GnRH dose at initiation of a 5-day CO-Synch + P4 for fixed time artificial insemination in suckled beef cows.

The objective of the present study was to evaluate the effect of GnRH dose administered at initiation (GnRH-1) of a 5-day CO-Synch + P4 protocol on ovulatory response, expression of estrus, and fertility in suckled beef cows. Suckled beef cows (n = 1101) at four locations were randomized to receive either 100 or 200 µg of gonadorelin acetate at initiation (D-8) of a 5-day CO-Synch + P4 protocol concurrently with insertion of an intravaginal progesterone (P4) device. On D-3 the P4 device was removed, two doses of prostaglandin F2α were administered concurrently and a patch was applied to evaluate expression of estrus. Artificial insemination was performed 72 h after P4 device removal (D0) simultaneously with the administration of 100 µg of gonadorelin acetate (GnRH-2). Increasing GnRH dose at initiation of a 5-day CO-Synch + P4 did not enhance ovulatory response (P = 0.57) to GnRH-1, expression of estrus (P = 0.79), nor pregnancies per AI (P/AI; P = 0.91). Both follicle size (quadratic) and circulating P4 (linear) affected (P < 0.01) ovulatory response to GnRH-1 independent of dose. Cows that had ovulation to GnRH-1 had smaller (P < 0.001) follicle size on D-3 and reduced (P = 0.05) expression of estrus compared to cows that did not have ovulation to GnRH-1, however, P/AI did not differ (P = 0.75). In conclusion, increasing the dose of GnRH-1 in the 5-day CO-Synch + P4 protocol did not enhance ovulatory response, expression of estrus, or P/AI in suckled beef cows.

Decreasing the interval between GnRH and PGF2alpha from 7 to 5 days and lengthening proestrus increases timed-AI pregnancy rates in beef cows.

Four experiments were conducted in postpartum beef cows to evaluate the influence of reducing the interval from GnRH to PGF(2alpha) from 7 to 5d in a Select-Synch + CIDR or CO-Synch + CIDR estrous synchronization program. In Expt 1, cows (n=156) were treated with either a 7 or 5d Select-Synch + CIDR program. A second PGF(2alpha) treatment was given to all cows in all experiments at 12h after the initial PGF(2alpha) (to ensure that luteolysis occurred with the 5d program). Estrous response, interval to estrus, conception rate, and first service AI pregnancy rates were similar between treatments. In Expt 2, cows (n=223) were treated with either a 7 or 5d CO-Synch + CIDR program, with timed-AI concomitant with GnRH at 60 h after PGF(2alpha). Timed-AI pregnancy rates were similar between treatments. In Expt 3 (n=223) and 4 (n=400) cows were treated with either a 7 or 5d CO-Synch + CIDR program with timed-AI concurrent with GnRH at either 60 h (7d) or 72 h (5d) after CIDR withdrawal. Timed-AI pregnancy rates were 13.3% (P<0.05; Expt 3) and 9.1% (P<0.05; Expt 4) greater for the 5 than 7d program. In conclusion, timed-AI pregnancy rates were improved with a 5d CO-Synch + CIDR program with timed-AI at 72 h after CIDR withdrawal, compared to a 7d CO-Synch + CIDR program with timed-AI at 60 h after CIDR withdrawal.

Role of progesterone concentrations during early follicular development in beef cattle: I. Characteristics of LH secretion and oocyte quality.

Objective was to investigate the effect of different progesterone (P4) concentrations during early follicular development on luteinizing hormone (LH) secretion and oocyte characteristics in beef cows. Primiparous cows (n = 24) were estrous pre-synchronized and follicular ablation was performed (d 0) 6 days following the time of ovulation. At the time of follicular ablation, cows were assigned to either: 1) high P4 treatment - HiP4; a new CIDR was inserted on d 0 to supplement P4 from the existing corpus luteum [CL], or 2) low P4 treatment - LoP4; a previously-used CIDR and two doses of PGF 8 to 12 h apart were given on d 0. Concentrations of P4 were greater (P < 0.01) in the cows of the HiP4 than LoP4 group on d 1.5, 2.5, and 3.5. Peripheral concentrations of E2 were greater (P < 0.05) in the cows of the LoP4 than HiP4 group on d 2.5 and 3.5. Frequency of LH pulses was greater (P <  0.05) in the LoP4 than HiP4 group on d 2.5, but mean LH concentration and pulse amplitude did not differ between treatments. Number of follicles aspirated per cow, total oocytes recovered, recovery rate, percentage of oocytes graded 1 to 3, oocyte diameter, percentage BCB+ oocytes, and relative abundance of oocyte mRNA for FST did not differ (P >  0.10) between treatments. In conclusion, lower P4 concentrations during early follicular development resulted in increased LH pulse frequency and E2 concentrations, but did not affect characteristics of oocyte developmental competence.

Role of progesterone concentrations during early follicular development in beef cattle: II. Ovulatory follicle growth and pregnancy rates.

Two experiments were conducted to investigate the role of relatively lesser and greater progesterone (P4) concentrations during early follicular development on ovulatory follicle growth and pregnancy rate in beef cattle. In Experiment 1, time of ovulation was synchronized with the 5 d CO-Synch + CIDR (Controlled Internal Drug Release) program in multiparous cows (n = 241). Six days after the 2nd GnRH injection of the pre-synchronization program (d 0), ablation of follicles ≥ 5 mm in the ovaries was performed and cows were assigned to receive either a previously used CIDR and 2x-25 mg PGF2α doses 8 h apart (LoP4), or a new CIDR (HiP4). On d 5, CIDR were removed from all cows, 2x-25 mg PGF2α were administered, and estrous detection tail paint was applied. Timed artificial insemination (TAI) was performed on d 8. On d 5, P4 concentrations were greater (P <  0.01) in the HiP4 (4.9 ± 0.13 ng/mL) than LoP4 (1.0 ± 0.06 ng/mL) treatment group. Conversely, d 5 estradiol (E2) concentrations and follicular diameter were greater (P <  0.01) in the LoP4 (5.0 ± 0.23 pg/mL and 8.9 ± 0.20 mm) than HiP4 (1.5 ± 0.12 pg/mL and 7.4 ± 0.15 mm) treatment group. Follicular diameter at TAI (12.0 ± 0.12 mm, Table 1) and TAI pregnancy rate did not differ (P >  0.10) between treatment groups. In Experiment 2, a new follicular wave was induced with estradiol benzoate on d -7, and cows (n = 275) were assigned on d 0 to receive 25 mg PGF2α and either have the CIDR replaced with a new CIDR (HiP4) or the used CIDR was left in place (LoP4).Furthermore, all cows received GnRH on d 0. The CIDRs were removed from all cows on d 5 and two doses of -25 mg PGF2α were administered. Estrous detection combined with AI 12 h later (Estrus-AI) was performed for 60 h after CIDR removal with TAI coupled with GnRH administration at 72 h if estrus was not detected. The concentrations of P4 on d 5 were greater (P <  0.01) in the HiP4 (2.8 ± 0.10 ng/ml) than LoP4 (1.7 ± 0.05 ng/mL) treatment group. For cows that were detected in estrus after PGF2α administration, estrous response (83.5%) and interval to estrus (55.0 ± 0.5 h) did not differ between treatment groups. Pregnancy rate (combined Estrus-AI and TAI) that resulted from breeding at the time of the synchronized time of estrus was similar between treatment groups (HiP4: 77.1%; LoP4: 82.3%). In conclusion, differences in P4 concentrations during early follicular development do not effect pregnancy rate in beef cows when the cows are inseminated at the time of a synchronized estrus if the cows have similar intervals of proestrus.

Steroidogenic changes and steady state amount of messenger RNA encoding steroidogenic enzymes, gonadotropin receptors and cell-death signalling in the dominant ovarian follicle during estradiol-induced atresia in cattle.

Changes in steroidogenic function and associated gene expression were characterized in dominant ovarian follicles (DF) of cattle where follicles were induced to become atretic by systemic administration of estradiol benzoate (EB). In experiment 1, follicular fluid (FF) steroid concentrations in the DF were measured at 12-hourly time points for 48 h in heifers treated with 1 mg EB i.m./500 kg body weight (EB; n=20) as compared with untreated controls (C; n=19). Treatment with EB promoted a transient reduction in circulating FSH, a rapid (12 h) and sustained reduction in FF estradiol, a rapid (12 h) but transient reduction in FF progesterone and a delayed (36 h) increase in FF testosterone concentrations. In experiment 2, whole follicular wall tissue was collected from DF of mature non-lactating cows allocated to a 0 h control group (0 HC: n=7), a 24h control group (24 HC; n=7) or an EB-treated group where tissue was collected 24 h after administration of 1 mg EB i.m./500 kg body weight (EB; n=8). As for experiment 1, EB promoted a transient reduction in circulating FSH, a pronounced reduction in FF estradiol and a smaller but significant reduction in FF progesterone concentrations. Semi-quantitative RT-PCR on follicular wall tissue revealed that the loss in estrogen activity at 24 h after EB was associated with two-fold reduction in aromatase mRNA, with an apparent acceleration in loss of 17alpha-hydroxylase mRNA. Expression of genes for gonadotropin receptors (LHR and FSHR) and a cell-death signalling pathway (Fas antigen and Fas ligand) were unchanged during the initial 24h of EB-induced atresia. These results suggest that EB initiates atresia in dominant ovarian follicles through a rapid suppression of follicular estradiol synthesis, an effect associated with down-regulation of the aromatase gene. A transient suppression in circulating FSH following administration of EB appears to have initiated these events, and it is suggested that subsequent processes involved in atresia follow this loss in estrogenic function.

Comparative response of rams and bulls to long-term treatment with gonadotropin-releasing hormone analogs.

The objective was to compare the relative response between rams and bulls in characteristics of LH, FSH and testosterone (T) secretion, during and after long-term treatment with GnRH analogs. Animals were treated with GnRH agonist, GnRH antagonist, or vehicle (Control) for 28 days. Serial blood samples were collected on day 21 of treatment, and at several intervals after treatment. Injections of natural sequence GnRH were used to evaluate the capacity of the pituitary to release gonadotropins during and after treatment. Treatment with GnRH agonist increased basal LH and T concentrations in both rams and bulls, with a greater relative increase in bulls. Endogenous LH pulses and LH release after administration of GnRH were suppressed during treatment with GnRH agonist. Treatment with GnRH antagonist decreased mean hormone concentrations, LH and T pulse frequency, and the release of LH and T after exogenous GnRH, with greater relative effects in bulls. Rams previously treated with antagonist had a greater release of LH after administration of GnRH compared with control rams, while rams previously treated with agonist showed a reduced LH response. Bulls previously treated with agonist had reduced FSH concentrations and LH pulse amplitudes compared with control bulls while bulls previously treated with antagonist had greater T concentrations and pulse frequency. The present study was the first direct comparison between domestic species of the response in males to treatment with GnRH analogs. The findings demonstrated that differences do occur between rams and bulls in LH, FSH and testosterone secretion during and after treatment. Also, the consequences of treatment with either GnRH analog can persist for a considerable time after discontinuation of treatment.

Effects of maturity of the potential ovulatory follicle on induction of oestrus and ovulation in cattle with oestradiol benzoate.

The effect of maturity of the dominant follicle (DF) on the capacity of oestradiol benzoate (ODB) to induce oestrus and ovulation was examined in cattle. In experiment 1, 31 prepubertal heifers each received an intravaginal progesterone insert (IPI) and 1mg ODB i.m./500kg BW (ODB1). Daily ovarian ultrasonography detected emergence of a new follicular wave 3.1+/-0.1 days after ODB1. The IPI was removed when newly emerged DF were "young" (1.3+/-0.1 days after emergence; YDF; n=15) or "mature" (4.2+/-0.1 days; MDF; n=16), and 24h later, heifers received 0.75mg ODB/500kg BW (ODB2; n=16) or no further treatment (NoODB2; n=15). Most of the heifers receiving ODB2 were observed in oestrus (15/16) and ovulated (12/16), as compared to 0/15 and 1/15 in the NoODB2 group, respectively (P<0.01). In experiment 2, 32 heifers received ODB1 on day 6 of the oestrous cycle, and new follicular wave emergence was detected 3.2+/-0.1 days later. Heifers received an injection of prostaglandin-F2alpha (PGF) when the DF was young (1.1+/-0.1 days after emergence; YDF; n=16) or mature (4 days; MDF; n=16), and then ODB2 24h later or no further treatment (NoODB2). The interval from PGF to oestrus was greater (P<0.01) in the YDF-NoODB2 (70+/-3.9h) as compared to MDF-NoODB2 group (57+/-1.8h). Inclusion of ODB2 reduced (P<0.01) this interval to 47.0+/-0.7h without regard to the maturity of the DF (maturityxODB2, P<0.05) and also reduced (P<0.05) the interval to ovulation. In experiment 3, 21 suckling anoestrous cows received an IPI and ODB1 at 29.3+/-1.7 days postpartum. The IPI were removed either 1 day (YDF; n=9) or 3.9+/-0.1 days (MDF; n=9) after emergence of a new follicular wave and every cow received ODB2. Oestrus was subsequently detected in all but one animal. Ovulation of the newly emerged DF was detected within 48h of ODB2 in nine of nine cows of the MDF group, and in four of nine of the YDF group (P<0.05). During the subsequent ovulatory cycle, luteal size and plasma concentrations of progesterone were greater (P<0.01) in the MDF group compared to the YDF group. We conclude that behavioural oestrus is readily induced by 0.75mg ODB i.m./500kg BW. Maturity of the DF appeared to have little influence on the ability of the DF to ovulate in heifers. In contrast, young DF in lactating anoestrous cows were less likely to respond to the ovulatory cue provided, and luteal development was compromised in those that did ovulate.

Estradiol benzoate delays new follicular wave emergence in a dose-dependent manner after ablation of the dominant ovarian follicle in cattle.

Administration of estradiol benzoate (EB) induces atresia of the dominant follicle (DF) in the ovaries of cattle within 36 h but emergence of a new wave of follicular development is delayed by 3-5 days. The present study investigated the role of EB in determining timing of emergence of a new follicular wave after removing the influence of the DF. At 6.4+/-0.2 days after ovulation in Angus and Angus/Simmental cattle (n=26), aged 4.9+/-0.6 years and weighing 634+/-20 kg, all ovarian follicles > or =5mm in diameter were aspirated with a 17-gauge needle using an ultrasound-guided transvaginal approach (Day 0 or Hour 0) and animals immediately received 0 (0EB), 1 (1EB), 2 (2EB) or 4 (4EB) mg EB i.m./500 kg body weight (n=6 or 7 per treatment). Ovarian structures were monitored by ultrasonography on a daily basis until emergence of a new wave of follicular development. Concentrations of estradiol (E2) were different among all treatments between Hours 24 and 72, increasing (P<0.01) with greater doses of EB administered. Hour of peak follicle-stimulating hormone (FSH) was 29.3+/-4.0, 53.3+/-4.5, 81.1+/-15.5, and 91.4+/-8.2 for the 0EB, 1EB, 2EB, and 4EB treatments, respectively, and emergence of a new wave of follicular development occurred on Days 1.5+/-0.2, 3.3+/-0.3, 4.0+/-0.6 and 4.4+/-0.4, respectively. Timing of peak FSH and emergence of a new wave of follicular development was earliest (P<0.05) in the 0EB treatment, similar (P>0.1) among the 1EB and 2EB treatments, and most delayed (P<0.05) in the 4EB treatment when compared to the 0EB or 1EB treatments. The overall mean interval from peak FSH to emergence of a new wave of follicular development was 15.7+/-3.3 h and was not affected by treatment. Concentrations of E2 at 24 h before new emergence were not different among EB-treated animals (20.2+/-5.5 pg/ml), but lower (P<0.01) in the 0EB treatment (1.6+/-0.2 pg/ml). In a dose-dependent manner, EB delayed the pre-emergence surge in FSH that stimulates new follicular development after the DF has ceased to be functional. The importance of using an 'optimal' dose of EB in hormonal regimens using this agent to strategically regulate follicular development is emphasized by the outcomes of this study.

Comparison of follicular dynamics and hormone concentrations between the 7-day and 5-day CO-Synch + CIDR program in primiparous beef cows.

The objectives were to compare follicular dynamics, preovulatory estradiol concentrations, and progesterone concentrations between the 7-day (7CO, n = 15) and 5-day (5CO, n = 13) CO-Synch + controlled internal drug release device (CIDR) program in primiparous suckled beef cows. On Day -7 (7CO) or Day -5 (5CO), GnRH (100 µg) was administered (GnRH-1) and a CIDR was inserted. On Day 0, hour 0, CIDR was removed and cows received PGF2α (25 mg) at hours 0 and 12. Animals were administered GnRH (100 µg, GnRH-2) at either hour 60 (7CO) or 72 (5CO). Follicular growth and ovulation to both GnRH-1 and GnRH-2 were evaluated using ultrasonography. Concentrations of estradiol were determined in blood samples taken at hours 0, 36, 60, and 72 (5CO). Blood samples were collected on Days 5, 8, and 14 for progesterone quantification. Ovulation rate to GnRH-1 did not differ between the 7CO (11/15) and 5CO (8/13) treatments, and for all dependent variables the statistical model included treatment, ovulation to GnRH-1, and their interaction. Diameter (mm) of the ovulatory follicle did not differ between treatments (13.4 ± 0.3) but was greater (P < 0.05) in cows that responded to GnRH-1 (13.8 ± 0.3) than those did not (12.6 ± 0.6). Maximum estradiol concentrations tended (P = 0.06) to be greater in the 5CO (7.3 ± 0.5 pg/mL) than 7CO (6.1 ± 0.7 pg/mL) treatment and tended to be greater (P = 0.08) in cows that responded to GnRH-1 (7.1 ± 0.5 pg/mL) than those did not (5.6 ± 0.9 pg/mL). Three cows in the 7CO treatment failed to develop a CL after GnRH-2. There was a treatment by response to GnRH-1 interaction (P < 0.05) for progesterone concentrations. In cows that did not respond to GnRH-1 in the 7CO treatment, progesterone concentrations were less (P < 0.05) than in those that responded to GnRH-1 in the 7CO treatment and tended (P = 0.09) to be less than in cows in the 5CO treatment that did not respond to GnRH-1. In conclusion, these findings demonstrate that failure to respond to GnRH-1 is detrimental to estradiol and progesterone concentrations with a 7-day interval between GnRH-1 and PGF2α but of little consequence when this interval is shortened to 5 days.

Effect of follicle age on conception rate in beef heifers.

The objective of this study was to determine the effect of age of the ovulatory follicle on fertility in beef heifers. Ovulation was synchronized with the 5 d CO-Synch + controlled intravaginal drug release (CIDR) program in heifers in Montana (MT; n = 162, Hereford and Angus Crossbred) and Ohio (OH; n = 170, Angus Crossbred). All heifers received estradiol benzoate (EB; 1 mg/500 kg BW, [i.m.]) 6 d after the final GnRH of the synchronization program to induce follicular atresia and emergence of a new follicular wave (NFW) followed by prostaglandin F2α (PGF(2α); 25 mg, i.m.) administration either 5 d ("young" follicle [YF]; n = 158) or 9 d ("mature" follicle [MF]; n = 174) after EB. Estrous detection was performed for 5 d after PGF(2α) with AI approximately 12 h after onset of estrus. Ovarian ultrasonography (MT location only) was performed in YF and MF at EB, 5 d after EB, PGF(2α), and AI. Heifers in MT (n = 20) and OH (n = 18) that were not presynchronized or did not initiate a NFW were excluded from further analyses, resulting in 142 and 152 heifers in MT and OH, respectively. Heifers from the MF treatment in MT that initiated a second NFW after EB but before PGF(2α) (MF2; n = 14) were excluded from the primary analysis. In the secondary analysis, the MF2 group was compared to MF and YF treatments in MT. Estrous response was similar (90%; 252/280) between treatments and locations. Proestrus interval (from PGF(2α) to estrus) and age of the ovulatory follicle at AI were similar for MF heifers between locations (54.6 ± 1.7 h and 8.3 ± 0.07 h) but were greater (P < 0.01) for YF heifers in OH (78.5 ± 1.4 h and 5.3 ± 0.06 h) than MT (67.4 ± 1.6 h and 4.8 ± 0.06 h; treatment × location, P < 0.01). However, conception rate did not differ for MF (63.8%; 74/116) and YF (67.0%; 91/136) treatments. In the MT heifers, follicle size and follicle age at AI in the YF treatment (10.4 ± 0.15 mm and 4.8 ± 0.06 d, respectively) was less (P < 0.01) than in the MF treatment (11.0 ± 0.18 mm and 8.3 ± 0.11 d, respectively), but conception rate to AI did not differ between treatments in MT. In the MF2 group proestrus interval was greater (P < 0.01); hence, diameter of the ovulatory follicle and age were similar to that for the YF treatment. Conception rate to AI did not differ between MF2, MF, and YF (61.5, 63.3, and 64.7%, respectively) in MT. In conclusion, manipulation of age of the nonpersistent ovulatory follicle at spontaneous ovulation did not influence conception rate.

The requirement of GnRH at the beginning of the five-day CO-Synch + controlled internal drug release protocol in beef heifers.

The objective of this study was to determine if the omission of GnRH at controlled internal drug release device (CIDR) insertion would impact pregnancy rates to timed AI (TAI) in beef heifers enrolled in a 5-d CO-Synch + CIDR protocol that used 1 PGF2α dose given at CIDR removal. Yearling beef heifers in Ohio in 2 consecutive breeding seasons (2011, n = 151, and 2012, n = 143; Angus × Simmental), Utah (2012, n = 265; Angus × Hereford), Idaho (2012, n = 127; Charolais), and Wyoming (2012, n = 137; Angus) were enrolled in the 5-d CO-Synch + CIDR protocol. At CIDR insertion (d -5), heifers were randomly assigned either to receive 100 µg GnRH (GnRH+; n = 408) or not to receive GnRH (GnRH-; n = 415). At CIDR removal (d 0 of the experiment), 25 mg PGF2α was administered to all heifers. All heifers were inseminated by TAI and given 100 µg GnRH 72 h after PGF2α (d 3). In heifers at the Ohio locations (n = 294), presence of a corpus luteum (CL) at CIDR insertion (d -5) was determined via assessment of progesterone concentrations (2011) and ovarian ultrasonography (2012). Subsequently, in both years, ovarian ultrasound was conducted on d 0 to determine the presence of a new CL. In this same subgroup of heifers, blood samples for progesterone analysis were collected on d 3 to assess luteal regression. Pregnancy diagnosis was performed between 32 and 38 d after TAI. At CIDR withdrawal, presence of a new CL was greater (P < 0.05) in the GnRH+ (55.8%, 82/147) than GnRH- (26.5%, 39/147) treatment. Incidence of failed luteal regression did not differ between the GnRH+ (3.4%) and GnRH- (0.7%) treatments. Pregnancy rate to TAI did not differ between the GnRH+ (50.5%) and GnRH- (54.9%) treatments. In conclusion, although the incidence of a new CL at CIDR removal was increased in the GnRH+ treatment, omission of the initial GnRH treatment in the 5-d CO-Synch + CIDR protocol did not influence TAI pregnancy rate in yearling beef heifers. In addition, a single dose of PGF2α at CIDR removal was effective at inducing luteolysis in yearling beef heifers enrolled in the 5-d CO-Synch + CIDR protocol, regardless of whether or not the initial GnRH treatment was given.

Impact of preovulatory estradiol concentrations on conceptus development and uterine gene expression.

This experiment was conducted to determine the effect of altering preovulatory estradiol concentrations, through manipulation of length of proestrus, on peripheral progesterone concentrations, conceptus development, interferon tau (IFNT) production and uterine gene expression in cattle. Approximately 6 days after a time-synchronized ovulation, all antral follicles (≥5 mm) were ablated from the ovaries in beef heifers. To manipulate preovulatory estradiol concentrations, the length of proestrus prior to the GnRH-induced LH surge was altered between treatments. Heifers were administered PGF(2α) either -2.5 days (2.5 days of proestrus; HiE2; n=5) or -1.5 days (1.5 days of proestrus; LoE2; n=5) prior to GnRH (Day 0 of the experiment; 6.75 days after follicle ablation). Follicular dynamics and estradiol concentrations were evaluated during proestrus and progesterone concentrations were analyzed in the subsequent estrous cycle. On Day 7, embryos were transferred into all heifers using standard procedures. On Day 15.5 heifers were slaughtered, the reproductive tract was flushed to collect the conceptus and uterine flush media, and the uterine tissue was processed for subsequent analyses. Peripheral progesterone concentrations, conceptus development and IFNT production were similar between treatments. However, amount of nuclear progesterone receptor in the deep glandular epithelium and mRNA concentrations for estradiol receptor alpha (ESR1) in the uterine endometrium were less in the LoE2 than HiE2 treatment. These changes in uterine characteristics in heifers with lower preovulatory estradiol concentrations were not related to aspects of conceptus development monitored, however, it is speculated that the alterations in mRNA and receptor protein detected may contribute to pregnancy failure subsequent to day 15.5 of gestation.

Controlling the dominant follicle in beef cattle to improve estrous synchronization and early embryonic development.

Estrous synchronization and timed ovulation programs that permit AI at a predetermined time (timed AI) rather than as determined by detection of spontaneous estrus are requisite for increased adoption of AI in the beef cattle industry. In the past two decades, significant progress has been achieved in developing programs that synchronize ovulation to address this need. While this progress has been driven by a multitude of fundamental discoveries in reproductive biology, the greatest impact in the past two decades has been the result of enhanced understanding of the pattern of ovarian follicle growth in cattle and development of technologies to coordinate growth and ovulation of the dominant follicle. At present, estrous synchronization programs that result in acceptable timed AI pregnancy rates are available for beef cattle. The capacity to control growth of the dominant follicle and evaluate the impact of various approaches on fertility has resulted in greater understanding of the factors that influence maturity of ovulatory follicles. Modifications to the standard industry breeding programs, with the aim of lengthening and/or increasing the gonadotropic stimulus and estradiol production by preovulatory follicles, have been shown to substantially increase timed AI pregnancy rate in beef cattle. Associations between characteristics of follicular development and fertility have surfaced from application of estrous and ovulation synchronization technologies and led to investigation of the fundamental mechanisms that underlie these relationships.

Influence of the length of proestrus on fertility and endocrine function in female cattle.

Previous research from our laboratory in beef cattle suggests that ovarian follicle maturity and subsequent fertility is influenced by length of proestrus across a range of follicle sizes. To test this hypothesis an animal model was used in which ovulation from similar sized follicles was induced following either a long (LPE; approximately 2.25 days) or short (SPE; approximately 1.25 days) proestrus (interval from PGF(2alpha) administration to a GnRH-induced LH surge). Specific objectives were to compare pregnancy rates and luteal phase concentrations of progesterone (Experiment 1) and to characterize preovulatory concentrations of estradiol, the GnRH-induced LH surge, and concentrations of progesterone in the subsequent estrous cycle (Experiment 2) between the LPE and SPE treatments. In Experiment 1, ovulation from follicles that were previously synchronized using follicular aspiration was induced with GnRH (Day 0) after either 2.25 days (LPE; n=40) or 1.25 days (SPE; n=38) of proestrus. Lactating and non-lactating cows were inseminated 12h following GnRH administration. Ovulatory follicle diameter was similar between treatments. Pregnancy rates to AI were greater (P<0.01) in the LPE (50.0%) compared to the SPE (2.6%) treatment. The proportion of cows having a short luteal phase in the subsequent estrous cycle was greater (P<0.01) in the SPE than LPE treatment. In cows with a luteal phase of normal length, timed-AI pregnancy rates and concentrations of progesterone in the subsequent luteal phase were greater (P<0.05) in the LPE than SPE treatment. In Experiment 2, a similar experimental approach was taken with non-lactating beef cows and ovulation was induced following either 2.2 days (LPE; n=8) or 1.2 days (SPE; n=8) of proestrus. Ovulatory follicle diameter was similar between treatments. Concentrations of estradiol during the proestrus period were greater (P<0.05) in the LPE than SPE treatment from Days -1.9 to Day 0 (GnRH administration). Concentration of LH during the GnRH-induced LH surge and concentrations of progesterone in the subsequent estrous cycle did not differ between treatments although there was a tendency (P=0.10) for increased incidence of short luteal phases in the SPE treatment. In conclusion, decreasing the length of proestrus before induction of ovulation of a large follicle resulted in lesser pregnancy rates and an increased incidence of short luteal phases. The impact of a shortened proestrus on concentrations of progesterone in cows with luteal phases of normal length varied among experiments. Decreased circulating concentrations of estradiol during the preovulatory period and/or reduced concentrations of progesterone during the subsequent estrous cycle in cows that do not experience a shortened luteal phase may represent the mechanism responsible for reduced fertility with the SPE treatment.

Influence of premature induction of a luteinizing hormone surge with gonadotropin-releasing hormone on ovulation, luteal function, and fertility in cattle.

We tested the hypothesis that luteal function and fertility would be reduced in cattle induced to ovulate prematurely compared with those ovulating spontaneously. Estrus was synchronized in 56 beef cows (24 that were nonlactating and 32 that were nursing calves). At 6.4 +/- 0.1 d after estrus, all follicles > or = 5 mm were aspirated (day of aspiration = d 0) with a 17-gauge needle using the ultrasound-guided transvaginal approach. On d 1.5 and 2, cows were administered 2 luteolytic doses of PGF2alpha. Ovarian structures were monitored by transrectal ultrasonography from d -2 to 12, or ovulation. Emergence of a new follicular wave occurred on d 1.7 +/- 0.1. When the largest follicle of the newly emerged wave was 10 mm in diameter (d 4.8 +/- 0.1), cows were assigned on an alternating basis to receive 100 microg of GnRH (GnRH-10; n = 29) to induce ovulation or, upon detection of spontaneous estrus, to the spontaneous (SPON) treatment (n = 24). Cows were bred by AI at 12 h after GnRH (GnRH-10) or 12 h after the onset of estrus (SPON) as detected using an electronic surveillance system. Blood samples were collected every other day beginning 2 d after ovulation until pregnancy diagnosis 30 d after AI. Ovulation and AI occurred in 29/29 cows in the GnRH-10 and in 24/24 cows in the SPON treatment. Ovulation occurred later (P < 0.05) in the SPON (d 7.7 +/- 0.1) than GnRH-10 (d 6.8 +/- 0.1) treatment. Double ovulations were detected in 47% of cows, resulting in 1.5 +/- 0.1 ovulations per cow. Diameters of the ovulatory and the second ovulatory (in cows with 2 ovulations) follicles were greater (P < 0.05) in the SPON (12.0 +/- 0.3 mm and 10.5 +/- 0.4 mm, respectively) than in the GnRH-10 (10.7 +/- 0.1 mm and 9.2 +/- 0.3 mm) treatment. Cross-sectional areas of luteal tissue and plasma concentrations of progesterone during the midluteal phase were greater (P < 0.05) in the SPON (3.62 +/- 0.2 cm2 and 6.4 +/- 0.3 ng/mL) than in the GnRH-10 (3.0 +/- 0.2 cm2 and 5.4 +/- 0.2 ng/mL) treatment. The conception rate to AI in the SPON (100%) treatment was greater (P < 0.05) than in the GnRH-10 (76%) treatment. The animal model used in this study resulted in unusually high conception rates and double ovulations. In conclusion, premature induction of the LH surge reduced the diameter of ovulatory follicle(s), the luteal function, and the conception rate to AI.

Induction of precocious puberty in heifers I: enhanced secretion of luteinizing hormone.

In beef heifers weaned between 3 and 4 mo of age and fed a high-concentrate diet, approximately 50% reach puberty before 300 d of age (precocious puberty). The objectives of this experiment were 1) to determine whether precocious puberty could be induced experimentally by weaning heifers early and feeding a high-concentrate diet, and 2) to determine the dynamics of secretion of LH associated with precocious puberty. Crossbred Angus and Simmental heifer calves were weaned at 73 +/- 3 d of age and 115 +/- 3 kg of BW and fed a high-concentrate (60% corn; HI, n = 9) or control diet (30% corn; CONT, n = 9). Heifers were fed individually, and target BW gains were 1.50 and 0.75 kg/d for the HI and CONT treatments, respectively. Heifers were weighed every 2 wk. Blood samples were collected weekly and assayed for progesterone concentration to determine age at puberty. Serial blood samples were collected at 20-min intervals for 24 h at mean ages of 102, 130, 158, 172, 190, 203, 217, 231, and 259 d and assayed for LH concentration to evaluate the dynamics of secretion of LH. Heifers fed the HI diet exhibited greater BW gain (P < 0.01) than CONT heifers (1.27 +/- 0.05 vs. 0.85 +/- 0.05 kg/d, respectively). As a result, BW in the HI treatment was greater (P < 0.01) than in the CONT treatment by 188 d of age and remained different through the end of the experiment. Precocious puberty occurred in 8 of 9 heifers fed the HI diet and 0 of 9 heifers fed the CONT diet. Age at puberty was reduced in the HI (P < 0.01) compared with the CONT heifers (262 +/- 10 vs. 368 +/- 10 d of age, respectively). Body weight at puberty was also reduced in the HI (P < 0.05) compared with the CONT treatment (327 +/- 17 vs. 403 +/- 23 kg, respectively). Heifers attaining puberty during the experiment continued with subsequent luteal phases as evidenced by cyclic patterns of progesterone concentrations. Frequency of pulses of LH (pulses/24 h) increased with age (P < 0.01) for both treatments. Heifers in the HI treatment exhibited a greater number of pulses of LH (P < 0.01) than those in the CONT treatment by 190 d of age and in all subsequent collection periods (treatment x age, P < 0.05). Mean LH concentrations also increased with age (P < 0.01) for both treatments but did not differ between treatments. In conclusion, precocious puberty induced by early weaning and feeding of a high-concentrate diet is preceded by increasing frequency of pulses of LH.

Induction of precocious puberty in heifers III: hastened reduction of estradiol negative feedback on secretion of luteinizing hormone.

Precocious puberty (<300 d of age) can be induced in beef heifers by early weaning and feeding a high-concentrate diet. The objective of this experiment was to determine whether precocious puberty occurs as a result of a hastened reduction of estradiol negative feedback on secretion of LH. Thirty crossbred Angus and Simmental heifers were weaned at 83 +/- 2 d of age and 114 +/- 3 kg of BW, blocked by BW, and randomly assigned to receive a high-concentrate (60% corn; H) or control (30% corn; C) diet and to receive ovariectomy (OVX), OVX plus an estradiol implant (OVXE), or to remain intact (INT). Residual ovarian tissue after OVX necessitated withdrawal of 6 heifers during the course of the experiment, resulting in the following treatment groups: OVX-C, n = 3; OVX-H, n = 5; OVXE-C, n = 4; OVXE-H, n = 2; INT-C, n = 5; INT-H, n = 5. To determine concentrations of progesterone and estradiol, blood samples were collected weekly beginning at a mean age of 160 d. To characterize LH concentrations, serial blood samples were collected at 12-min intervals for 12 h at mean ages of 119, 149, 188, 217, 246, 281, 323, 365, 407, and 449 d. By a mean age of 202 d, heifers fed the H diet were heavier (P < 0.05) than those fed the C diet. Heifers in the INT-H treatment attained puberty earlier (P < 0.05) than in the INT-C treatment (275 +/- 30 vs. 385 +/- 14 d of age, respectively). Overall mean concentrations of estradiol did not differ between OVXE-H and OVXE-C, between INT-H and INT-C, or between OVXE and INT treatments. The OVX treatments exhibited greater LH pulse frequency than the OVXE and INT treatments by the first serial blood collection (treatment x age, P < 0.05). The frequency of LH pulses was greater (P < 0.05) in the INT-H than the INT-C treatment by a mean age of 246 d and was greater (P < 0.05) in the OVXE-H than the OVXE-C treatment by a mean age of 281 d. In the OVXE-H treatment, LH secretion increased and subsequently "escaped" from estradiol negative feedback (detection of > or = 1 LH pulse/h) earlier (P < 0.05) than in the OVXE-C treatment (307 +/- 30 and 420 +/- 21 d of age, respectively). It is concluded that advancing the reduction of estradiol negative feedback on secretion of LH is the mechanism by which early weaning and feeding a high-concentrate diet results in precocious puberty in heifers.

Induction of precocious puberty in heifers II: advanced ovarian follicular development.

Precocious puberty can be induced in a majority of heifers weaned early and fed a high-concentrate diet. The objective of this experiment was to determine whether induction of precocious puberty is associated with an acceleration of ovarian maturation in heifers. Crossbred Angus and Simmental heifer calves were weaned at 104 +/- 2 (n = 18; early weaned) or 208 +/- 3 (n = 10; normal-weaned, NW) d of age. The early weaned heifers were fed a high-concentrate (60% corn; EWH, n = 9) or control diet (30% corn; EWC, n = 9). The NW heifers were also fed the control diet after weaning. Daily transrectal ultrasonography was performed to characterize a complete follicular wave beginning at a mean age of 126, 161, 196, 224, and 252 (EWH and EWC), or 224 and 252 (NW) d. Blood samples were collected daily during periods of ultrasonography to determine estradiol concentrations and weekly beginning at mean ages of 153 (EWH and EWC) or 216 (NW) d to be analyzed for progesterone concentrations. Heifers in the EWH treatment were heavier (P < 0.01) than EWC heifers from a mean age of 175 d through the end of the study (treatment x age; P < 0.05). Body weights did not differ between EWC and NW. At mean ages of 196 and 224 d, the maximum diameter of the dominant follicle (MaxDF) was greater (P < 0.05) in EWH than EWC heifers. At a mean age of 224 d, MaxDF was greater (P < 0.05) in EWC than NW heifers but was not different by a mean age of 252 d. All EWH, 5 of 9 EWC, and 5 of 10 NW heifers attained puberty at less than 300 d of age (precocious puberty). Age at puberty was less (P < 0.05) in EWH (252 +/- 9 d) than in EWC and NW (308 +/- 26 and 330 +/- 25 d, respectively) treatments. Across all heifers, MaxDF and duration of follicular waves increased with age (P < 0.05), mean number of follicles during follicular waves decreased with age (P < 0.05), and peak concentrations of estradiol during follicular waves increased until a mean age of 224 d. To further characterize aspects of precocious puberty, heifers were compared across treatments between those that experienced precocious puberty and those that did not. In heifers that experienced precocious puberty, BW at puberty was less (P < 0.01) and MaxDF, follicular wave duration, and peak estradiol concentrations were greater (P < 0.05) compared with heifers that did not experience precocious puberty. Ovarian maturation was accelerated in heifers that were weaned early and fed a high-concentrate diet and was associated with precocious onset of puberty.

Testicular development of Zebu bulls after chronic treatment with a gonadotropin-releasing hormone agonist.

The objective was to compare testis characteristics of Zebu bulls treated with the GnRH agonist, deslorelin, at different times and for different durations during their development. An additional objective was to determine the usefulness of a stain for the transcription factor GATA-binding protein 4 (GATA-4) as a specific marker for Sertoli cell nuclei in cattle. Bulls (54) were allocated to nine groups (n = 6) and received s.c. deslorelin implants as follows: G1 = from birth to 3 mo of age; G2 = from 3 to 6 mo; G3 = from 6 to 9 mo; G4 = from 9 to 12 mo; G5 = from birth to 15 mo; G6 = from 3 to 15 mo; G7 = from 6 to 15 mo; G8 = from 12 to 15 mo; and G9 (control) = no implant. Bulls were castrated at 19 mo of age. Paraffin sections (10 microm) were subjected to quantitative morphometry and GATA-4 immunohistochemistry. At castration, all bulls in the control group (6/6) had attained puberty (scrotal circumference > or = 28 cm), whereas a smaller proportion (P < 0.05) had reached puberty in G2 (2/5) and G6 (1/6). Bulls in G2 and G6 also had a lesser (P < 0.05) testis weight compared with the control group. Total volume of seminiferous epithelium and total daily sperm production in G2 and G6 were only half that observed in the control group. Spermatids were observed in less than 50% of seminiferous tubules in G2, G6, and G7 compared with 82% in the control group (P < 0.05). Staining for GATA-4 was specific for and abundant in the Sertoli cell nucleus in both pre- and postpubertal bulls, and no other cell nucleus inside the seminiferous tubule was positive for GATA-4. Total number of Sertoli cells was not affected by treatment (P = 0.45), but nuclear volume was smaller in G2 and G6 (P < 0.05) compared with the control group. In conclusion, treatment of Zebu bulls with deslorelin had no apparent beneficial effect on testis development and delayed puberty when treatment was initiated at 3 mo of age. Staining for GATA-4 was a useful method for identifying and quantifying Sertoli cell nuclei in both pre- and postpubertal bulls.

Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles.

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.