduced growth rate of each dominant follicle in carriers of the Trio allele. Finally, FSH concentrations were, on average, greater (P = 0.01 with combined FSH data in Figure 4) in Trio carriers, but more importantly analysis of FSH in relation to key stages during follicle development revealed a subtle but significantly greater FSH concentration (Figure 5) in Trio carriers during the 24 h encompassing deviation. These exceptional results allow us to add depth and insight into current models of follicle growth, follicle selection, and, in particular, the dynamic processes that occur during selection of multiple dominant follicles in Trio, and perhaps other, high-fecundity animal models. Recently, Juengel et al [5] proposed an intriguing model for how mutations in TGFB family members result in a high ovulation rate. They proposed that ewes that are heterozygous for mutations in either GDF9 or BMP15 or their receptors have reduced granulosa cell proliferation but an earlier acquisition of LH receptivity in the granulosa cells in follicles of smaller size. The recently discovered high-fecundity bovine genotype Trio [27] has been clearly linked to overexpression of SMAD6 in granulosa cells of heterozygous Trio carriers [28]. This result clearly places this bovine high-fecundity genotype within a similar functional category as the high-fecundity ovine mutations that affect the oocyte-derived TGFB family members, BMP15 and GDF9, and their intracellular signal transduction pathways [5]. Our first hypothesis when designing this study was that carriers of the high-fecundity allele would ovulate smaller-sized follicles compared to noncarriers, although the total ovulatory follicular volume would be similar. This hypothesis was supported by the observations that Trio carriers ovulate multiple follicles of smaller size, but the total ovulatory follicle volume was similar in carriers and noncarriers. Mean dominant follicles in Trio carriers were smaller in diameter from first detection at 12 h after the last follicle aspiration, through deviation and until ovulation. The finding of smaller follicle size in carriers of the Trio allele is in agreement with previous results for ewes that were carriers of high-fecundity alleles as determined by terminal studies or by ultrasound monitoring [40,41,43]. The USDA-MARC twinner cattle population has also been shown to have smaller preovulatory follicles as ovulation rate increases, although diameters appear to be larger than those observed in this study in Trio carriers [44]. The most revealing information, however, has resulted from the consideration of follicle size from a volume perspective, where individual follicle volume in Trio carriers was significantly smaller but total dominant follicle volume, calculated as the sum of each individual dominant follicle, was similar to the volume observed in noncarrier controls. Traditionally, follicle growth has been viewed from a two-dimensional perspective and this has served well for most of the research related to follicle selection and it is extremely easy to understand since follicles are viewed in two dimensions on the ultrasound screen. However, as obvious as it may seem, the follicle is a three-dimensional structure and thus the use of the volume of a sphere may better represent the relationship between different follicle sizes and the components of the follicle such as granulosa cell numbers. The smaller individual follicle but similar total follicle volume observed in Trio carrier heifers emphasize the relationship between follicle size and ovulation rate, as Trio carriers had ∼4-fold greater number of ovulations than controls but their preovulatory follicles were individually ∼4-fold smaller in volume than in controls; thus, the total preovulatory follicle volume was not different. Assuming that the follicle volume is related to the number of granulosa cells, then total hormonal output would be similar whether it was coming from one large follicle or four smaller follicles [40–42]. The smaller size of ovulatory follicles was due to a slower growth rate, ∼65% slower follicle growth rate in carriers compared to single-ovulating, noncarrier heifers. These results support our second hypothesis that follicles in the carrier animals would grow at a slower rate than follicles of noncarriers. Previous research demonstrated reduced proliferation of granulosa cells from Booroola FecB carrier ewes, as measured by [3H] thymidine uptake under basal conditions, as compared to granulosa cells from wild-type ewes [45,46] and this is likely to be the case in carriers of Trio. These ideas are consistent with a model in which a reduction in granulosa cell proliferation underlies the smaller follicle sizes that have been reported in previous results with high-fecundity ewes [40–42,47] and the reduced follicle growth rate observed in our study. Our third hypothesis was that concentrations of FSH would be greater in carriers than in noncarriers, particularly near the time of follicular deviation. Circulating FSH concentrations have a key regulatory role in follicle growth as demonstrated by the clear FSH surge associated with the emergence of each follicular wave and the subsequent decrease in FSH concentrations associated with diameter deviation, as proposed for two way coupling between follicles and FSH [48–50]. The use of follicle aspiration in this study allowed for the synchronization of the follicular wave at a self-appointed time, and both Trio carriers and noncarrier controls had similar FSH surges in response to follicle aspiration with similar peak FSH concentrations as observed in previous reports [32]. The evidence for a relationship between circulating FSH, follicle selection, and ovulation rate has been provided by several studies [49,51–53]. For example, the administration of low doses of exogenous FSH before diameter deviation prolonged the occurrence of deviation and stimulated the growth of the presumptive future SFs [51]. Moreover, the administration of exogenous FSH preparations has become the basis for superstimulation protocols used for superovulation and multiple embryo production [54]. The natural occurrence of multiple ovulations in cattle has also been associated with changes in FSH, as shown by the greater FSH concentrations immediately before deviation in lactating dairy cows with multiple codominant follicles [52]. Conversely, circulating concentrations of FSH were found to be similar between USDA-MARC twinner cattle and controls, although in this case FSH patterns were not normalized in relation to wave emergence or deviation [55,56]. In this study, circulating FSH was, on average, greater in Trio carriers; however, detailed analysis performed in relation to observed deviation indicated that FSH was greater only in the 24 h encompassing deviation. The finding of this subtle but significant difference in FSH encompassing deviation is in agreement with one of the proposed models for the mechanisms underlying multiple ovulation proposed originally by Baird in 1987 [57] and recently reviewed and updated by Scaramuzzi et al [58]. It is also consistent with the two-way FSH-follicle growth coupling hypothesis proposed by Ginther et al [50]. Based on these models, Trio carriers in this study exhibited greater FSH concentrations during the time encompassing deviation, which is consistent with the idea of widening the gate to allow more follicles to achieve dominance. Thus, the primary mechanism that allows selection of multiple dominant follicles in carriers of the Trio allele may be that FSH is not completely suppressed until multiple follicles undergo deviation and provide the final suppression of FSH that determines the number of “selected” follicles. The role for greater FSH near deviation in selection of multiple follicles is physiologically reasonable, based on our current understanding of follicle selection, and is clearly supported by association between increased FSH near deviation and selection of multiple dominant follicles. There has been an ongoing controversy about the role for circulating FSH in increasing multiple ovulations in high-fecundity ovine genotypes. It seems possible that the role for greater FSH near deviation in selection of multiple follicles may differ in different high-fecundity genotypes. It also seems possible that changes in FSH may be subtle making it difficult to detect without precise normalization of follicular dynamics, particularly normalization of FSH to the precise time of follicle deviation in each individual animal. Our fourth and final hypothesis was that follicular deviation would occur at smaller follicle sizes in carrier animals than in noncarriers. This hypothesis was also clearly supported by our ultrasound evaluations. Of particular importance, diameter deviation in Trio carriers was observed at a similar time as observed in noncarriers and despite smaller-sized individual follicles, the total volume of all future dominant follicles was not different between genotypes at the time of deviation. The measurements of intrafollicular estradiol concentrations in experiment 3 were planned to provide information on the acquisition of dominance in follicles of Trio carriers compared to noncarriers. The use of estradiol concentrations is based on the premise that the rise in estradiol concentrations within the follicular fluid is probably one of the most consistent findings in relation to the acquisition of dominance [59–63]. The decision to collect follicular fluid from follicles when the largest follicle reached 7 mm was based on the idea that follicles in noncarriers would not yet have acquired dominance by that size [59,60]. In agreement with our ultrasound results demonstrating follicular deviation prior to 7 mm in Trio carriers, estradiol concentrations were greatly elevated in all three follicles from Trio carriers, consistent with a dominant phenotype [60], and much greater than estradiol concentrations in any of the three follicles of noncarriers. These findings, coupled with the observation that deviation occurs when the largest follicle is ∼6 mm in diameter, provide support for our hypothesis that follicles of Trio carriers acquire dominance at a smaller size than observed in noncarriers. In conclusion, Trio carriers had smaller-sized follicles, which developed at a reduced growth rate during the follicular wave, and underwent diameter deviation at a similar time but at smaller size than observed in half-sibling noncarriers. The relationship between the number of ovulations and the individual follicle volume is such that, when taken together, the total preovulatory follicle volume and subsequent total luteal tissue volume are similar to single ovulating controls, and as a result circulating P4 is not different. The smaller follicle size at which follicles acquire dominance appears to be associated with greater circulating FSH concentrations for a short time period near deviation that could allow for more follicles to acquire dominance and this may be a key part of the mechanisms that produce multiple ovulations in carriers of the Trio allele. Supplementary data Supplementary data are available at BIOLRE online. Supplemental Figure S1. Growth profile of the largest follicle (F1) diameter in heifers (left) and cows (right) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared based on number of days from the last follicle aspiration until the removal of the intravaginal P4 implant. Each point represents mean (±SEM). *Indicates significant differences between genotype for a given time point (P < 0.05). G, genotype; D, day; G*D, genotype by day interaction. Supplemental Figure S2. Circulating P4 in heifers (A) and cows (B) that are Trio carriers or age-matched, half-sib noncarrier controls (experiment 1). Data were compared during the days in which the CIDR was in place and each point represents mean (±SEM). In heifers, genotype P = 0.84; day P = 0.13; genotype by day P = 0.80. In cows, genotype P = 0.18; day P = 0.10; genotype by day P = 0.53. Acknowledgments Special thanks to A. Sallam, R.V. Barletta, M. Z. Toledo, C. Gamarra, E. Trevisol, P. L. Monteiro, J. Levandowski and E. Walleser for technical assistance; and the staff of Arlington Research Station Beef Grazing unit and the Lancaster Agricultural Research Station for animal handling and production. A patent (20150007358) assigned to the Wisconsin Alumni Research Foundation has been awarded to BWK related to determining the Trio haplotype. Footnotes † Grant support: Funding was provided by WI Experiment Station as Hatch Project WIS01240 to MCW and as Hatch Project WIS01648 and WIS01932 to BWK. References 1. Spearow JL. Major genes control hormone-induced ovulation rate in mice. Reproduction  1988; 82: 181– 186. 2. Rocha JL, Eisen EJ, Siewerdt F, Van Vleck LD, Pomp D. A large-sample QTL study in mice: III. Reproduction. Mamm Genome  2004; 15: 878– 886. Google Scholar CrossRef Search ADS PubMed  3. Sugiura K, Su Y-Q, Eppig JJ. Does Bone Morphogenetic Protein 6 (BMP6) affect female fertility in the mouse? Biol Reprod  2010; 83: 997– 1004. Google Scholar CrossRef Search ADS PubMed  4. 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Biology of ReproductionOxford University Press

Published: Mar 1, 2018

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