Effects of progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI in beef cattle

Three experiments were conducted to determine the effects of low-dose progesterone presynchronization and eCG on pregnancy rates to GnRH-based, timed-AI (TAI) in beef cattle (GnRH on Day 0, PGF_2α on Day 7, with GnRH and TAI on Day 9, 54-56 h after PGF_2α). Experiments 1 and 2 were 2 × 2 factorials with presynchronization (with or without a once-used CIDR; Days −15 to 0 in Experiment 1 and Days −7 to 0, with PGF_2α at insertion, in Experiment 2), and with or without 400 IU eCG on Day 7 in suckled cows. In Experiment 3, suckled cows and nulliparous heifers were either presynchronized with a twice-used CIDR (Days −5 to 0) and PGF_2α at insertion, or no treatment prior to insertion of a new CIDR (Days 0-7). Presynchronization increased (P < 0.05) ovulation rate to GnRH on Day 0 (75.0% vs 48.7%, 76.7% vs 55.0%, and 60.0% vs 36.1% for Experiments 1, 2, and 3, respectively), increased the diameter of the preovulatory follicle in Experiments 1 and 2, and increased the response to PGF_2α (regardless of parity) in Experiment 1 (P < 0.01), and in primiparous cows in Experiment 2 (P < 0.01). Effects of presynchronization on pregnancy rates (53.4% vs 54.1%, 57.7% vs 45.3%, and 54.3% vs 44.4% for Experiments 1, 2, and 3, respectively) were influenced by parity and eCG (P < 0.05). Treatment with eCG had no effect (P > 0.05) on the diameter of the preovulatory follicle (Experiment 1), or the response to PGF_2α (Experiments 1 and 2), but tended (P = 0.08) to improve pregnancy rates, especially in primiparous cows that were not presynchronized (P < 0.01). However, the effects of eCG and presynchronization were not additive.     

Effect of purified llama ovulation-inducing factor (OIF) on ovarian function in cattle

Two experiments were designed to determine the effect of purified ovulation inducing factor (OIF) on ovarian function in cattle. In Experiment 1, prepubertal heifers (n = 11 per group) were treated on Day 5 (Day 0 = day of follicular wave emergence) of the follicular wave with an intramuscular dose of saline (1 mL), GnRH (100 μg), or purified OIF (1 mg/100 kg body weight). Ovulation occurred in 9/11 heifers treated with GnRH, and 1/11 heifers in each of the OIF- and saline-treated groups (P < 0.05). Compared to saline-treated controls, OIF treatment was associated with a smaller dominant follicle diameter (P < 0.01), a rise in plasma FSH concentration (P < 0.1), and earlier emergence of the next follicular wave (P < 0.05). In Experiment 2, sexually mature heifers were given either GnRH or purified OIF on Days 3, 6 or 9 of the first follicular wave (i.e., early growing, early static, or late static phase of the dominant follicle; n = 5 per group per day), or were untreated (n = 10). In heifers treated with OIF on Day 6, the dominant follicle diameter profile tended to be smaller than in controls, and was associated with a rise (P < 0.05) in plasma FSH concentrations. A similar rise in FSH was detected after OIF treatment on Day 9. Compared to untreated controls, treatment with OIF and GnRH was associated with a larger CL diameter (Days 3 and 6 groups; P < 0.05) and a greater concentration of plasma progesterone (Days 6 and 9 groups; P < 0.05). Treatment with purified OIF did not induce ovulation in heifers, but hastened new follicular wave emergence in prepubertal heifers, influenced follicular dynamics in a phase-specific manner in mature heifers, and was luteotrophic.     

Ovarian follicular dynamics, follicle deviation, and oocyte yield in Gyr breed (Bos indicus) cows undergoing repeated ovum pick-up

The objective of this study was to evaluate ovarian follicular dynamics during intervals between successive ovum pick-up (OPU) and determine its effects on the number and quality of recovered cumulus-oocyte complexes (COCs) in Zebu cows (Bos indicus). Pluriparous nonlactating Gyr cows (Bos indicus; n = 10) underwent four consecutive OPU sessions at 96-h intervals. The dynamics of ovarian follicular growth between OPU sessions was monitored by twice-daily ultrasonographic examinations. A single dominant follicle (DF) or two codominant (CDF) follicles (>9 mm) were present in 63.3% (19 of 30) of intervals studied, with follicle deviation beginning when the future dominant follicle (F1) achieved a diameter of 6.2 ± 0.3 mm. The phenomenon of codominance was observed in four (13.3%) of the inter-OPU intervals. The remaining intervals (36.6%, 11 of 30) were characterized by a greater follicular population, lower rate of follicular growth, and a smaller diameter F1 (P < 0.0001). There was a tendency (P = 0.08) toward an increase in the number of recovered COCs when dominant follicles were not present (NDF). The quality of COCs was not affected by the presence of a single dominant follicle, but codominant follicles resulted in recovery of a lower proportion of viable embryos (40.0%, 62.1%, and 63.6%; P < 0.05) and higher proportions of degenerate COCs (56.0%, 30.3%, and 28.6%; P < 0.05) for CDF, NDF, and DF respectively. We concluded that, in Zebu cows, (a) repeated follicle aspirations altered ovarian follicular dynamics, perhaps by increasing follicular growth rate; (b) follicular dominance could be established in cows undergoing twice-a-week OPU; and (c) the presence of a dominant follicle during short inter-OPU intervals may not affect COC quality, except when a codominant follicle was present.     

Suppression of dominant and subordinate ovarian follicles by a proteinaceous fraction of follicular fluid in heifers

Nulliparous Holstein heifers were examined ultrasonically once daily during an interovulatory interval (ovulation = Day 0). Follicles with a diameter >/=4 mm were sequentially identified. Heifers were randomized into four groups (n = 4 heifers per group): untreated control heifers and those treated on Days 0 to 3, Days 3 to 6, or Days 6 to 11. Heifers designated for treatment were given an intravenous injection, twice daily, of a proteinaceous fraction of follicular fluid (PFFF; 16 ml) prepared by extracting bovine follicular fluid with activated charcoal. Mean cessation of growth of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 5.5) than in heifers treated on Days 0 to 3 (Day 1.5) or Days 3 to 6 (Day 3.5). Mean onset of regression of the dominant follicle of Wave 1 was later (P<0.005) in control heifers (Day 12.0) than in heifers treated on Days 0 to 3 (Day 5.0) or Days 3 to 6 (Day 7.5). Mean cessation of growth of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 3.0) than in heifers treated on Days 0 to 3 (Day 1.2). Mean onset of regression of the largest subordinate follicle of Wave 1 was later (P<0.05) in control heifers (Day 7.0) than in heifers treated on Days 0 to 3 (Day 4.8). In heifers treated on Days 6 to 11, cessation of growth and onset of regression of the dominant follicle (means, Days 5.2 and 12.0, respectively) were not significantly different from those of the controls. The hypothesis that PFFF treatment on Days 0 to 3 would cause suppression of all follicles of Wave 1 was supported. The hypothesis that PFFF treatment on Days 3 to 6 would not alter growth of the dominant follicle of Wave 1 was not supported. The mean day of detection of the dominant follicle of Wave 2 was different (P<0.005) in control heifers (Day 8.5) than in heifers treated on Day 0 to 3 (Day 5.5) or Days 6 to 11 (Day 14.2). The mean length of the interovulatory interval was shorter (P<0.05) in control heifers (20.5 d) than in heifers treated on Days 6 to 11 (23.2 d). The hypothesis that PFFF treatment on Days 6 to 11 would delay the emergence of Wave 2 was supported. The proportion of heifers with 2-wave interovulatory intervals was 3 4 for control heifers and 0 4 , 1 4 , and 4 4 for heifers treated on Days 0 to 3, Days 3 to 6, and Days 6 to 11, respectively (3 4 vs 0 4 , P<0.05); the remaining heifers had 3-wave interovulatory intervals. On average, in PFFF-treated heifers, follicles stopped growing 1 d after treatment was started, and Wave 2 was detected 3 d after treatment was stopped.     

Effect of progesterone concentrations,follicle diameter,timing of artificial insemination,and ovulatory stimulus on pregnancy rate to synchronized artificial insemination in postpubertal Nellore heifers

Two experiments were designed to evaluate the effects of treatments with low versus high serum progesterone (P₄) concentrations on factors associated with pregnancy success in postpubertal Nellore heifers submitted to either conventional or fixed timed artificial insemination (FTAI). Heifers were synchronized with a new controlled internal drug release device (CIDR; 1.9 g of P₄ [CIDR1]) or a CIDR previously used for 18 days (CIDR3) plus 2 mg of estradiol (E₂) benzoate on Day 0 and 12.5 mg of prostaglandin F2α on Day 7. In experiment 1 (n = 723), CIDR were removed on Day 7 or 9 and heifers were inseminated after estrus detection. In experiment 2 (n = 1083), CIDR were all removed on Day 9 and FTAI was performed either 48 hours later in heifers that received E₂ cypionate (ECP) on Day 9 (0.5 mg; E48) or 54 or 72 hours later in conjunction with administration of GnRH (100 μg; G54 or G72). Synchronization with CIDR1 resulted in greater serum P₄ concentrations and smaller follicle diameters on Days 7 and 9 in both experiments. In experiment 1, treatment with CIDR for 9 days decreased the interval from CIDR removal to estrus (Day 7, 3.76 ± 0.08 days vs. Day 9, 2.90 ± 0.07; P < 0.01) and improved conception (Day 7, 57.1% vs. Day 9, 65.8%; P = 0.05) and pregnancy rates (Day 7, 37.6% vs. Day 9, 45.3%; P = 0.04). In experiment 2, treatment with ECP improved (P < 0.01) the proportion of heifers in estrus (E48, 40.9%^a; G54, 17.1%^c; and G72, 32.0%^b), but the pregnancy rate was not affected (P = 0.64) by treatments (E48, 38.8%; G54, 35.5%; G72, 37.5%). Synchronization with CIDR3 increased follicle diameter at FTAI (CIDR1, 11.07 ± 0.10 vs. CIDR3, 11.61 ± 0.10 mm; P < 0.01), ovulation rate (CIDR1, 82.8% vs. CIDR3, 88.0%; P < 0.01) and did not affect conception (CIDR1, 42.2 vs. CIDR3, 45.1%; P = 0.38) or pregnancy rates (CIDR1, 34.7 vs. CIDR3, 39.4%; P = 0.11). In conclusion, length of treatment with P₄ affected the fertility of heifers bred based on estrus detection. When the heifers were submitted to FTAI protocol, follicle diameter at FTAI (≤10.7 mm, 23.6%; 10.8–15.7 mm, 51.5%; ≥15.8 mm, 30.0%; P < 0.01) was the main factor that affected conception and pregnancy rates.     

Intrafollicular effect of IGF1 on development of follicle dominance in mares

The effect of an injection of a supraphysiologic dose of rhIGF1 into the second-largest ovarian follicle (F2) at the expected beginning of deviation (F1, > or =20 mm; Day 0) on development of dominance by F2 was studied in mares (n=16; controls, n=8). F1 became dominant (> or =28 mm) in 8 of 8 and 15 of 16 follicles in the controls and treated groups, respectively. The incidence of dominance (P<0.001) and ovulation (P<0.02) for F2 was greater for the IGF1 group (13 of 16 and 10 of 16) than for the controls (1 of 8 and 1 of 8). There were day effects but no group effects or group-by-day interactions for systemic FSH, LH, estradiol, or ir-inhibin during the 4 days after treatment. In another experiment, treatment of every follicle, excluding F1, when it reached > or =20mm after the expected beginning of deviation resulted in dominance by 8 of 12 follicles treated with rhIGF1 on Days 1-3 (n=8 mares). Results demonstrated that the IGF1 system plays a pivotal intrafollicular role in the deviation mechanism without altering systemic concentrations of the gonadotropins and ovarian follicular hormones.     

Effect of progesterone supplementation in the first week post conception on embryo survival in beef heifers

Progesterone is essential for establishment and maintenance of pregnancy in mammals. The objective of this study was to examine the effect of elevating progesterone during the different physiological stages of early embryo development on embryo survival. Estrus was synchronized in cross-bred beef heifers (n = 197, ∼2-years old) and they were inseminated 12-18 h after estrus onset (=Day 0). Inseminated heifers were randomly assigned to 1 of 3 treatments: (1) Control, n = 69; (2) progesterone supplementation using a Controlled Internal Drug Release Device (CIDR) from Day 3 to 6.5, n = 64; or (3) progesterone supplementation using a CIDR from Day 4.5 to 8, n = 64. Body condition (BCS) and locomotion scores (scale of 1-5) were recorded for all animals. Animals with a locomotion score ≥4 (very lame) were excluded. Embryo survival rate was determined at slaughter on Day 25. Conceptus length and weight were recorded and the corpus luteum (CL) of all pregnant animals was dissected and weighed. Supplementation with exogenous progesterone increased (P < 0.05) peripheral progesterone concentrations, but did not affect embryo survival rate compared with controls. Mean CL weight, conceptus length and conceptus weight were not different between treatments. There was a positive relationship (P < 0.04) between the increase in progesterone concentrations from Days 3 to 6.5 and embryo survival rate in treated heifers and a similar trend existed between the increase from Days 4.5 to 8 (P < 0.06). There was also a positive relationship (P < 0.05) between the progesterone concentration on Day 6.5 and the embryo survival rate in treated heifers. A direct correlation was seen between locomotion score and embryo survival rate, with higher (P < 0.05) early embryo survival rates in heifers with a lower locomotion score. In conclusion, supplementation with progesterone at different stages of early embryo development increased peripheral progesterone concentration and resulted in a positive association between changes in progesterone concentration during the early luteal phase and embryo survival rate. Supplementation with progesterone had no effect on either CL weight or conceptus size in pregnant animals. Lameness had a significant negative effect on early embryo survival.     

Age-related dynamics of follicles and hormones during an induced ovulatory follicular wave in mares

An ovulatory follicular wave was induced by ablation of follicles ≥6 mm and treatment with prostaglandin F2α (PGF) on Day 10 (ovulation = Day 0). Follicle and hormone dynamics of the induced waves were compared among three age groups: young (5-6 y, n = 14 waves), intermediate (10-14 y, n = 16), and old (≥18 y, n = 15). During the common-growth phase of the induced wave (Days 12-17), diameter of the future ovulatory follicle was not different among ages, but the young group had more (P < 0.05) follicles that reached ≥10 mm. The number was correlated (r = +0.7; P < 0.0001) within mares between consecutive interovulatory intervals, indicating repeatability. Concentrations of LH increased in all age groups during Days 12-17, but were greatest (P < 0.002) in the young group and continued to be greater (P < 0.0001) throughout the ovulatory LH surge. During several days before Day −1, there were no age-related effects on systemic estradiol concentrations, diameter of the preovulatory follicle, or B-mode echo texture or color-Doppler signals of blood flow in the follicle wall. Interpretations were: (1) greater number of follicles in the young group reflected a greater follicle reserve, (2) greater LH concentrations throughout the ovulatory surge in the young group reflected a more positive response to an extraovarian/environmental influence after removal of the negative effect of progesterone, and (3) lower LH concentrations in the older groups were adequate for the preovulatory changes in the follicle.     

Role of LH in luteolysis and growth of the ovulatory follicle and estradiol regulation of LH secretion in heifers

The role of LH in luteolysis and development of the ovulatory follicle and the involvement of GnRH receptors in estradiol (E2) stimulation of LH secretion were studied in heifers. A pulse of PGF_2α, as indicated by a metabolite, was induced by E2 treatment on Day 15 (Day 0 = ovulation) and LH concentration was reduced with a GnRH-receptor antagonist (acyline) on Days 15, 16, and 17. Blood samples were collected every 6 h on Days 14-17 and hourly for 10 h beginning at the Day-15 treatments. Four groups were used (n = 6): control, acyline, E2, and E2/acyline. The number of LH pulses/heifer during the 10 h posttreatment was greater (P < 0.0002) in the E2 group (2.3 ± 0.4, mean ± SEM) than in the acyline group (0.2 ± 0.2) and was intermediate in the E2/acyline group (1.4 ± 0.2). Concentrations of progesterone in samples collected every 6 h on Day 15 showed a group-by-hour interaction (P < 0.02); concentrations decreased in the acyline group but not in the control group. The 12 heifers in the combined acyline and E2/acyline groups had three follicular waves compared to two waves in 10 of 12 heifers in the combined control and E2 groups. Results (1) supported the hypothesis that LH delays the progesterone decrease associated with luteolysis, (2) supported the hypothesis that LH has a positive effect on the continued development and growth of the selected ovulatory follicle, and (3) indicated that E2 stimulates LH production through an intracellular pathway that involves GnRH receptors on the gonadotropes and a pathway that does not involve the receptors.     

Expression of receptors for BMP15 is differentially regulated in dominant and subordinate follicles during follicle deviation in cattle

Bone morphogenetic proteins are known to be involved in determining ovulation rate in mammals. The mechanisms through which these proteins determine follicle fate are incompletely understood. In the present study, we used cattle as a model to evaluate the regulation of BMP15 and GDF9 receptors in granulosa cells during dominant follicle (DF) selection. Before follicular deviation (day 2 of the follicular wave), BMPR2 mRNA abundance tended to be higher in the second largest follicles (F2; P < 0.1) compared to the future dominant follicle (F1). At the expected time of follicular deviation (day 3), BMPR2 and BMPR1B mRNA levels were higher in subordinate follicles (SFs; P < 0.05) compared to dominant follicles (DFs). After deviation (on day 4), BMPR1B mRNA and protein were significantly more abundant in atretic SFs (as assessed by cleaved caspase 3) than in DFs. The fact that BMPR1B is more expressed in atretic follicles was further confirmed by using intrafollicular treatment with two agents known to induce atresia, namely an estradiol receptor antagonist (fulvestrant) and FGF10. In conclusion, the fact that BMPR-1B and -2 are more expressed in the second largest follicles before and at the expected time of follicular deviation is indicative of their inhibitory role in follicle differentiation and steroidogenesis. BMPR1B also seems to have a pivotal role during follicle regression since it is upregulated in advanced atretic follicles.     

A combination of nutrition and genetics is able to reduce age at puberty in Nelore heifers to below 18 months

Nelore heifers usually begin their reproductive life at ⩾24 months of age mainly due to suboptimal nutritional conditions and genetics. This study aimed to determine the effect of expected progeny difference (EPD) for age at first calving and average daily gain (ADG) on puberty in Nelore (Bos taurus indicus) heifers. A total of 58 weaned heifers (initial BW=174±6 kg; age=9±1 months) were allocated into 28 feedlot pens. Heifers were born from four sires, of which two had low EPD for age at first calving (L; n=33) and two had high EPD for age at first calving (H; n=25). Then, heifers of each EPD were randomly assigned to high ADG (HG; 0.7 kg) or low ADG (LG; 0.3 kg), resulting in four treatments: heifers from L sires were submitted to either HG (LHG; n=17) or LG (LLG; n=16), and heifers from H sires were submitted to either HG (HHG; n=12), or LG (HLG; n=13). The HG heifers were fed a 75% grain diet, whereas the LG heifers received 93% of forage in their diet. Blood samples were collected at 9, 14, 18, 24 and 28 months of age for IGF1 and leptin determination. There was a treatment effect (P<0.01) on the proportion of heifers that attained puberty by 18 (62%, 0%, 0% and 0%), 24 (100%, 6%, 54% and 0%) or 36 (100%, 100%, 100% and 38%) months of age for LHG, LLG, HHG and HLG treatments, respectively. In addition, mean age at puberty was different across treatments (P<0.01). Heifers from the LHG achieved puberty at the earliest age when compared with cohorts from other treatments (18.1, 28.9, 23.9 and 34.5 months for LHG, LLG, HHG and HLG, respectively). Serum IGF1 concentrations were higher for L heifers compared with H cohorts at 9, 14, 18, 24 and 28 months of age (P<0.01; treatment×age interaction), whereas circulating leptin concentrations were higher (P<0.01; age effect) as heifers became older, regardless of the treatments. In conclusion, only Nelore heifers with favorable genetic merit for age at first calving were able to attain puberty by 18 months of age. In heifers with unfavorable genetic merit for age at first calving, supplementary feeding to achieve high ADG was unable to shift the age at puberty below 24 months.     

Regulation of the proliferative activity of ovarian surface epithelial cells by follicular fluid

Despite critical roles of the ovarian surface epithelium (OSE) in ovulation and post-ovulatory wound repair, little is known about the physiological mechanism regulating OSE proliferation. A role of follicles and corpora lutea in locally regulating the proliferative activity of OSE has been suggested. In this study, the effects of follicular and luteal products on proliferation of cultured OSE cells were tested using cells obtained from seasonally anoestrous ewes. Follicular fluid but not luteal extracts induced OSE cell proliferation (2.5-fold relative to untreated controls; P < 0.0001). The response of OSE cells was not affected by follicle size or previous charcoal-extraction of follicular fluid (P > 0.1). Treatment with IGF-1 (2.2-fold; P < 0.01), EGF (1.9-fold; P < 0.01) and, to a lesser extent, FSH (P < 0.05) also induced OSE cell proliferation. In contrast, oestradiol or progesterone did not induce cell proliferation or enhance the effects of FSH on proliferation (P > 0.1). It was concluded that follicular fluid can directly stimulate ovine OSE cell proliferation and that this effect is attributable to non-steroidal mitogens.     

Effect of bovine follicular fluid administration during or after norgestomet treatment on the fate of the proestrus dominant follicle in heifers

The objective was to examine the influence of follicle stimulating hormone (FSH) on the maintenance and ovulation of the proestrus dominant follicle (DF) in cattle. This was investigated by subjecting a proestrus DF, maintained by a single norgestomet (N) implant, to bovine follicular fluid (BFF) injections during N treatment and immediately after its removal. Earlier, we demonstrated that with an insertion of a single N implant the proestrus DF could be maintained for 9 days without affecting its ovulatory capacity. Eighteen cycling Holstein heifers were treated with a N implant at proestrus. The day of implant insertion was designated day 1 of the implant period and the implant was retained for 9 days. Heifers (n = 6 per group) were randomly allocated to receive saline for 4 days from day 5 to day 8 of the implant period and for 4 days from the day of implant removal to day 3 after removal (CONTROL) or BFF from day 5 to day 8 of implant period (BFF-DURING) or BFF from the day of implant removal to day 3 after implant removal (BFF-AFTER). Injections (10 ml) were given i.v. twice daily and the ovaries monitored by ultrasonography daily, throughout and after the implant period. All CONTROL heifers maintained the DF during treatment and ovulated following implant withdrawal. In all BFF-DURING heifers, the BFF injections caused regression of the DF and its disappearance. In three of the BFF-AFTER heifers, BFF injections caused regression of the DF. In the remaining three BFF-AFTER heifers, the DF ovulated. Mean plasma FSH concentrations did not differ (P > 0.05) between the CONTROL and BFF-DURING heifers. However, the mean plasma FSH concentrations were lower in BFF-AFTER heifers compared with CONTROLS (P < 0.05). Mean plasma LH concentrations did not differ among treatment groups (P > 0.05). In summary, BFF treatment caused atresia of the proestrus DF when maintained by N and this was not associated with suppression of circulating FSH. Administration of BFF after implant removal resulted in an equal chance of ovulation or regression of DF. Regression was associated with suppression of FSH and LH.     

Follicular populations during the estrous cycle in heifers. I. Influence of day

Ovarian follicles ⩾ 2 mm were studied in 22 Holstein heifers by daily ultrasound examinations. There were significant differences (P < 0.0001) among days of the estrous cycle for diameter of the largest and second largest follicles and in the numbers of follicles 2–3 mm, 4–6 mm, 7–10 mm, 11–13 mm, > 13 mm, and total number of follicles ⩾2 mm. Patterns of the mean profiles for all follicular endpoints except the number of follicles 4–6 mm and total number of follicles ⩾ 2 mm were bimodal. The days encompassed by the first and second portions, respectively, of the bimodal profiles were approximately: diameter of largest follicle, Days 0–14 and 15–21 (ovulation); diameter of second largest follicle, Days 0–7 and 8–20; number of follicles 2–3 mm, Days 1–11 and 12–20; number of follicles 7–10 mm, Days 0–6 and 7–18; number of follicles 11–13 mm, Days 0–8 and 9–20; and number of follicles > 13 mm, Days 2–14 and 16–21. Data for the various categories were recombined to demonstrate relationships between the numbers of follicles 2–3 mm and ⩾ 4 mm during the interovulatory interval. There were significant differences (P < 0.0001) among days in both 2–3 mm and ⩾ 4 mm follicular categories. Differences appeared due to periods of higher mean numbers of follicles 2–3 mm which began between Days 2 or 3 and Days 15 or 16 and reached maximum levels on Day 7 and Day 19, respectively. There was an inverse relationship between the number of follicles 2–3 mm vs ⩾ 4 mm and between the diameters of largest and second largest follicles. The process of selection of the follicle destined to ovulate appeared to become manifest as selective growth of the preovulatory follicle with concurrent decrease in diameter of the second largest follicle and regression of the other follicles in the various follicular categories. A similar process apparently occurred early in the interovulatory interval. There was apparently selective growth of a follicle to preovulatory size by Day 6, coincident decrease in diameter of the second largest follicle, and apparent regression of other follicles in the ultrasonically detectable pool. The only apparent difference was that the follicle which attained preovulatory diameter early in the interovulatory interval remained in the ovary for 5 or 6 days, then regressed, while the follicle which attained preovulatory diameter at approximately Day 18–20 ovulated.     

Effect of follicular wave stage on potential fertility predictors and their repeatability coefficient in prepubertal Bos indicus (Nellore) and Bos taurus (Caracu) heifers

In an attempt to develop accessible methods for the precocious individual selection based on phenotypic attributes related to reproductive superiority, this study evaluated the effects of follicular wave stages (emergence and dominance) and the repeatability coefficients of potential fertility predictors (vulvar morphometry, uterine biometry, echogenicity and echotexture, ovarian biometry, and antral follicle count – AFC) in two breeds of purebred prepubertal heifers. Nellore (n = 30) and Caracu (n = 28) heifers were submitted to a sequence of 11 evaluations conducted every 48 h (D0 – random day of the antral follicular wave until D20) to study potential fertility indicators under natural conditions. The data obtained were compared according to breed and follicular wave stage. Statistical analysis included the fixed effects of breed, evaluation day, and their statistical interaction and was performed using MIXED, GENMOD, GLM, and CORR procedures of the SAS program. Breed was found to influence rima height, ovarian area, and AFC, which were greater in Nellore heifers (P < 0.02). The follicular wave stage also influenced most of the potential predictors, highlighting AFC which was higher in the presence of a dominant follicle in both breeds (P < 0.0001). The repeatability coefficients for vulvar width (0.76 and 0.66), ovarian area (0.70 and 0.62), and AFC (0.76 and 0.74) were considered to be high in Nellore and Caracu heifers, respectively. Only ovarian biometry was able to predict AFC (Pearson correlation coefficient ≥ 0.66; P < 0.0001) in prepubertal heifers. The results indicate that most of the phenotypic reproductive parameters analyzed can be characterized throughout prepuberty using a single measure since they are intrinsic attributes of the individual.     

Active immunization of post-pubertal heifers against prostaglandin F2α to suppress oestrous behaviour: effect of adjuvant and dose of immunogen

Two experiments were performed to develop an effective prostaglandin F_2α immunization protocol to suppress oestrous behaviour in beef heifers. In Experiment 1, a 3 × 2 factorial plan (n=4–5 per treatment) was used to test three doses (3.3, 10 and 30 mg) of a prostaglandin F_2α- human serum albumin (PGF-HSA) conjugate as the immunogen and two adjuvants, GNE (proprietary product; Intervet, The Netherlands) and diethylaminoethyl (DEAE)-dextran. Heifers (n=5) in a control group were untreated. Booster immunizations were given on Days 42 and 145 after the primary immunization (Day 0) and data collection for statistical purposes ended on Day 297. After Day 42 the incidence of oestrous behaviour was: (1) greater (P < 0.05) for control than immunized heifers (4.3 and 2.2, respectively), (2) greater (P < 0.05) for heifers immunized using GNE than for heifers immunized using DEAE-dextran (2.6 and 1.9, respectively), and (3) greater for heifers immunized with 30 mg of immunogen than for those immunized with either 3.3 or 10 mg (3.1, 1.7 and 1.9, respectively). Suppression of oestrous behaviour was accompanied by formation of a persistent corpus luteum (CL). Persistent CL were formed in ten of the 28 immunized heifers and the mean (± standard error of the mean) duration of persistence was 397 ± 85 days. In Experiment 2, a 2 × 2 factorial plan (n=6–7 per treatment) was used to test two doses (1 and 10 mg) of the PGF-HSA conjugate as the immunogen and two adjuvants, non-ulcerative Freund's adjuvant (NUFA) and DEAE-dextran. A control group was untreated (n=6). Booster immunization was given on Day 183 after the primary immunization (Day 0) and the experiment finished on Day 384. Antibody titres were higher (P < 0.05) in NUFA-treated heifers than in DEAE-dextran-treated (1 mg) heifers in the 183- to 283-day period. After Day 183, oestrous behaviour was suppressed in 26 out of the 27 immunized heifers. Persistent CL were maintained for longer (P < 0.05) in NUFA-treated heifers (245 days) than in DEAE-dextran-treated heifers (166 days) but there was no difference due to dose of immunogen (208 and 203 days, 1 and 10 mg, respectively). It is concluded that immunization against PGF-HSA results in suppression of oestrous behaviour in heifers due to prolongation of the life-span of the CL; however, efficacy of response is dependent on the immunization regime used.     

Effects of a dominant follicle on ovarian follicular dynamics during the oestrous cycle in heifers

Two hypotheses were tested: (1) a dominant follicle causes regression of its subordinate follicles, and (2) a dominant follicle during its growing phase suppresses the emergence of the next wave. Cyclic heifers were randomly assigned to one of four groups (6 heifers/group): cauterization of the dominant follicle of Wave 1 or sham surgery (control) on Day 3 or Day 5 (day of ovulation = Day 0). Ultrasonic monitoring of individually identified follicles was done once daily throughout the interovulatory interval. The onset of regression (decreasing diameter) of the largest subordinate follicle of Wave 1 was delayed (P less than 0.01) by cauterization of the dominant follicle of Wave 1 on Day 3 compared to controls (mean onset of regression, Days 10.8 +/- 2.1 vs 4.3 +/- 0.4). Cauterization of the dominant follicle of Wave 1 on Days 3 or 5 caused early emergence (P less than 0.01) of Wave 2 when compared to controls (Day-3 groups: Days 5.5 +/- 0.4 vs 9.6 +/- 0.7; Day-5 groups: Days 7.0 +/- 0.3 vs 9.1 +/- 0.4). The results supported the two hypotheses. In addition, cauterization of the dominant follicle of Wave 1 on Days 3 or 5 increased the incidence of 3-wave interovulatory intervals.     

Activin A,estradiol, and free insulin-like growth factor I in follicular fluid preceding the experimental assumption of follicle dominance in cattle

In cattle, the two largest follicles of a wave (F1, F2) begin to deviate into a dominant follicle and a subordinate follicle when F1 is a mean of 8.5 mm in diameter. After the beginning of deviation, F1 and F2 are diameter-defined dominant and subordinate follicles. Changes associated with the conversion of F2 into a future dominant follicle were studied by ablating F1 at the expected beginning of deviation (F1, 8.5 mm; Hour 0) and assessing the follicular-fluid factors in F2. Follicles were designated F1C and F2C in controls and F2A in F1-ablated heifers. Follicular-fluid collections were made at Hours 0, 4, 8, or 12 (n = 7 heifers per hour; fluid from F1C, F2C, and F2A; experiment 1) or at Hours 4, 6, 8, 10, or 12 (n = 9 heifers per hour; fluid from F2A; experiment 2). Postablation concentrations of circulating FSH increased (P < 0.05) between Hours 2 and 6. Diameter of F2A increased (P < 0.05) after Hour 8 in both experiments so that the diameter of F2A at Hours 10 or 12 was not different (P > 0.1) from the diameter of F1 at Hour 0. A transient elevation (P < 0.05) in follicular-fluid activin A occurred in F2A at Hour 8 in both experiments. Concentrations of estradiol (P < 0.05) and insulin-like growth factor I (IGF-I; P < 0.1) decreased in F2C by Hour 8. In F2A, the concentrations of both factors began to increase (P < 0.05) after Hours 4 or 8 so that there was no difference (P > 0.1) between F1C and F2A at Hour 12. Concentrations of IGF-I and IGF binding protein 2 (IGFBP-2) in F2A changed in opposite directions at the same hours. No differences between follicles were found for concentrations of progesterone, androstenedione, inhibin A, and inhibin B. The order of events in the conversion of a future subordinate follicle to a future dominant follicle was an increase in systemic FSH, a transient elevation in follicular-fluid activin A, and a simultaneous increase in follicular-fluid estradiol and restoration of an apparent growth-compatible balance of free IGF-I and IGFBP-2.     

Intraovarian effect of dominant follicle and corpus luteum on number of follicles during a follicular wave in heifers

The intraovarian relationships among dominant follicle (DF), corpus luteum (CL), and number of follicles between Days 0 to 5 (Day 0 = ovulation) in wave 1 (n = 65 waves) and Days 9 to 13 in wave 2 (n = 62) were analyzed in separate experiments in Bos taurus heifers. Ovaries were grouped into intraovarian patterns of DF–CL, DF alone, CL alone, and neither DF nor CL. In wave 1, the pattern frequencies of DF–CL or neither DF nor CL (34% each) were greater (P < 0.0004) than for DF alone or CL alone (16% each). The number of growing follicles ≥5.0 mm, was greater (P < 0.0001) in ovaries with the DF, even when the DF was removed from the tally (P < 0.03). In a factorial analysis of wave 1, there was a positive main effect of DF (3.9 ± 0.2 vs. 2.2 ± 0.2 follicles; P < 0.0001), but the main effect of CL and the interaction of DF and CL were not significant. In a factorial analysis of wave 2, there were more (P < 0.0001) follicles greater than 6 mm in ovaries with a DF when the DF was included and an approaching difference (P < 0.09) when the DF was excluded. The main effect of CL and the interaction of DF and CL were not significant. The hypothesis that both the DF and CL have a positive intraovarian effect on number of follicles in waves 1 and 2 was only partly supported; the DF, but not the CL, had an effect in the factorial analyses. Previous reports in cattle and sheep of a positive intraovarian effect of CL on number of follicles are questionable in that location of the DF was not considered.     

Evaluation of treatments with hCG and carprofen at embryo transfer in a demi-embryo and recipient virgin heifer model

An in vivo model, combining a low developmental competence embryo (demi-embryo) and a high-fertility recipient (virgin dairy heifer) was used to evaluate the effects of treatment with human chorionic gonadotropin (hCG) and carprofen at embryo transfer (ET) on plasma progesterone (P₄) concentrations of recipients and on embryonic growth and survival. Embryos were bisected and each demi-embryo was transferred to a recipient on Day 7 of the estrous cycle. At ET, heifers (n = 163) were randomly allocated to treatment with hCG (2500 IU im), carprofen (500 mg iv), hCG plus carprofen or to untreated controls. Plasma P₄ concentrations were measured on Days 0, 7, 14 and 21 of all recipients plus on Days 28, 42 and 63 of pregnant recipients. Pregnancy was presumed to be present in recipients with luteal plasma P₄ concentrations until Day 21 and confirmed by using transrectal ultrasonography on Days 28, 42 and 63. Embryonic measurements (crown–rump length and width) were obtained on Day 42. Treatment with hCG induced formation of secondary corpora lutea (CL) in 97% of heifers and increased (P < 0.01) mean plasma P₄ concentrations of non-pregnant recipients on Day 14 and of pregnant heifers on Days 14 to 63. This was associated to a significant decrease in early embryonic mortality. In contrast, subsequent embryonic losses resulted in a non-significant numerical increase by 8% of pregnancies maintained to Day 63. Therefore, treatment with hCG significantly rescued embryos through the maternal recognition of pregnancy window but was not able to support development thereafter. Treatment with carprofen at ET had no significant effects on plasma P₄ concentrations and rate of embryo mortality. Treatment with hCG plus carprofen at ET induced formation of secondary CL in 90% of heifers but decreased the luteotrophic effect of hCG, resulting in no effect on embryo survival. Low developmental competence embryos showed an intrinsic deficiency in overcoming the maternal recognition of pregnancy challenge and in proceeding to further development until Day 28 of pregnancy, whereas mortality beyond this point was residual. Results on pregnancy rates should be confirmed in further experiments involving a larger sample size.