Selection of a single dominant follicle from a cohort of growing follicles is a unique biological process, a key step in female reproductive function in monovular species, and lies at the core of reproductive technologies in cattle. Follicle growth and the number of follicles that ovulate are regulated by precise endocrine, paracrine, and autocrine mechanisms. Most of our current understanding about follicle selection focuses on the role of FSH, LH, and the IGF family in follicle growth and selection of the dominant follicle. However, more recently the role of members of the TGF-ß family has been highlighted, particularly in high fecundity genotypes in sheep. Intercellular signaling between the oocyte and granulosa cells (GC) regulates proliferation and differentiation due to actions of bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) within the follicle. Mutations that either knockout or reduce the activity of BMP15 or GDF9 have been found to increase ovulation rate in heterozygotes and generally cause severe follicle abnormalities in homozygotes. A mutation in the intracellular kinase domain of the BMPR1B receptor (Booroola fecundity gene) increases ovulation rate in heterozygotes with further increases in ovulation in homozygotes. The physiological mechanisms linking these mutations to increased ovulation rates are still not well defined. A recently identified high fecundity bovine genotype, Trio, causes increased expression of SMAD6, an intracellular inhibitor of the BMP15/GDF9 signalling pathways. This bovine model has provided insights into the mechanisms associated with selection of multiple dominant follicles and multiple ovulations in carriers of fecundity alleles. The present review focuses on the mechanisms involved in follicle selection in ruminants with a special emphasis on the contribution made by multiple ovulation models in both cattle and sheep. The evaluation of multiple ovulation models in ruminants has allowed us to construct a new physiological model that relates changes in the BMP15/GDF9 signalling pathways to the physiological changes that result in selection of multiple dominant follicles. This model is characterized by acquisition of dominance at a smaller follicle size but at a similar time in the follicular wave with multiple follicles acquiring dominance in a hierarchal sequence, delaying FSH suppression and, thus allowing additional follicles to continue to grow and acquire dominance.

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Heifers typically have a reduced ovulation rate following gonadotrophin-releasing hormone (GnRH) application at initiation of a CO-Synch + controlled internal drug release (CIDR) protocol. Thus, the objective of the present study was to determine whether increasing the dose of GnRH at initiation of a 5-day CO-Synch protocol in beef heifers would improve ovulation rate and therefore increase pregnancies per AI (P/AI). Angus yearling heifers (n=299) at five locations in Ohio (United States) were randomised to receive either 100µg (single; n=149) or 200µg (double; n=150) of gonadorelin acetate (Gonabreed, Parnell) at initiation of a 5-day CO-Synch. On Day −8, heifers received a new intravaginal progesterone-releasing device (1.38g of progesterone; CIDR, Zoetis) and either a single or double dose of GnRH as described above. Five days later (Day −3), devices were removed, 1000µg of cloprostenol sodium (Estroplan, Parnell) was administered, and an oestrous detection patch was applied (Estrotect, Rockway Inc.). Sixty hours after device removal, AI was performed concurrently with the administration of 100µg of GnRH. Pregnancy was determined using ultrasonography 35 days after AI. Ovaries from a subset of animals (n=178) were examined on Days −8 and −3 using ultrasonography to determine the presence of corpora lutea (CL) and the size of the largest follicle. Data were analysed using the GLIMMIX procedure of SAS ver. 9.4 (SAS Institute Inc.). Oestrous expression was similar (P=0.50) between heifers treated with a single (49.0%) or double (52.7%) dose of GnRH. Overall, P/AI was similar (P=0.35) between heifers receiving a single (43.6%; 65/149) or double (38.7%; 58/150) dose of GnRH at initiation of the protocol. However, increasing the dose of GnRH resulted in a greater (P=0.04) ovulation rate in heifers in the double-dose group (40.9%; 36/88) compared with those in the single-dose group (26.1%; 23/88). In addition, heifers with a CL at the time of treatment had reduced ovulatory response to GnRH treatment (16.0%) compared with heifers without a CL (53.7%; P=0.001); however, there was no treatment×CL presence interaction (P=0.69). Heifers that did not ovulate to the initial GnRH treatment had a greater (P=0.0008) diameter of the largest follicle on Day −3 compared with heifers that did ovulate (11.4±0.2 vs. 10.0±0.3). Furthermore, heifers that did ovulate after the initial GnRH had greater (P=0.04) P/AI (52.5%) than heifers that did not ovulate (40.2%), and heifers with a CL on Day −8 tended (P=0.07) to have greater P/AI (47.9%) than heifers without a CL (40.2%). In addition, heifers with a CL present on Day −3 had greater (P=0.04) P/AI (48.2%) than heifers without a CL (31.7%). In summary, increasing the dose of GnRH at initiation of a 5-day CO-Synch did not affect fertility to fixed-time AI but enhanced ovulation rate in beef heifers. Furthermore, heifers that did ovulate at initiation of the protocol or that had a CL at device insertion or removal had greater fertility to fixed-time AI. Thus, alternative strategies that maximise ovulation at initiation of the synchronisation protocol are needed.

Colour Doppler ultrasonography (CDU) of the corpus luteum (CL) has the potential to be used for early pregnancy diagnosis in order to improve reproductive efficiency and increase the use of fixed-time AI (FTAI) in beef cattle. The objective of the present study was to determine the sensitivity and specificity of CDU of the CL for pregnancy diagnosis in beef heifers with or without a CIDR at different days after FTAI. Angus-cross beef heifers (n=84) were synchronized using a 5-day Co-Synch with AI at 60h after CIDR removal. On Day 15 post-AI, heifers were randomly assigned to receive a CIDR (Eazi-Breed CIDR, Zoetis, Parsippany-Troy Hills, NJ) for 9 days or remain as untreated controls. Heifers were evaluated by transrectal CDU (MyLab Delta, Esoate, Genoa, Italy; 7.5-MHz linear array probe, pulse repetition frequency=960Hz) at 15, 17, 20, and 22 days post-AI. Heifers were determined to be pregnant by CDU if colour pixels covered >10% of the periphery of the CL and contained at least 2 colour internal tracts penetrating toward the centre of the CL. Heifers were evaluated by B-mode ultrasonography on Day 28 to determine true pregnancy status. Differences between days and treatments were evaluated using generalized estimating equations. Pregnancies per AI at Day 28 after FTAI were 53.6% (45/84) and were not different between CIDR (52.4%; 22/42) and control (54.8%; 23/42) heifers (P=0.83). Sensitivity and specificity for CIDR and control heifers at different days are shown in Table 1. There was no effect of treatment (P=0.49), day (P=0.99), or treatment by day interaction (P=0.99) on test sensitivity. Specificity was different (P<0.01) between days; however, no treatment (P=0.91) or treatment by day interaction (P=0.82) was identified. Specificity was lowest on Days 15 and 17 and increased to reach its maximum at Day 22. Although no differences between treatments were observed, specificity on Day 22 was numerically greater in CIDR-treated heifers. Positive and negative predictive values for CDU at Day 22 were 84 and 94.1%, respectively, for CIDR-treated heifers and 79.3 and 100%, respectively, for control heifers. Cohen’s Kappa indicated slight (0.19), fair (0.27), moderate (0.50), and substantial (0.73) agreement between conventional ultrasound at Day 28 and CDU at Days 15, 17, 20, and 22, respectively. In summary, CDU showed excellent sensitivity between Days 15 and 22, indicating a very low rate of false-negative results. However, high specificity (low false-positive rate) was achieved only at Day 22. Thus, pregnancy diagnosis by CDU at Day 22 after FTAI coupled with the use of a CIDR may be an effective strategy to identify nonpregnant heifers and attempt their prompt resynchronization. Table 1.Sensitivity and specificity (%) for pregnancy diagnosis by CDU in beef heifers with or without a CIDR at different days after FTAI

Ovulatory response to the initial gonadotrophin-releasing hormone (GnRH) of the CO-Synch protocol is affected by circulating progesterone (P4) and follicle size. In addition, heifers that ovulate to the initial GnRH treatment have greater fertility after AI. Thus, this study determined the effect of (1) presynchronization (Presynch) before a 6-day CO-Synch protocol and (2) circulating [RCE1] (P4) on ovulatory response, oestrus expression, and pregnancies per AI (P/AI) in beef heifers. Yearling beef heifers (n=233) at three locations were randomly assigned in a 2×2 factorial design to the following treatments: (1) Presynch+6-day CO-Synch with a new P4 device; (2) Presynch+6-day CO-Synch with a once-used P4 device; (3) 6-day CO-Synch with a new P4 device; and (4) 6-day CO-Synch with a once-used P4 device. Presynch consisted of insertion of a new P4 intravaginal device (1.38g of P4) on Day −17 and removal of P4 device on Day −11 concurrently with 500µg of cloprostenol sodium (PGF). On Day −9, all heifers received either a new (New) or once-used (Used) CIDR and 100µg of gonadorelin acetate (GnRH). Six days later (Day −3), CIDRs were removed, 1000µg of PGF was administered and an oestrous detection patch applied (Estrotect). At 72h after CIDR removal, AI was performed concurrently with administration of 100µg of GnRH. Pregnancy was determined by transrectal ultrasonography 31 days after AI. A subset of heifers (n=155) was examined on Day −9 and Day −3 by ultrasonography to determine ovulation to Day −9 GnRH. Data were analysed using generalized linear mixed models (SAS 9.4; SAS Institute Inc.). Presynch heifers had larger follicle diameter on Day −9 (12.7±0.3 vs. 10.1±0.3 mm; P<0.001), greater ovulatory response to Day −9 GnRH (82.5%; 66/80 vs. 56%; 42/75; P<0.001), greater expression of oestrus (90.6%; 106/117 vs. 78.4%; 91/116; P<0.02), and earlier oestrus (49.8±1 vs. 53.1±1 h; P<0.01) compared with controls. There was a treatment×CIDR interaction on oestrous expression, such that a lesser (P<0.05) percentage of control heifers with new CIDR expressed oestrus compared with all other groups (Table 1). Heifers with a used CIDR during the 6-day CO-Synch tended (P=0.08) to have greater P/AI (52.1%; 61/117) than those with a new CIDR (40.5%; 47/116). In conclusion, presynchronization before initiation of a 6-day CO-Synch increased follicle diameter, enhanced ovulatory response and oestrous expression, but did not affect fertility. The earlier onset of oestrus in presynchronized heifers suggests that the timing of AI may need to be modified. Table 1. Oestrous expression and pregnancy per AI (P/AI) in beef heifers with or without presynchronization and treated with a new or used CIDR during a 6-day CO-Synch Treatment CIDR Oestrus (%; n/n) Time of oestrus (h) P/AI (%; n/n) Control New 67.8a (40/59) 53.7±1.5a 33.9 (20/59) Used 89.5b (51/57) 52.7±1.6a 50.9 (29/57) Presynch New 94.7b (54/57) 50.9±1.4b 47.4 (27/57) Used 86.7b (52/60) 48.7±1.3b 53.3 (32/60) P-value Treatment 0.03 0.01 0.21 CIDR 0.62 0.19 0.08 Interaction 0.003 0.75 0.38 a,bValues with different superscripts differ (P<0.05).