Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage.

OBJECTIVE: The adamalysin metalloproteinase 15 (ADAM15) has been shown to protect against development of osteoarthritis in mice. Here, we have investigated factors that control ADAM15 levels in cartilage. DESIGN: Secretomes from wild-type and Adam15 -/- chondrocytes were compared by label-free quantitative mass spectrometry. mRNA was isolated from murine knee joints, either with or without surgical induction of osteoarthritis on male C57BL/6 mice, and the expression of Adam15 and other related genes quantified by RT-qPCR. ADAM15 in human normal and osteoarthritic cartilage was investigated similarly and by fluorescent immunohistochemistry. Cultured HTB94 chondrosarcoma cells were treated with various anabolic and catabolic stimuli, and ADAM15 mRNA and protein levels evaluated. RESULTS: There were no significant differences in the secretomes of chondrocytes from WT and Adam15 -/- cartilage. Expression of ADAM15 was not altered in either human or murine osteoarthritic cartilage relative to disease-free controls. However, expression of ADAM15 was markedly reduced upon aging in both species, to the extent that expression in joints of 18-month-old mice was 45-fold lower than in that 4.5-month-old animals. IL-13 increased expression of ADAM15 in HTB94 â€‹cells by 2.5-fold, while modulators of senescence and autophagy pathways had no effect. Expression of Il13 in the joint was reduced with aging, suggesting this cytokine may control ADAM15 levels in the joint. CONCLUSION: Expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging human and murine joints, possibly due to a concomitant reduction in IL-13 expression. We thus propose IL-13 as a novel factor contributing to increased osteoarthritis risk upon aging.

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.

Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls II: Endocrinology, puberty attainment, and mature sperm production in Holstein bulls.

The use of genomic testing in the cattle industries has renewed an interest in hastening bull puberty. In prepubertal males, FSH facilitates Sertoli cell proliferation and testis maturation. The aim of this study was to determine the effect of prepubertal administration of a timed-release FSH (delivered in a hyaluronan solution) on hormone secretion, puberty attainment, and mature sperm production in Holstein bulls in an AI center. Bulls (n = 29) were randomly assigned to one of two treatment groups based on birth date and pedigree. Beginning at 62 days of age (Day 62), bulls were injected im every 3.5 days with either 30 mg FSH (Folltropin-V; NIH-FSH-P1 units) in a 2% hyaluronan solution (FSH-HA, n = 17) or saline (control, n = 12) until Day 170.5. Blood samples to assess FSH, activin A, and testosterone were collected prior to each treatment. Scrotal circumference (SC) and BW were measured monthly. Puberty assessment (ability to ejaculate 5 × 107 sperm, 10% motile) was initiated at Day 244. Average mature daily sperm production (3× wk collection, combined 2 ejaculates) was assessed from Day 571-627. In blood collected every 3.5 days, FSH concentrations within FSH-HA bulls were increased (P < 0.05) over initial Day 62 concentration from Day 93.5-170.5. Concentrations of FSH did not differ between treatments from Day 62-93.5, but were greater (P < 0.05) in FSH-HA than control bulls from Day 97-170.5. Concentrations of activin A assessed for Day 62, 86.5, 107.5, 139, and 170.5 were greater (P < 0.05) in FSH-HA than control bulls on Day 86.5 and 107.5. Treatments did not differ (P > 0.1) in testosterone, BW, or SC. FSH-HA bulls attained puberty at a younger age than control bulls (278 ± 7.7 vs. 303 ± 9.1 days of age, P < 0.05), but mature daily sperm production was not different when measured from Day 571-627 (average 5.84 ± 0.11 billion cells/day, P = 0.5). In summary, FSH administration every 3.5 days from Day 62-170.5 resulted in an increase in FSH concentration beginning at 97 days of age and a hastened age of puberty. We propose this exogenous FSH delivered in hyaluronan initiates a positive feedback loop that includes an increase in activin A production observed on Day 86.5 and 107.5. However, differences in mature sperm production were not realized in this experiment.

Impact of a timed-release FSH treatment from 2 to 6 months of age in bulls I: Endocrine and testicular development of beef bulls.

In prepubertal males, FSH facilitates Sertoli cell proliferation and testis maturation. The study aimed to determine the effect of an exogenous FSH treatment on hormone secretion and testis development in Angus bulls. Bulls (n = 22) weaned at 53 ± 3.8 days of age were randomized into two treatment groups based on age and pedigree. Beginning at Day 59, bulls were injected im every 3.5 days with either 30 mg FSH (Folltropin-V; NIH-FSH-P1 units) in a 2% hyaluronan solution (FSH-HA, n = 11) or saline (control, n = 11) until Day 167.5. Blood samples to assess FSH, activin A, and testosterone were collected prior to each treatment. To determine how FSH profiles surrounding treatment were affected, three intensive blood sampling periods, each encompassing two treatment administrations, began at Day 66, 108, and 157, and blood was collected at 0, 6, 12, 18, 24, 36, 60, and 84 h respective to time of treatment. Scrotal circumference (SC) and BW were measured monthly. Bulls were castrated at Day 170 to measure testis size, seminiferous tubule diameter, and the number of Sertoli and germ cells per tubule cross-section. During intensive FSH sampling, FSH-HA bulls experienced an increase (P < 0.05) in FSH over control bulls for at least 18 h post-injection in all instances. In blood collected every 3.5 days, FSH concentrations in FSH-HA bulls were increased (P < 0.05) over initial Day 59 concentration from Day 97.5-167.5. FSH concentrations did not differ between treatments from Day 59-90.5, but were greater (P < 0.05) in FSH-HA from Day 94-167.5. Concentrations of activin A assessed for Day 59, 83.5, 94, 129, and 167.5 were greater (P < 0.05) in FSH-HA than control bulls on Day 83.5 and 94. The treatments did not differ (P > 0.1) in testosterone, BW, SC, testis size, tubule diameter, or number of germ cells per tubule. However, the number of Sertoli cells per tubule was greater in FSH-HA than control bulls (45.2 ± 1.4 vs. 41.6 ± 0.9 cells, P < 0.05). In summary, FSH-HA treatment every 3.5 days from Day 59-167.5 maintained elevated FSH for a minimum of 18 h post-injection, likely attributable to the addition of HA. We propose the exogenous FSH-HA treatment initiates a positive feedback loop that includes an increased density of Sertoli cells per tubule cross-section, which is related to increased activin A concentrations on Day 83.5 and 94. Furthermore, this activin A increase preceded an increase in endogenous FSH from Day 94-167.5 in FSH-HA bulls.

Impact of a timed-release follicle-stimulating hormone treatment from one to three months of age on endocrine and testicular development of prepubertal bulls.

In prepubertal bulls, FSH facilitates testis maturation and a transient proliferation of Sertoli cells. Two experiments examined the effects of exogenous FSH on hormone secretion and testis development in Angus bulls. Exogenous FSH treatment consisted of an intramuscular injection (i.m.) of 30 mg FSH (Folltropin-V) in a 2% hyaluronic acid solution (FSH-HA). In Exp. 1, bulls (50 ± 6.5 d of age) received either FSH-HA ( = 5) or saline (control; = 5) on d 50 and 53.5. Blood samples were collected via jugular venipuncture to assess FSH concentrations every 6 h for 24 h after treatment and every 12 h until 84 h. After each treatment, peripheral FSH concentrations were greater ( < 0.05) in the FSH-HA-treated bulls than in the control bulls 6 h after treatment and tended to be greater ( ≤ 0.08) 12 h after treatment. The FSH concentration from 18 to 84 h after treatment did not differ between treatments. In Exp. 2, bulls were treated with FSH-HA ( = 11) or saline (control; = 11) every 3.5 d from 35 to 91 ± 2 d of age. Blood samples were collected before each treatment to quantify FSH, testosterone, and activin A concentrations. Scrotal circumference (SC) and BW were measured weekly. Bulls were castrated at 93 ± 2 d of age. Seminiferous tubule diameter, testis composition, and the number of Sertoli cells per tubule cross section (GATA-4 positive staining) were determined from fixed and stained histological sections. Follicle-stimulating hormone concentrations within the FSH-HA-treated bulls increased ( < 0.05) on d 70 from prior sampling and remained elevated. The FSH concentration did not differ between treatments from 35 to 66.5 d of age but were greater ( < 0.05) in the FSH-HA-treated bulls than in the control bulls from 70 to 91 d of age. Serum concentration of activin A on d 35, 70, and 91 did not differ between treatments. The FSH-HA and control bulls did not differ ( > 0.1) in BW, SC, testis weight, testis volume, percent of parenchyma composed of tubules, tubule diameter, and concentration of testosterone. The number of Sertoli cells per tubule cross section was greater in the FSH-HA-treated bulls than in the control bulls (33.35 ± 0.9 vs. 28.27 ± 0.9 cells; ˂ 0.05). In summary, the FSH-HA treatment from 35 to 91 d of age resulted in increased endogenous FSH from 70 to 91 d and increased numbers of Sertoli cells at 93 d of age. Exogenous FSH altered endocrine mechanisms regulating endogenous FSH secretion and augmented Sertoli cell proliferation in young bulls, but this effect was apparently not caused by increased activin A concentration in the FSH-HA-treated bulls.

Fibulin-3 promotes muscle-invasive bladder cancer.

Urothelial carcinoma is the most common type of bladder cancer and can be categorized as either non-muscle-invasive (Ta-T1) or muscle-invasive (⩾T2). The majority of bladder cancers are non-muscle-invasive at presentation; however, the recurrence rate for these tumors is high and a subset can progress to T2. In this study, we aimed to identify genes differentially expressed between T1 vs T2 bladder cancer to identify key regulators of bladder cancer progression and/or invasion. We performed RNA-Seq on T1 and T2 bladder cancer tissues and used publicly available bladder cancer profiling studies to prioritize differentially expressed genes for validation and functional assessment. This integrative approach nominated an extracellular matrix glycoprotein, fibulin-3 (FBLN3, also known as EFEMP1), as being highly expressed in T2 vs T1 bladder cancer and aggressive vs indolent disease. We confirmed the overexpression of fibulin-3 in ⩾T2 vs non-muscle-invasive bladder cancer (NMIBC) by quantitative reverse transcriptase-PCR. Consistent with these findings, fibulin-3 expression level correlated with the invasive ability of several bladder cancer cell lines and modulation of fibulin-3 expression directly affected invasion. Fibulin-3 knockdown in bladder cancer cells decreased the incidence of MIBCs in a murine orthotopic bladder cancer model and decreased the expression of insulin-like growth factor-binding protein-5 (IGFBP5). Restoring IGFBP5 in these cells rescued their invasive and migratory potential. These results indicate that fibulin-3 serves as a pro-invasive factor in bladder cancer, which may be mediated through modulation of IGFBP5 expression. This also suggests fibulin-3 and IGFBP5 may have potential as biomarkers of aggressive bladder cancer or therapeutic targets.

Effects of dietary energy on sexual maturation and sperm production in Holstein bulls.

In prepubertal bulls and heifers of dairy and beef breeds, puberty can be induced to occur earlier than typical with targeted high-energy diets due to precocious activation of the endocrine mechanisms that regulate puberty. Precocious activation of puberty in bulls intended for use in the AI industry has the potential to hasten and perhaps increase sperm production. It was hypothesized that feeding bulls a high-energy diet beginning at 8 wk of age would advance the prepubertal rise in LH and lead to advanced testicular maturation and age at puberty. From 58 to 230 ± 0.3 d of age, Holstein bulls received either a high-energy diet (HE;n = 9; targeted ADG 1.5 kg/d) or a control diet (CONT;n = 10; targeted ADG 0.75 kg/d). Thereafter, all bulls were fed a similar diet. The HE treatment increased LH secretion at 125 d of age, testosterone concentrations from 181 to 210 d, and scrotal circumference (SC) from 146 to 360 d of age relative to the CONT treatment. Beginning at 241 ± 5 d of age, semen collection (artificial vagina) was attempted every 14 d in bulls from the HE (n = 8) and CONT (n = 7) treatment until each bull attained puberty (ejaculate containing 50 × 10 spermatozoa with 10% motility). To assess semen production as mature bulls, semen was collected thrice weekly beginning at 541 ± 5 d of age until slaughter at 569 ± 5 d of age. After slaughter, epididymal and testicular measurements were collected and testicular tissue was fixed to determine seminiferous tubule diameter. Age at puberty did not differ between treatments (310 ± 35 d). Although testis and epididymal weight and testis volume were greater (P < 0.05) in the HE than the CONT treatment, sperm production of mature bulls did not differ between treatments. Diameter of seminiferous tubules also did not differ between treatments. We conclude that the HE advanced aspects of sexual maturation and increased testes size, but this was not reflected in hastened puberty or sperm production in the present experiment.

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.

Effect of interval from induction of puberty to initiation of a timed AI protocol on pregnancy rate in Nellore heifers.

Prepubertal Bos indicus heifers (n = 774) were submitted to an E2/P4-based timed artificial insemination (TAI) protocol at three different intervals after induction of their pubertal ovulation by insertion of an intravaginal progesterone (P4) device for 12 days. Heifers were randomly assigned to start the TAI protocol at 10 (group 10; n = 253), 12 (group 12; n = 265), or 14 (group 14; n = 256) days after the P4 device was removed. The TAI protocol consisted of the following: insertion of intravaginal device containing P4 (Controlled internal drug release [CIDR]; previously used twice for 9 days each) + estradiol benzoate (2 mg) on Day 0, CIDR withdrawal + estradiol cypionate (0.5 mg) and PGF2α (12.5 mg) on Day 9, and TAI on Day 11. A subgroup of heifers (n = 472) was evaluated by ultrasound on Days 9 and 11 to evaluate the ovaries and to determine P4 concentrations on Day 9. On Day 9, more (P < 0.05) CLs were present, and follicular diameter was smaller (P < 0.05) for group 10 than for groups 12 and 14 (38.4%, 29.3%, and 23.3% with CL and 9.4 ± 0.1, 9.9 ± 0.1, and 9.8 ± 0.1 mm diameter, respectively), but P4 concentrations did not differ (P > 0.1) between treatments (2.4 ± 0.06 ng/mL). Follicular diameter at TAI (11.08 ± 0.09 mm) and ovulation rate (88.4%) did not differ between treatments (P > 0.1). However, conception and pregnancy rates for all heifers were greater (P < 0.05) in group 12 (50.4% and 45.5%, respectively) than in group 10 (38.2% and 33.7%, respectively), with group 14 intermediate to other treatments (45.6% and 40.6%, respectively). The final pregnancy rate did not differ between treatments (80.9%). In conclusion, a 12-day interval from the end of the puberty induction protocol to the start of the TAI protocol resulted in greater conception and pregnancy rates in prepubertal Nellore heifers.

The effect of follicle age on pregnancy rate in beef cows.

The effect of the age of the ovulatory follicle on fertility in beef cows was investigated. Multiparous (n = 171) and primiparous (n = 129) postpartum beef cows in 2 groups (G1 and G2) received estradiol benzoate (EB; 1 mg/500 kg BW, intramuscular [i.m.]) 5.5 d (G1; n = 162) and 6.5 d (G2; n = 138) after the final GnRH of a synchronization program (5d CO-Synch + CIDR) to induce emergence of a new follicular wave (NFW), followed by prostaglandin F2α (PGF(2α); 25 mg, i.m.) administration either 5.5 d ("young" follicle, YF; n = 155) or 9.5 d ("mature" follicle, MF; n = 145) after EB. Estrous detection coupled with AI 12 h later (estrus-AI) was performed for 60 h (MF) and 84 h (YF) after PGF(2α); cows not detected in estrus within this period received timed AI (TAI) coupled with GnRH at 72 and 96 h, respectively. Within the first 72 h after PGF(2α), more (P < 0.01) cows in the MF (76.3%) than YF treatment (47.7%) exhibited estrus, but through 96 h, the proportion detected in estrus (P < 0.05) and interval from PGF(2α) to estrus (P < 0.01) were greater in the YF than MF treatment (88.6% vs. 76.3%, 78.9 ± 0.8 vs. 57.5 ± 1.6 h, respectively). Age of the ovulatory follicle at AI was greater (P < 0.01) in the MF (9.32 ± 0.04 d) than YF (6.26 ± 0.02 d) treatment, but follicle diameter at AI and pregnancy rates did not differ between MF (13.1 ± 0.2 mm; 72.0%) and YF (12.9 ± 0.1 mm; 67.1%) treatments. Regardless of treatment, the diameter of the ovulatory follicle at AI and pregnancy rate were greater (P < 0.01) with estrus-AI (13.1 ± 0.1 mm; 75.0%) than TAI (12.6 ± 0.2 mm; 55.4%). Cows in the MF treatment that initiated a second NFW after EB but before PGF(2α) (MF2; n = 47) were induced to ovulate with GnRH and TAI at 72h, when ovulatory follicles were 4 d old and 10.2 ± 0.2 mm in diameter. Pregnancy rate for TAI (51.1%) in MF2 did not differ from TAI pregnancy rate (55.4%) across the MF and YF treatments. In summary, the age of the ovulatory follicle affected interval to estrus and AI but did not influence pregnancy rate in suckled beef cows.

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.

Effect of intrauterine administration of gonadotropin releasing hormone with glycerol on serum LH concentrations in lactating dairy cows.

The objectives of the study were to assess: (1) preovulatory serum LH concentrations and (2) synchrony of ovulation after im or iu administration of GnRH with or without the addition of glycerol. Cows were presynchronized with 2 injections of PGF2α given 14d apart (starting at 26±3DIM) followed by Ovsynch (OV; GnRH-7d-PGF2α-48h-GnRH) 12d later. At the time of the second GnRH of OV (GnRH2), cows were blocked by parity and randomly allocated to 1 of 4 treatments: (1) control (CON; n=8) received 2mL of sterile water im; (2) im (IM; n=8) received 100µg of GnRH im; (3) cows were infused with 200µg GnRH into the uterus (IU; n=9); and (4) iu administration of 200µg GnRH plus glycerol 7% v/v (IUG; n=8). Serum circulating progesterone concentrations at hour 0 did not differ (P>0.05) among groups. Concentrations of LH were greater (P<0.05) in IM than IU, IUG, and CON cows at hours 1, 1.5, 2, and 3. All cows ovulated within 48h in the IM (8/8) group followed by IU (6/9) and IUG (4/8) groups, and only two out of eight cows ovulated in the CON group. Although iu administration of GnRH in the IU and IUG groups resulted in lower serum concentrations of LH than IM cows, IU or IUG cows were able to ovulate within 48h after GnRH2 administration.

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 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.

Triennial Reproduction Symposium: deficiencies in the uterine environment and failure to support embryonic development.

Pregnancy failure in livestock can result from failure to fertilize the oocyte or embryonic loss during gestation. The focus of this review is on cattle and factors affecting and mechanisms related to uterine insufficiency for pregnancy. A variety of factors contribute to embryonic loss and it may be exacerbated in certain animals, such as high-producing lactating dairy cows, and in some cattle in which estrous synchronization and timed AI was performed, due to reduced concentrations of reproductive steroids. Recent research in beef cattle induced to ovulate immature follicles and in lactating dairy cows indicates that deficient uterine function is a major factor responsible for infertility in these animals. Failure to provide adequate concentrations of estradiol before ovulation results in prolonged effects on expression and localization of uterine genes and proteins that participate in regulating uterine functions during early gestation. Furthermore, progesterone concentrations during early gestation affect embryonic growth, interferon-tau production, and uterine function. Therefore, an inadequate uterine environment induced by insufficient steroid concentrations before and after ovulation could cause early embryonic death either by failing to provide an adequate uterine environment for recognition of embryo signaling, adhesion, and implantation or by failing to support appropriate embryonic growth, which could lead to decreased conceptus size and failed maternal recognition of pregnancy.

Triennial Reproduction Symposium: the obstacle course to successful establishment of pregnancy in domestic livestock species.

The 2012 Triennial Reproduction Symposium (TRS), "Impediments to Fertility in Domestic Animals," was held immediately before the Joint Annual Meeting of the American Society of Animal Science, American Dairy Science Association, Canadian Society of Animal Science, Western Section of the American Society of Animal Science, and the Asociación Mexicana de Producción Animal in Phoenix, AZ, in July, 2012. The theme of the symposium highlighted key impediments or opportunities in the process of creating a pregnancy, beginning with male and female gametes and ending with a viable fetus. The 2012 TRS was designed to focus on areas of current and exciting investigation across a variety of species and to include 8 presentations from a mix of established and early-career scientists. The TRS was also the venue for presentation of the 2012 L. E. Casida Award for Graduate Education; the recipient was R. D. Randel (Texas A&M University). The symposium provided an excellent opportunity for reproductive biologists to consider the broad spectrum of factors that limit fertility in domestic species and contemplate the current status of knowledge relative to several of the significant obstacles to pregnancy.

Progesterone-based strategies to induce ovulation in prepubertal Nellore heifers.

Four experiments were conducted to evaluate hormonal strategies to induce ovulation in Nellore heifers. In experiment 1, heifers (N = 1039) received a controlled internal drug release (CIDR) of fourth use (CIDR-4) on Day -12 or no CIDR (CIDR-0). The CIDR was removed on Day 0 in the CIDR-4 treatment, and estrus detection and AI were performed from Days 1 to 7. On Day 8, heifers not detected in estrus were evaluated for CL presence and received the same treatment again, followed by estrus detection and AI from Days 21 to 27. All heifers in experiments 2 (N = 896), 3 (N = 839), and 4 (N = 948) received the CIDR-4 treatment on Day -12. In experiment 2, heifers were randomly assigned to a control group (no additional treatment) or to receive equine chorionic gonadotropin (eCG; 200 IU eCG im) on Day 0. In experiment 3, heifers received the same treatments as in experiment 2, or a treatment that included eCG and estradiol cypionate (ECP) (eCG+ECP; 200 IU im eCG plus 0.5 mg ECP im) on Day 0. In experiment 4, heifers received the treatments described in experiment 3 or only ECP (0.5 mg) on Day 0. In experiments 2 and 3, estrus detection and AI was performed from Days 1 to 7 and on Day 8, heifers not detected in estrus were evaluated for CL presence. In experiment 4, heifers were evaluated for presence of a CL between Days 10 and 14. In experiment 1 heifers treated with CIDR-4 had greater estrus detection, ovulation induction, and pregnancy rates than in the CIDR-0 group. In experiment 2, heifers treated with eCG had greater estrus detection, ovulation induction, and pregnancy rates in 7 days than heifers in the control group. In experiment 3, heifers treated with eCG+ECP had greater estrus detection, ovulation induction, and pregnancy rates than the control and eCG treatments. In experiment 4, ovulation induction was greater for heifers treated with eCG and eCG+ECP relative to control, but did not differ from the ECP treatment. In conclusion, the use of a CIDR of fourth use for 12 days and the addition of eCG and/or ECP at CIDR removal efficiently induced ovulation and increased pregnancy rates in prepubertal Nellore heifers.

Changing expression of chloride channels during preimplantation mouse development.

Plasma membrane chloride channels (ClCs) play important roles in a broad range of cellular processes including cell volume regulation, proliferation, and transepithelial transport, all of which are critical during preimplantation embryonic development. In this study, the molecular and functional expression of voltage-gated ClCs was analyzed throughout preimplantation development of the mouse conceptus. mRNA transcripts for all Clcn genes were detected. Only Clcn1 mRNA showed differential expression in the blastocyst, being detected in the trophectoderm but not in the inner cell mass. CLCN3 protein was detected at low levels in the cytoplasm and plasma membrane in 4-cell embryos and was localized to the apical plasma membrane of the trophoblasts in the blastocyst. Whole-cell patch-clamp recordings demonstrated the presence of a DIDS-sensitive, outwardly rectifying Cl(-) current throughout development, with this conductance being large at the 1-cell, morula and blastocyst stages. A second DIDS-insensitive Cl(-) current, which was inactivated by membrane depolarization, was present in cells differentiating into the trophoblast lineage and during blastocyst expansion. Inhibition of the DIDS-sensitive current and the DIDS-insensitive current, with 9-AC, prevented blastocyst expansion.

Triennial Reproduction Symposium: influence of follicular characteristics at ovulation on early embryonic survival.

Reproductive failure in livestock can result from failure to fertilize the oocyte or embryonic loss during gestation. Although fertilization failure occurs, embryonic mortality represents a greater contribution to reproductive failure. Reproductive success varies among species and production goals but is measured as a binomial trait (i.e., pregnancy), derived by the success or failure of multiple biological steps. This review focuses primarily on follicular characteristics affecting oocyte quality, fertilization, and embryonic health that lead to pregnancy establishment in beef cattle. When estrous cycles are manipulated with assisted reproductive technologies and ovulation is induced, duration of proestrus (i.e., interval from induced luteolysis to induced ovulation), ovulatory follicle growth rate, and ovulatory follicle size are factors that affect the maturation of the follicle and oocyte at induced ovulation. The most critical maturational component of the ovulatory follicle is the production of sufficient estradiol to prepare follicular cells for luteinization and progesterone synthesis and prepare the uterus for pregnancy. The exact roles of estradiol in oocyte maturation remain unclear, but cows that have lesser serum concentrations of estradiol have decreased fertilization rates and decreased embryo survival on d 7 after induced ovulation. When length of proestrus is held constant, perhaps the most practical follicular measure of fertility is ovulatory follicle size because it is an easily measured attribute of the follicle that is highly associated with its ability to produce estradiol.