Toll-like receptor and related cytokine mRNA expression in bovine corpora lutea during the oestrous cycle and pregnancy.

Improving our understanding of the mechanisms controlling the corpus luteum (CL) and its role in regulating the reproductive cycle should lead to improvements in the sustainability of today's global animal industry. The corpus luteum (CL) is a transient endocrine organ composed of a heterogeneous mixture steroidogenic, endothelial and immune cells, and it is becoming clear that immune mechanisms play a key role in CL regulation especially in luteolysis. Toll-like receptors (TLR) mediate innate immune mechanisms via the production of pro-inflammatory cytokines, especially within various tissues, although the role of TLR within CL remains unknown. Thus, the objectives of this study were to characterize TLR mRNA expression in the CL during the oestrous cycle and in pregnancy (day 30-50), and to examine the role of TLR signalling in luteal cells. Corpora lutea were collected at various stages of the cycle and pregnancy and analysed for TLR and cytokine mRNA expression. In addition, luteal cells were cultured with the TLR4 ligand (lipopolysaccharide, LPS) for 24 h to evaluate the role of TLR4 in regulating luteal function. Toll-like receptors 1, 2, 4, 6, tumour necrosis factor alpha (TNF), interferon gamma (IFN-G), and interleukin (IL)-12, mRNA expressions were greatest in regressing CL compared with earlier stages (p < .05), whereas no change was observed for IL-6 mRNA expression. Cytokine mRNA expression in cultured luteal cells was not altered by LPS. Based on these data, one or more of the TLRs found within the CL may play a role in luteolysis, perhaps via pro-inflammatory cytokine mRNA expression.

Bone morphogenetic protein 6 promotes FSH receptor and anti-Müllerian hormone mRNA expression in granulosa cells from hen prehierarchal follicles.

A growing body of literature provides evidence of a prominent role for bone morphogenetic proteins (BMPs) in regulating various stages of ovarian follicle development. Several actions for BMP6 have been previously reported in the hen ovary, yet only within postselection (preovulatory) follicles. The initial hypothesis tested herein is that BMP6 increases FSH receptor (FSHR) mRNA expression within the granulosa layer of prehierarchal (6-8 mm) follicles (6-8 GC). BMP6 mRNA is expressed at higher levels within undifferentiated (1-8 mm) follicles compared with selected (≥9 mm) follicles. Recombinant human (rh) BMP6 initiates SMAD1, 5, 8 signaling in cultured 6-8 GC and promotes FSHR mRNA expression in a dose-related fashion. In addition, a 21 h preculture with rhBMP6 followed by a 3 h challenge with FSH increases cAMP accumulation, STAR (StAR) expression, and progesterone production. Interestingly, rhBMP6 also increases expression of anti-Müllerian hormone (AMH) mRNA in cultured 6-8 GC. This related BMP family member has previously been implicated in negatively regulating FSH responsiveness during follicle development. Considering these data, we propose that among the paracrine and/or autocrine actions of BMP6 within prehierarchal follicles is the maintenance of both FSHR and AMH mRNA expression. We predict that before follicle selection, one action of AMH within granulosa cells from 6 to 8 mm follicles is to help suppress FSHR signaling and prevent premature granulosa cell differentiation. At the time of selection, we speculate that the yet undefined signal directly responsible for selection initiates FSH responsiveness. As a result, FSH signaling suppresses AMH expression and initiates the differentiation of granulosa within the selected follicle.

Progesterone inhibits cytokine/TNF-α production by porcine CL macrophages via the genomic progesterone receptor.

In pigs, luteolytic sensitivity to PGF-2α (=LS) is delayed until d 13 of the estrous cycle. While the control of LS is unknown, it is temporally associated with macrophage (MAC; which secretes tumor necrosis factor [TNF]-α) infiltration into the corpora lutea (CL), and previous studies have shown that TNF-α induces LS in porcine luteal cells (LCs) in culture. This study was designed to explore the control of LS by CL macrophage (CL MAC)/TNF-α by progesterone (P4), and to examine the hypothesis that P4 acting via the genomic P4 receptor (PGR) inhibits CL MAC TNF-α and thus plays a key role in regulating LS during the pig estrous cycle. In experiment 1, the effects of LCs on CL MAC cytokine/TNF-α mRNA expression in co-culture were examined (MID cycle; ~d 7-12; no LS); results showed that LC was inhibitory to cytokine/TNF-α. In experiment 2, the effects of P4 or R5020 (PGR-agonist) on CL MAC cytokine/TNF-α mRNA expression were examined (MID cycle; ~d 7-12; no LS); results showed that both P4 and R5020 dose-dependently inhibited TNF-α. In experiment 3, CL MACs were isolated from CL at MID (~d 7-12; no LS) and LATE (~d 13-18; + LS) cycle, and TNF-α/PGR mRNA measured. Results indicated that while TNF-α mRNA was 4.2-fold greater in CL MACs from LATE vs MID cycle, PGR mRNA was 4.5-fold greater in CL MACs from MID vs LATE cycle. These data support our hypothesis and suggest that progesterone, acting via PGR, plays a critical physiological role in the control of TNF-α production by CL MACs and LS during the pig estrous cycle.

Low peripheral progesterone and late embryonic/early fetal loss in suckled beef and lactating dairy cows.

Pregnancy failure during placentation in lactating dairy cows was associated with low concentrations of serum progesterone. Beef cows have greater serum progesterone and less pregnancy failure. Experiment 1 determined that reduction of serum progesterone affected late embryonic/early fetal loss in suckled beef cows. Cows (n=40) received progesterone from two new or used controlled internal drug releasing devices, replaced every 5d, beginning on Day 28 of gestation (mating=Day 0); CL were enucleated on Day 29. Retention of pregnancy was 77% in treated cows and 97% in 78 control cows (P<0.05). Experiment 2 determined how pregnant, lactating dairy cows with high or low progesterone concentrations during Days 28-34 differed in luteal function or in serum progesterone during replacement therapy. Luteal tissue from such cows was assayed for progesterone and expression of mRNA for genes of endothelin and prostaglandin (PG) systems. Secretion of progesterone and prostaglandins by dispersed luteal cells was determined during incubation with LH, endothelin-1, or arachidonic acid. Neither luteal progesterone nor mRNAs for endothelin or prostaglandin systems differed. Endothelin-1 inhibited secretion of progesterone more (P<0.05) in luteal cells from cows with low versus high serum progesterone, when incubated with arachidonic acid. Secretion of prostaglandin F(2)alpha was increased and that of 6-keto-PGF(1)alpha decreased by endothelin-1 in vitro. Serum progesterone during replacement was lower (P<0.05) for cows with low than high serum progesterone at lutectomy. Thus, clearance, more than luteal production, determined peripheral progesterone in pregnant, lactating dairy cows.

Site of PGF2α injection does not alter effectiveness of the Select Synch + controlled internal drug release and timed artificial insemination protocol.

Beef Quality Assurance programs have contributed to significant improvements in the wholesomeness of beef available for consumption. Injection site blemishes in the round have declined since the promotion of administering intramuscular injections in the neck. Unfortunately, many producers continue to administer estrus synchronization (ES) drugs in the rump. The objective of this study was to compare the effectiveness of injection site of PGF2α, in ES protocols, on steroid hormone concentrations and pregnancy rates. A Select Synch + 7-day controlled internal drug release ES protocol was conducted with the site of PGF2α injection alternated between neck and rump in beef cattle (n = 312) at the Ohio State University Agricultural Technical Institute and North Carolina State University. Blood samples (n = 75) were collected at controlled internal drug release insertion and at the time of artificial insemination (AI) to determine if progesterone (P4) and estrogen (E2) concentrations varied due to PGF2α injection site. All cattle were confirmed pregnant by ultrasonography at approximately 30 and 90 days after insemination in North Carolina and approximately 70 days after insemination in Ohio. Data were analyzed as randomized complete block designs in PROC GLIMMIX with animal as the experimental unit. Differences were declared significant at P < 0.05. Site of PGF2α injection, in either the neck or rump, did not affect (P > 0.05) overall conception rates in response to AI (58.4% and 55.6%, respectively). Altering PGF2α injection site did not impact P4, E2 concentrations, or the P4:E2 ratio at AI (P > 0.05). However, cattle inseminated after displaying estrus had greater (P < 0.05) pregnancy rates than timed AI (67.8 vs. 47.5%, respectively). First service conception rates and pregnancy rates were consistent with previous reports. Overall, altering the location of the PGF2α injection during ES did not change circulating hormone concentrations at AI or pregnancy rates; therefore, cattle producers should follow Beef Quality Assurance guidelines when administering ES protocols.

Ability of induced corpora lutea to maintain pregnancy from the third month of gestation to term in cattle.

The local relationship between the pregnant uterine horn and the CL during maternal recognition of pregnancy is well-documented. It continues beyond that time; pregnancies were maintained in lutectomized cows when CL were induced on the ovary ipsilateral, but not contralateral, to the uterine horn of pregnancy during Days 28-53. This study evaluated factors affecting maintenance of pregnancy by CL induced after Day 53, in lutectomized cows that had received exogenous progesterone from Day 29 to 15 days after induction of a CL. Twenty-four suckled beef cows were lutectomized on Day 29 of gestation; pregnancy was maintained with progesterone from two controlled internal drug releasing (CIDR) inserts, exchanged every 5 days. Beginning on Day 53, ovaries and viability of pregnancy were evaluated by ultrasonography every 5 days. When a follicle >or=10 mm in diameter was present ipsilateral to the fetus, each cow received 1,000 IU of hCG. Following induction of a CL (20 of 24), progesterone was reduced to a single CIDR for 5 days, then removed. Retention of pregnancy was confirmed by rectal palpation and calving. Cows with induced CL maintained pregnancy to term, including four with the CL contralateral to the fetus. Three cows failed to form normal CL by Day 98 and lost pregnancy after removal of exogenous progesterone. One cow that did not respond to hCG lost pregnancy during exogenous progesterone. In conclusion, CL induced after Day 53 maintained pregnancy to term, even when induced contralateral to the pregnant uterine horn.

Comparison of pregnancy rates in beef cattle after a fixed-time AI with once- or twice-used controlled internal drug release devices.

The use of fixed-time artificial insemination (FTAI) provides producers with numerous benefits including the use of superior genetics, shorter breeding and calving seasons, and a more uniform calf crop. However, the cost of implementing FTAI protocols is one of the several drawbacks hindering their use in the beef industry. Potential injection-site lesions from intramuscular injections of the hormones necessary for estrus synchronization are also a cause of concern for carcass quality. The objectives of this experiment were to (1) determine whether or not a twice-used controlled internal drug release (CIDR) device would be effective in an FTAI protocol without adversely affecting pregnancy rate and (2) whether or not the subcutaneous administration of PGF2α affects pregnancy rate. Nulliparous females (n = 99) between 13 and 27 months of age and multiparous cows (n = 43) between 48 and 74 months of age were synchronized for estrus using the 7-day CO-Synch + CIDR protocol. The females were randomly assigned to one of the two treatments: (1) a once-used CIDR (control) or (2) a twice-used CIDR device (treatment) incorporated into their synchronization protocol. The females were also randomly assigned to have their injection of PGF2α administered either intramuscularly or subcutaneously. Blood was taken in a random subset of nulliparous females (n = 52) just before device removal and assayed for concentration of progesterone. The concentration of progesterone was higher (P = 0.01) in the animals that received once-used CIDR devices than that in those received twice-used CIDR devices (3.4 ± 0.5 and 1.4 ± 0.5 ng/mL, respectively). There was no significant effect of parity (P = 0.82), artificial insemination technician (P = 0.60), PGF2α administration (P = 0.83), or treatment (P = 0.67) on pregnancy rates to artificial insemination which were 75.4 ± 6.0% and 71.7 ± 6.4%, for animals that received once- and twice-used CIDR devices, respectively. This study provides evidence that although concentration of progesterone is decreased in animals treated with a twice-used CIDR device, there is still a sufficient release of progesterone from the device to effectively synchronize estrus without adversely affecting the fertility of a herd.

Effect of protein supplementation and forage allowance on the growth and reproduction of beef heifers grazing stockpiled tall fescue.

Stockpiled tall fescue can provide adequate winter forage for beef cattle, although unsupplemented replacement heifers may display marginal performance before breeding. The objective of this study was to determine if protein supplementation and/or additional forage improves growth and reproductive performance of replacement heifers grazing stockpiled fescue. Cattle averaging 272 ± 1.59 kg were stratified by BW and then randomly assigned to 1 of 4 plots within a pasture replication. Treatment combinations were assigned in a 2 × 2 factorial arrangement and included 1) a conservative forage allocation ("normal," targeting 85% forage use) and mineral supplement (normal forage allocation with mineral supplement [FM]), 2) normal forage allocation with protein tub (FT), 3) more liberal forage allocation ("extra," targeting 70% forage use) and mineral supplement (extra forage allocation with mineral supplement [EM]), and 4) "extra forage allocation with protein tub (ET). Treatments were administered for 8 wk from early November to early January. Heifers were fed fescue hay for 1 wk before breeding in late January. Heifers were synchronized with the 7-d CO-Synch + controlled internal drug release device protocol and inseminated in late January. Heifers were checked for pregnancy by ultrasonography at 35 and 90 d after AI. Main and interaction effects between the 2 treatments were determined. Total supplement intake was greater for protein tub than mineral supplement (0.36 vs. 0.11 kg·heifer·d, respectively; < 0.0001), and the additional dietary protein in the tub groups resulted in greater serum urea N concentrations ( < 0.0001; 8.15 vs. 10.4 mg/dL for mineral and protein tub, respectively). Forage utilization efficiency was greater for normal than extra forage allocation (74.7 vs. 65.8%, respectively; < 0.0001). Main effects of both treatments on ADG were significant ( < 0.0001; 0.28, 0.43, 0.43, and 0.51 kg·heifer·d for FM, FT, EM, and ET, respectively). There was an interaction effect of the 2 treatments on change in BCS ( < 0.05; 0.12, 0.10, 0.18, and 0.31 for FM, FT, EM, and ET, respectively). Reproductive tract scores, pelvic area, and AI pregnancy rates were not different between treatments ( > 0.05). Overall, feeding a protein supplement or providing extra forage increased gain and interacted to increase BCS but did not have an effect on reproductive performance. Supplementing with protein and providing extra forage are strategies that can increase gain in heifers, which could aid heifers in reaching puberty before estrous synchronization.

Effects of service sire on prenatal mortality and prolificacy in ewes.

Ability to select service sires that minimize partial or complete losses of pregnancy could have major economic impacts in sheep production systems. This study tested the null hypothesis that survival of potential progeny did not vary with breed type of service sire or among individual rams. Data included 980 ewes on 10 farms; each ewe was pregnant to 1 of 67 rams of 12 breeds. Number of conceptuses was estimated once during pregnancy by ultrasonography, either transrectal (embryos) or transabdominal (fetuses), and was compared with number of lambs born to estimate losses. Data were examined first for number of lambs born and second for documented losses. Individual service sires affected number born (P < 0.001), which varied from 0.70 to 2.45 lambs per pregnant ewe. The main effects of breed type on lambs born were not significant, but breed types of both service sires (P < 0.0002) and ewes (P < 0.001) interacted with diagnosed number of conceptuses. Lambs born varied with ewe age (P < 0.0001) and among farms (P < 0.0001), and statistically, farms interacted with number of diagnosed conceptuses (P < 0.0001); season had no effect. In documented losses, there were both main effects of individual service sire and a service sire × number of diagnosed embryos interaction (P < 0.005). Thus, ewes bred to some rams were more apt to lose single pregnancies, whereas ewes bred to other rams were more apt to lose 1 or more embryos or fetuses from multiple pregnancies. Breed type of service sire affected (P < 0.05) prenatal death. Complete losses of single conceptuses tended to be greater in ewes bred to black-faced or hair-type rams (service sire breed type × number of diagnosed conceptuses; P < 0.09). Breed type of ewes also varied in incidence of complete losses (P < 0.05); hair-type ewes (46%) lost more (P < 0.02) documented conceptuses from examination to birth than black-faced (27%), white-faced (20%), or dairy-type (25%) ewes. Greater losses of singles than of multiples occurred in black-faced (37% vs. 18%) and hair-type (64% vs. 27%) ewes than in other breeds (ewe breed type × number of conceptuses; P < 0.03) per ewe. Surprisingly, purebred conceptuses were lost less often (24%) than crossbreds (36.4%; P < 0.002). Selection of rams based on records of prenatal losses in ewes they serviced may be a method to decrease embryonic and fetal wastage. However, further study to determine repeatability of differences among service sires from year to year will be required.

Adipogenic differentiation state-specific gene expression as related to bovine carcass adiposity.

Genetic regulation of the site of fat deposition is not well defined. The objective of this study was to investigate adipogenic differentiation state-specific gene expression in feedlot cattle (>75% Angus; <25% Simmental parentage) of varying adipose accretion patterns. Four groups of 4 steers were selected via ultrasound for the following adipose tissue characteristics: low subcutaneous-low intramuscular (LSQ-LIM), low subcutaneous-high intramuscular (LSQ-HIM), high subcutaneous-low intramuscular (HSQ-LIM), and high subcutaneous-high intramuscular (HSQ-HIM). Adipose tissue from the subcutaneous (SQ) and intramuscular (IM) depots was collected at slaughter. The relative expression of adipogenic genes was evaluated using quantitative PCR. Data were analyzed using the mixed model of SAS, and gene expression data were analyzed using covariate analysis with ribosomal protein L19 as the covariate. No interactions (P > 0.10) were observed between IM and SQ adipose tissue depots for any of the variables measured. Therefore, only the main effects of high and low accretion within a depot and the effects of depot are reported. Steers with LIM had smaller mean diameter IM adipocytes (P < 0.001) than HIM steers. Steers with HSQ had larger mean diameter SQ adipocytes (P < 0.001) than LSQ. However, there were no differences (P > 0.10) in any of the genes measured due to high or low adipose accretion. Preadipogenic delta-like kinase1 mRNA was greater in the IM than the SQ adipose tissue; conversely, differentiating and adipogenic genes, lipoprotein lipase, PPARγ, fatty acid synthetase, and fatty acid binding protein 4 were greater (P < 0.001) in the SQ than the IM depot. Intramuscular adipocytes were smaller than SQ adipocytes and had greater expression of the preadipogenic gene, indicating that more hyperplasia was occurring. Meanwhile, SQ adipose tissue contained much larger (P < 0.001) adipocytes that had a greater expression (P < 0.001) of differentiating and adipogenic genes than did the IM adipose tissue, indicating more cells were undergoing differentiation and hypertrophy. Adipogenic differentiation state-specific gene expression was not different in cattle with various phenotypes, but adipogenesis in the SQ and IM adipose tissues seems to occur independently.

Pregnancy outcome in dairy and beef cattle after artificial insemination and treatment with seminal plasma or transforming growth factor beta-1.

Reduced capability of the uterus to support pregnancy in the absence of its interaction with secretions from male accessory glands has been demonstrated in rodents and to some extent in pigs. However, in cattle, the role of postmating inflammatory response on pregnancy success has not been studied. The current study examined the influence of uterine presensitization with seminal antigens at breeding on pregnancy outcome in cows. Lactating beef (n=1090) and dairy (n=800) cows received 0.5 mL seminal plasma (SP), 40 ng recombinant human transforming growth factor-beta1 (rhTGF-beta1), or 0.5 mL bovine serum albumin (BSA), or were left untreated before or at insemination. Semen was deposited into the anterior cervix using a second insemination gun. Pregnancy was diagnosed at 35 to 40 d postinsemination by transrectal ultrasonography or from records of calves born the subsequent calving season. Pregnancy rates in beef cows did not differ among treatments but differed among trials (69.8%, 52.5% vs. 40.3%; P<0.05). In trials where average pregnancy rates were below 50%, treatments with TGF-beta1 but not SP tended (P<0.07) to increase pregnancy rates in beef cows. In dairy cows, SP and TGF-beta1 improved pregnancy outcome by 10 percentage points, but these increments did not achieve statistical significance. In conclusion, this study did not find any conclusive evidence for the effect of TGF-beta1 or seminal plasma on pregnancy outcome in lactating dairy or beef cows but realized marginal improvements when pregnancy rates were below 50% (compromised fertility).