Prevalence and risk factors related to anovular phenotypes in dairy cows.

The objective of the current study was to evaluate the relationship of body condition score (BCS) at 35 d in milk (DIM), milk production, diseases, and duration of the dry period with prevalence of anovulation at 49 DIM and then, specifically, with the prevalence of each anovular phenotype. We hypothesized that anovular follicular phenotypes, classified based on maximal size of the anovular follicle, have different etiologies. A total of 942 lactating Holstein cows (357 primiparous and 585 multiparous) from 1 herd had ovaries evaluated by ultrasonography at 35 ± 3 and 49 ± 3 DIM to detect the absence of a corpus luteum (CL), and to measure the diameter of the largest follicle. Cows were classified as cyclic at 49 DIM if a CL was observed in at least 1 of the 2 examinations, or anovular if no CL was observed at either examination. Cows considered anovular were divided into 3 groups based on the largest diameter of the largest follicle as follows: ranging from 8 to 13 mm, 14 to 17 mm, or ≥18 mm. Cows were evaluated for the following diseases: retained placenta, metritis, hyperketonemia, mastitis, lameness, respiratory problem, and digestive problem. At 35 DIM, BCS was determined, and milk yield for individual cows was recorded. A total of 28.5% (268/942) of cows were classified as anovular. Anovular cows had longer dry periods (90 vs. 71 d) and smaller BCS than cyclic cows (2.83 vs. 2.99). Cows with a single disease or multiple diseases had 2 and 3-fold increase in odds of being anovular, respectively. Anovular cows had follicles that ranged from 4 to 50 mm. The prevalence of anovular phenotype, among anovular cows, that had the diameter of the largest follicle ranging from 8 to 13 mm, 14 to 17 mm, and ≥18 mm was 29.9 (79/264), 37.5 (99/264), and 32.6% (86/264), respectively. Anovular cows with follicles of 8 to 13 mm had longer dry periods than those with follicles ≥18 mm (104 vs. 74 d), whereas anovular cows with medium size follicles had intermediate days dry (99 d). Cows with small and medium anovular follicles had smaller BCS and greater prevalence of multiple diseases than cyclic cows. For almost all risk factors, the cows with large anovular follicles (≥18 mm) were similar to cyclic cows and different from cows with smaller anovular follicles (8-13 mm). Thus, longer dry periods, less BCS at 35 DIM, and diseases were risk factors for anovulation. Moreover, the risk factors for the 3 distinct anovular follicle phenotypes differed.

Short communication: Heat stress does not affect induced luteolysis in Holstein cows.

Heat stress (HS) has deleterious effects on bovine reproduction, including prolongation of the luteal phase in Holstein cows, perhaps due to compromised luteolysis. The objective was to characterize effects of HS on luteolytic responses of nonlactating Holstein cows given 25 or 12.5 mg of PGF2α on d 7 of the estrous cycle. Cows were randomly distributed into 2 environments: thermoneutral (n = 12; 25°C) or HS (n = 12; 36°C). In each environment, cows were treated with 2 mL of saline, 25 or 12.5 mg of PGF2α (n = 4 cows per group). The HS environment induced a significant increase in rectal temperature and respiratory rate compared with the thermoneutral environment. Heat stress did not have significant effects on luteolytic responses or circulating progesterone concentrations. Rapid and complete luteolysis occurred in all cows given 25 mg of PGF2α and in 4 of 8 cows given 12.5 mg; the other 4 cows given 12.5 mg had partial luteolysis, with circulating progesterone concentrations initially suppressed, but subsequently rebounding. Therefore, we conclude that HS does not change corpus luteum sensitivity to PGF2α.

Circulating progesterone concentrations in nonlactating Holstein cows during reuse of intravaginal progesterone implants sanitized by autoclave or chemical disinfection.

The aim of this study was to compare plasma progesterone (P4) concentrations in nonlactating, multiparous Holstein cows (n = 24) treated with 2 types of intravaginal implants containing either 1.0 or 1.9 g of P4 either at the first use or during reuse of the implants after sanitizing the implant by autoclave or chemical disinfection. In a completely randomized design with a 2 × 3 factorial arrangement and 2 replicates, every cow underwent 2 of 6 treatments. Two sources of P4 [controlled internal drug release (1.9 g of P4) from Zoetis (São Paulo, Brazil), and Sincrogest (1.0 g of P4) from Ourofino (Cravinhos, Brazil)] and 3 types of processing, new (N), reused after autoclave (RA), and reused after chemical disinfection (RC), were used. After inducing luteolysis to avoid endogenous circulating P4, the cows were randomized in 1 of 6 treatments (1.9 g of N, 1.9 g of RA, 1.9 g of RC, 1.0 g of N, 1.0 g of RA, and 1.0 g RC). Cows were treated with the implants for 8 d and during this period blood samples were collected at 0, 2, 12, 24, 48, 72, 96, 120, 144, 168, and 192 h. Statistical analyses were performed using Proc-Mixed and the mean ± standard error of the mean P4 concentrations were calculated using the Proc-Means procedures of SAS 9.4 (SAS Institute Inc., Cary, NC). No interaction between treatments was observed. Comparing types of implant, average P4 concentrations during treatments were greater for 1.9 g than 1.0 g (1.46 vs. 1.14 ± 0.04 ng/mL). When types of processing were compared, average P4 concentrations did not differ between autoclaved and new inserts (1.46 vs. 1.37 ± 0.05 ng/mL; respectively), but both were greater than chemically disinfected implants (1.09 ± 0.04 ng/mL). Within 1.9-g P4 inserts, P4 concentrations from autoclaved implants were greater than new, which were greater than chemically disinfected (1.67 ± 0.06 vs. 1.49 ± 0.07 vs. 1.21 ± 0.05 ng/mL; respectively). For 1.0-g P4 implants, P4 concentrations from autoclaved did not differ from new, but both were greater than chemically disinfected (1.20 ± 0.08 vs. 1.24 ± 0.06 vs. 0.97 ± 0.05 ng/mL; respectively). In conclusion, the mean plasma P4 concentration in nonlactating Holstein cows was greater for 1.9 than 1.0 g of P4 and regardless of the type of implant, the autoclaving process provided greater circulating P4 in relation to chemical disinfection, and similar or greater P4 concentrations compared with a new implant.

Follicular dynamics, circulating progesterone, and fertility in Holstein cows synchronized with reused intravaginal progesterone implants that were sanitized by autoclave or chemical disinfection.

This experiment aimed to compare circulating progesterone (P4), follicular dynamics, and fertility during reuse of intravaginal P4 implants that were sanitized by autoclave or chemical disinfection in lactating Holstein cows submitted to fixed-time artificial insemination (FTAI). For this, 123 primiparous and 226 multiparous cows from 2 farms, averaging (mean ± standard deviation) 163.9 ± 141.9 d in milk, 35.7 ± 11.3 kg of milk/d, and a body condition score of 2.9 ± 0.5, were enrolled in the study. Cows were randomly assigned to 1 of 2 treatments using a completely randomized design and each cow received a reused implant (1.9 g of P4; previously used for 8 d) that was either autoclaved (AUT; n = 177) or chemically disinfected (CHEM; n = 172) on d -10. Also on d -10, cows received 2 mg of estradiol benzoate and 100 µg of GnRH. On d -3, cows received 25 mg of dinoprost (PGF2α). A second PGF2α was given on d -2, along with 1 mg of estradiol cypionate and P4 implant removal. Cows received FTAI on d 0. A subset of cows (n = 143) was evaluated by ultrasound on d -10, -8, -6, -3, -2, 0, and 5 to identify ovarian structures, and blood was sampled on d -10, -3, and -2 for P4 concentrations by RIA. Pregnancy diagnoses were performed at d 32 and 60. Statistical analyses was performed using PROC-MIXED for continuous variables and PROC-GLIMMIX of SAS 9.4 (SAS Institute Inc., Cary, NC) for binomial variables. The treatments did not differ in circulating P4 on d -10 or -3, but P4 was greater on d -2 in CHEM cows. Ovulation to the treatments on d -10 was associated with lower circulating P4 on d -10 (2.0 vs. 3.1 ng/mL) and resulted in greater P4 on d -3 (4.0 vs. 2.4 ng/mL) and more cows with a corpus luteum on d -3 (100 vs. 40%) than nonovulating cows. Cows that ovulated to d -10 treatments were more likely to have a synchronized new follicular wave (97.9 vs. 63.2%) and had an earlier wave emergence (1.9 vs. 2.6 d), resulting in less cows ovulating a persistent follicle (0.0 vs. 35.7%). Type of P4 implant, corpus luteum presence on d -10, and ovulation to d -10 treatments did not affect fertility (pregnancy per AI; P/AI). However, P/AI on farm A was greater than on farm B at 32 (40.8 vs. 27.8%) and 60 d (35.8 vs. 24.3%), independent of treatment. In conclusion, P4 implants with different P4 release patterns did not produce detectable differences in follicular dynamics, synchronization rate, or P/AI. Nevertheless, presence of corpus luteum or ovulation at the beginning of the FTAI protocol affected reproductive variables, such as timing and synchronization of follicular wave emergence, and size of the ovulatory follicle. Beyond that, more overall synchronized cows became pregnant to the FTAI protocol.

Gene expression profile in heat-shocked Holstein and Nelore oocytes and cumulus cells.

The present study determined the transcriptome profile in Nelore and Holstein oocytes subjected to heat shock during IVM and the mRNA abundance of selected candidate genes in Nelore and Holstein heat-shocked oocytes and cumulus cells (CC). Holstein and Nelore cows were subjected to in vivo follicle aspiration. Cumulus-oocyte complexes were assigned to control (38.5°C, 22h) or heat shock (41°C for 12h, followed by 38.5°C for 10h) treatment during IVM. Denuded oocytes were subjected to bovine microarray analysis. Transcriptome analysis demonstrated 127, nine and six genes were differentially expressed between breed, temperature and the breed×temperature interaction respectively. Selected differentially expressed genes were evaluated by real-time polymerase chain reaction in oocytes and respective CC. The molecular motor kinesin family member 3A (KIF3A) was upregulated in Holstein oocytes, whereas the pro-apoptotic gene death-associated protein (DAP) and the membrane trafficking gene DENN/MADD domain containing 3 (DENND3) were downregulated in Holstein oocytes. Nelore CC showed increased transcript abundance for tight junction claudin 11 (CLDN11), whereas Holstein CC showed increased transcript abundance for antioxidant metallothionein 1E (MT1E) . Moreover, heat shock downregulated antioxidant MT1E mRNA expression in CC. In conclusion, oocyte transcriptome analysis indicated a strong difference between breeds involving organisation and cell death. In CC, both breed and temperature affected mRNA abundance, involving cellular organisation and oxidative stress.

Development of insulin resistance in dairy cows by 150 days of lactation does not alter oocyte quality in smaller follicles.

The objective of this study was to test the hypothesis that high-producing dairy cows become increasingly resistant to insulin throughout lactation and that, consequently, oocyte quality is compromised. We used Holstein cows at 50 (51.5±3.7; n=30), 100 (102.3±9.4; n=30), and 150 (154.5±18.9; n=30) days in milk (DIM). We measured circulating insulin and glucose and performed a glucose tolerance test (GTT) after 5h of fasting. To evaluate oocyte quality, we performed ovum pickup on the day before the GTT (581 oocytes). We performed statistical analyses using the MIXED procedure of SAS. The model included the fixed effects of DIM, period, time, parity, and an interaction between DIM and time. We observed no difference in the GTT between groups for any variable related to circulating glucose (for example, glucose peak=203.3±7.2, 208.8±6.3, and 194.3±5.9mg/dL). However, various measures of circulating insulin were different in cows at 150 DIM compared with 50 or 100 DIM: higher basal insulin (8.8±0.9, 8.8±0.8, and 11.9±0.8 µIU/mL), peak insulin (61.9±6.2, 69.1±5.7, and 89.0±6.1 µIU/mL), delta maximum insulin (51.1±5.5, 59.4±5.0, and 73.5±5.4 µIU/mL), and area under the curve 5-60 (1,874.8±171.0, 2,189.5±157.8, and 2,610.5±174.0 µIU/mL × min). Nevertheless, we observed no difference among groups in the number of viable oocytes (3.2±0.7, 3.9±0.7, and 3.6±0.7 per cow per ovum pickup) or percentage of viable oocytes (49.3, 52.2, and 51.8%). Increased circulating insulin before and throughout the GTT in cows at 150 DIM indicates that cows develop increasing insulin resistance with increasing DIM; however, increased insulin resistance was not associated with a detectable alteration in the quality of oocytes aspirated from small and medium-sized follicles.

Progesterone-based fixed-time artificial insemination protocols for dairy cows: Gonadotropin-releasing hormone versus estradiol benzoate at initiation and estradiol cypionate versus estradiol benzoate at the end.

Our objectives were to evaluate ovarian dynamics and fertility comparing 2 treatments at the start of a progesterone (P4)-based fixed-time artificial insemination (FTAI) protocol and 2 treatments at the end of the protocol. Thus, 1,035 lactating Holstein cows were assigned in a random phase of the estrous cycle to 1 of 4 treatments using a completely randomized design with a 2×2 factorial arrangement. At the beginning of the protocol (d -10), cows received GnRH or estradiol benzoate (EB) and, at the end, EB (d -1) or estradiol cypionate (ECP; d -2), resulting in 4 treatments: GnRH-EB, GnRH-ECP, EB-EB, and EB-ECP. All cows received an intravaginal P4 device on d -10, which was removed on d -2. Cows also received PGF2α on d -3 and -2. The FTAI was performed on d 0. Ovaries were evaluated by ultrasound for corpus luteum (CL) presence and regression (d -10 and -3) and follicle measurements (d -10 and 0), as well as the uterus for percentage pregnant per AI (P/AI; d 32 and 60). Blood samples were collected (d -10 and -3) for P4 measurements. Treatment with GnRH rather than EB tended to increase P/AI on d 32 (38.2 vs. 33.7%) and on d 60 (32.9 vs. 28.9%). More cows treated with GnRH had CL on d -3 compared with EB-treated cows (77.3 vs. 58.3%), due to less CL regression between d -10 and -3 (24.7 vs. 43.8%) and more cows with a new CL on d -3 (35.9 vs. 25.0%). Cows treated with GnRH also had greater P4 concentrations on d -3 than EB cows (3.4 vs. 2.0 ng/mL). Increased circulating P4 at the start of the protocol (d -10) decreased the probability of ovulation to EB or GnRH at that time. Cows from GnRH group also ovulated a larger-diameter follicle at the end of the protocol (15.5 vs. 14.7mm). No difference between EB and ECP in P/AI on d 32 (34.8 vs. 37.0) and 60 (30.8 vs. 31.0%) or in pregnancy loss (11.1 vs. 15.4%) was detected and we found no interaction between treatments for P/AI. Independent of treatment, cows with CL on d -10 and -3 had the greatest P/AI on d 60 (36.9%). In conclusion, treatments at the end of the protocol were similar for ECP or EB and we found no additive effect or interactions on P/AI between treatments. However, cows treated with GnRH rather than EB on d -10 had less luteolysis and tended to have greater P/AI, probably because P4 concentrations were greater during the protocol. Finally, regardless of treatments, cows with CL at the beginning of the protocol as well as at the time of PGF2α had greater fertility.

Increasing estradiol benzoate, pretreatment with gonadotropin-releasing hormone, and impediments for successful estradiol-based fixed-time artificial insemination protocols in dairy cattle.

With the objective to optimize fixed-time artificial insemination (FTAI) protocols based on estradiol benzoate (EB) and progesterone (P4), we performed 2 experiments (Exp.) in dairy cows. In Exp. 1 (n=44), we hypothesized that increased EB (EB3=3 mg vs. EB2=2 mg) on d 0 would improve synchronization of ovarian follicle wave emergence. Likewise, in Exp. 2 (n=82), we hypothesized that a GnRH treatment on d -3 (early in a follicular wave on d 0) versus d -7 (presence of a dominant follicle on d 0) would better synchronize wave emergence. Moreover, results from both experiments were combined to identify reasons for the lack of synchronization. All cows were treated with EB at the time of introduction of a P4 implant (d 0). On d 7, cows were given 25 mg of prostaglandin F2α; on d 8, the implant was removed and cows were given 1mg of estradiol cypionate. All cows received FTAI on d 10. In both experiments, daily ultrasound evaluations were performed and, in Exp. 2, circulating P4 was evaluated during the protocol. Pregnancy per artificial insemination (P/AI) was determined on d 31 and 59 after FTAI. In Exp. 1, EB dose did not change time to wave emergence, but EB3 compared with EB2 decreased the percentage of cows with a corpus luteum on d 7 (19.8 vs. 55.3%) and time to ovulation (10.4 vs. 10.9 d). In Exp. 2, although we detected a tendency for delayed follicle wave emergence after the start of the FTAI protocol in cows ovulating to GnRH given on d -7, there was no difference in percentage of cows with a synchronized wave emergence (~80%). Regardless of treatment, more cows with P4<0.1 ng/mL, compared with P4≥0.1 and <0.22 ng/mL at the time of AI, ovulated to the protocol (81.2 vs. 58.0%) and had increased P/AI (47.4 vs. 21.4%). An analysis of data from both experiments showed that only 73.8% (93/126) of cows had synchronized wave emergence, and only 77.8% (98/126) of cows ovulated at the end of the protocol. Fertility was much greater in cows that had emergence of a new wave synchronized and ovulated to end of the protocol [P/AI 61.3% (46/75)] compared with cows that failed to present one or both of the outcomes above [15.7% (8/51)]. Thus, although current FTAI protocols using EB and P4 produce P/AI between 30 and 40% for lactating dairy cows, there remains room for improvement because less than 60% (75/126) of the cows were correctly synchronized. Starting the FTAI protocol without the dominant follicle or increasing the dose of EB to 3mg was not effective in increasing synchronization rate.

Effect of injectable vitamin E on incidence of retained fetal membranes and reproductive performance of dairy cows.

The objectives were to evaluate the effects of injectable vitamin E during the last 3 wk prepartum on the incidence of retained fetal membranes (RFM) and reproductive performance. Dairy cows (n=890), 390 Holsteins (132 nulliparous and 258 parous) and 500 crossbred Holstein × Gyr (199 nulliparous and 301 parous), from 3 dairy farms in Brazil were assigned to the study. In all 3 farms, from October to March, prepartum cows grazed tropical grasses and received 2 kg/d of a mixture of finely ground corn, soybean meal, and minerals and vitamins. From April to September prepartum cows received a total mixed ration composed of corn silage, finely ground corn, soybean meal, and minerals and vitamins. During the prepartum period, cows were fed 280 (farm 1), 390 (farm 2), and 480 IU (farm 3) of supplemental vitamin E per day, and throughout postpartum, cows were fed 370 (farm 1), 500 (farm 2), and 600 (farm 3) IU of supplemental vitamin E. Within each farm, cows were randomly assigned to remain as untreated controls or to receive 3 i.m. injections of 1,000 IU each of dl-α-tocopherol administered at 19.2 ± 4.3, 12.9 ± 3.3, and 6.2 ± 2.9 d before calving (VitE). Blood was sampled from 141 cows immediately before enrollment to determine the α-tocopherol and cholesterol statuses. Blood was also sampled and analyzed for concentrations of cortisol and nonesterified fatty acids in the last 3 wk of gestation. The serum concentration of α-tocopherol or α-tocopherol:cholesterol ratio did not differ between treatments and averaged 2.97 ± 0.10 µg/mL and 4.46 ± 0.16 × 10(-3), respectively. In total, 53.2% of the cows had an inadequate concentration of serum α-tocopherol based on the 3.0 µg/mL cut-off for adequacy. The risk of RFM decreased as serum α-tocopherol increased. Milk production did not differ between controls and VitE cows. Treatment with injectable α-tocopherol decreased RFM from 20.1 to 13.5%, decreased incidence of stillbirth from 14.9 to 6.8%, and tended to decrease death by 200 d postpartum. VitE cows tended to have improved pregnancy per insemination at first AI (36.7 vs. 30.1%) because of decreased pregnancy loss from 31 to 62 d of gestation (12.5 vs. 20.5%). Despite a similar insemination rate, VitE cows had 22% greater pregnancy rate than control cows. Cows receiving vitamin E had decreased circulating cortisol and nonesterified fatty acids around calving. In summary, when cows were fed limited amounts of supplemental vitamin E, 28 to 48% of the recommendations, prepartum supplementation with injectable α-tocopherol decreased incidence of RFM and improved reproduction.

Characterization of allele-specific expression of the X-linked gene MAO-A in trophectoderm cells of bovine embryos produced by somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) may affect epigenetic mechanisms and alter the expression of genes related to embryo development and X chromosome inactivation (XCI). We characterized allele-specific expression of the X-linked gene monoamine oxidase type A (MAO-A) in the trophectoderm (TF) of embryos produced by SCNT. Total RNA was isolated from individual biopsies (N = 25), and the allele-specific expression assessed by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism. Both paternal and maternal alleles were expressed in the trophectoderm. However, a higher frequency of the mono-allelic expression of a specific allele was observed (N = 17; 68%), with the remaining samples showing the presence of mRNA from both alleles (N = 8; 32%). Considering that MAO-A is subject to XCI in bovine, our results suggest that SCNT may influence XCI because neither an imprinted (mono-allelic expression in all samples) nor a random (presence of mRNA from both alleles in all samples) pattern of XCI was observed in TF. Due to the importance of XCI in mammalian embryo development and its sensitivity to in vitro conditions, X-linked genes subject to XCI are candidates for use in the development of embryo quality molecular markers for assisted reproduction.

Effects of supplemental progesterone after artificial insemination on expression of interferon-stimulated genes and fertility in dairy cows.

The objectives of the current study were to evaluate the effects of supplemental progesterone after artificial insemination (AI) on expression of IFN-stimulated genes (ISG) in blood leukocytes and fertility in lactating dairy cows. Weekly cohorts of Holstein cows were blocked by parity (575 primiparous and 923 multiparous) and method of insemination (timed AI or AI on estrus) and allocated randomly within each block to untreated controls, a controlled internal drug release (CIDR) containing 1.38g of progesterone from d 4 to 18 after AI (CIDR4), or a CIDR on d 4 and another on d 7 after AI and both removed on d 18 (CIDR4+7). Blood was sampled to quantify progesterone concentrations in plasma and mRNA expression in leukocytes for the ubiquitin-like IFN-stimulated gene 15-kDa protein (ISG15) and receptor transporter protein-4 (RTP4) genes. Pregnancy was diagnosed on d 34±3 and 62±3 after AI. Treatment increased progesterone concentrations between d 5 and 18 after AI in a dose-dependent manner (control=3.42, CIDR4=4.97, and CIDR4+7=5.46ng/mL). Cows supplemented with progesterone tended to have increased luteolysis by d 19 after AI (control=17.2; CIDR4=29.1; CIDR4+7=30.2%), which resulted in a shorter AI interval for those reinseminated after study d 18. Pregnancy upregulated expression of ISG in leukocytes on d 19 of gestation, but supplementing progesterone did not increase mRNA abundance for ISG15 and RTP4 on d 16 after insemination and tended to reduce mRNA expression on d 19 after AI. For RTP4 on d 19, the negative effect of supplemental progesterone was observed only in the nonpregnant cows. No overall effect of treatment was observed on pregnancy per AI on d 62 after insemination and averaged 28.6, 32.7, and 29.5% for control, CIDR4, and CIDR4+7, respectively. Interestingly, an interaction between level of supplemental progesterone and method of AI was observed for pregnancy per AI. For cows receiving exogenous progesterone, the lower supplementation with CIDR4 increased pregnancy per AI on d 62 in cows inseminated following timed AI (CIDR4=39.2; CIDR4+7=27.5%); in those inseminated following detection of estrus, however, the use of a second insert on d 7 resulted in greater pregnancy per AI (CIDR4=26.9; CIDR4+7=31.5%). Pregnancy loss did not differ among treatments. Supplemental progesterone post-AI using a single intravaginal insert on d 4 was beneficial to pregnancy in cows inseminated following timed AI, but incremental progesterone with a second insert on d 7 did not improve fertility of dairy cows.

Effects of treatment with human chorionic gonadotrophin or intravaginal progesterone-releasing device after AI on circulating progesterone concentrations in lactating dairy cows.

Adequate circulating progesterone (P4) is important for pregnancy. Lactating dairy cattle have lower circulating P4, particularly when smaller follicles are ovulated during timed AI protocols. The aim of the present study was to determine the supplementation strategy that resulted in P4 concentrations in lactating dairy cattle similar to those in heifers. Lactating Holstein cows (n=61) were synchronised using the Double-Ovsynch method and, on Day 5, were randomly assigned to receive no treatment (control), controlled internal drug release (CIDR), human chorionic gonadotrophin (hCG; 3300 IU) or CIDR+hCG. Heifers after normal oestrus were followed as controls (n=10). Profiles of circulating P4 concentrations were compared using repeated-measures ANOVA. Heifers had greater P4 concentrations than control cows at all times after Day 5 (P<0.0001). Cows receiving CIDR had lower P4 concentrations than heifers (P=0.0037) on Days 8-16. Treatment with hCG generally caused ovulation and resulted in circulating P4 concentrations greater than those in control lactating cows by 3 days after treatment (Day 8 after AI), but the treatment×time interaction (P=0.01) showed that cows treated with hCG generally had lower P4 concentrations than heifers. Supplementation with CIDR+hCG resulted in P4 concentration profiles similar to those in heifers. Thus, the use of CIDR and the production of an accessory corpus luteum with hCG elevates P4 concentrations in lactating cows to those seen in heifers. This information may be useful for designing future trials into P4 supplementation and fertility.

Effect of treatment with human chorionic gonadotropin on day 5 after timed artificial insemination on fertility of lactating dairy cows.

Reproductive management programs that synchronize ovulation can ovulate a smaller than normal follicle, potentially resulting in inadequate progesterone (P4) concentrations after artificial insemination (AI). Ovulation of the dominant follicle of the first follicular wave with human chorionic gonadotropin (hCG) treatment can produce an accessory corpus luteum and increase circulating P4 concentrations. This manuscript reports the results of 2 separate analyses that evaluated the effect of hCG treatment post-AI on fertility in lactating dairy cows. The first study used meta-analysis to combine the results from 10 different published studies that used hCG treatment on d 4 to 9 post-AI in lactating dairy cows. Overall, pregnancies per artificial insemination (P/AI) were increased 3.0% by hCG treatment post-AI [34% (752/2,213) vs. 37% (808/2,184); Control vs. hCG-treated, respectively]. The second study was a field research trial in which lactating Holstein cows (n=2,979) from 6 commercial dairy herds were stratified by parity and breeding number and then randomly assigned to one of 2 groups: control (no further treatment, n=1,519) or hCG [Chorulon i.m.: 2,000 IU (in 3 of the herds) or 3,300 IU (in 3 herds); n=1,460] on d 5 after a timed AI (ovulation synchronized with Ovsynch, Presynch-Ovsynch, or Double-Ovsynch). In a subset of cows, the hCG profile and P4 changes were determined. Treatment with hCG increased P4 (4.3 vs. 5.3 ng/mL on d 12). Pregnancies per AI were greater in cows treated with hCG (40.8%; 596/1,460) than control (37.3%; 566/1,519) cows. Interestingly, an interaction among treatment and parity was observed; primiparous cows had greater P/AI after hCG (49.7%; 266/535) than controls (39.5%; 215/544). In contrast, older cows receiving hCG (35.7%; 330/925) had similar P/AI to controls (36.0%; 351/975).Thus, targeted use of hCG on d 5 after TAI enhances fertility about 3.0% (based on meta-analysis) to 3.5% (based on our field trial). Surprisingly, this fertility-enhancing effect of hCG was very large in first-lactation cows but not observed in older cows in the field study. Future research is needed to confirm these intriguing results and to determine why older cows did not have improved fertility after hCG treatment.

Food restriction reverses the hyper-muscular phenotype and force generation capacity deficit of the myostatin null mouse.

Food restriction has a great impact on skeletal muscle mass by inducing muscle protein breakdown to provide substrates for energy production through gluconeogenesis. Genetic models of hyper-muscularity interfere with the normal balance between protein synthesis and breakdown which eventually results in extreme muscle growth. Mutations or deletions in the myostatin gene result in extreme muscle mass. Here we evaluated the impact of food restriction for a period of 5 weeks on skeletal muscle size (i. e., fibre cross-sectional area), fibre type composition and contractile properties (i. e., tetanic and specific force) in myostatin null mice. We found that this hyper-muscular model was more susceptible to catabolic processes than wild type mice. The mechanism of skeletal muscle mass loss was examined and our data shows that the myostatin null mice placed on a low calorie diet maintained the activity of molecules involved in protein synthesis and did not up-regulate the expression of genes pivotal in ubiquitin-mediated protein degradation. However, we did find an increase in the expression of genes associated with autophagy. Surprisingly, the reduction on muscle size was followed by improved tetanic and specific force in the null mice compared to wild type mice. These data provide evidence that food restriction may revert the hyper-muscular phenotype of the myostatin null mouse restoring muscle function.

Review: Manipulation of follicle development to improve fertility of cattle in timed-artificial insemination programs.

The development of an ovulatory follicle is a fundamental premise for any reproductive management program that aims to optimize fertility in cattle. Controlling follicular development comprises the synchronized emergence of a new follicular wave, selection and growth of the dominant follicle, and synchronized ovulation of a high-quality oocyte. All these follicular events, primarily driven by gonadotropin secretion, occur under a very dynamic hormonal environment. In this sense, controlling follicular development demands essentially a precise manipulation of the hormonal environment to modulate gonadotropin secretion. Furthermore, the effectiveness of hormonal manipulation strategies in the management of follicular development depends on specific particularities of each situation, which can vary widely according to genetic groups (Bos taurus vs Bos indicus), nutritional, metabolic, and reproductive status. In this regard, the constant search for the refined synchrony between the hormonal treatments and reproductive events, considering these distinctions and particularities, have provided valuable information that contributed to the development of efficient reproductive programs. This manuscript discusses the physiological bases behind the development of fine-tuned timed-artificial insemination protocols for beef and dairy cattle that resulted in great improvements in reproductive efficiency of beef and dairy herds.

Unravelling the role of 17ß-estradiol on advancing uterine luteolytic cascade in cattle.

In cattle, 17ß-estradiol (E2) stimulates prostaglandin F2α (PGF2α) synthesis, which causes luteolysis. Except for the well-established upregulation of oxytocin receptor gene (OXTR), molecular mechanisms of E2-induced PGF2α release in vivo remain unknown. We hypothesized that E2-induced PGF2α release requires de novo transcription of components of the PGF2α synthesis machinery. Beef cows (n = 52) were assigned to remain untreated (Control; n = 10), to receive 50% ethanol infusion intravenously (Placebo; n = 21), or 3 mg E2 in 50% ethanol infusion intravenously (Estradiol; n = 21) on day 15 (D15) after estrus. We collected a single endometrial biopsy per animal at the time of the treatment (0h; Control B0h group), 4 hours (4h; Placebo B4h group and Estradiol B4h group), or 7 hours (7h; Placebo B7h group and Estradiol B7h group) post-treatment. Compared to the Placebo group, the Estradiol group presented significantly greater 13,14-dihydro-15-keto-PGF2α concentrations between 4h and 7h and underwent earlier luteolysis. At 4h, the qPCR analysis showed a lower abundance of ESR1, ESR2 and aldo-keto reductase family 1 member B1 (AKR1B1) genes in the Estradiol B4h group, and a greater abundance of OXTR compared to the Placebo B4h group. Similarly, the E2 treatment significantly reduced the abundance of AKR1B1, and AKR1C4 in the Estradiol B7h group, compared to the placebo group. Overall, E2-induced PGF2α release and luteolysis involved an unexpected and transient downregulation of components of the PGF2α-synthesis cascade, except for OXTR, which was upregulated. Collectively, our data suggest that E2 connects newly-synthesized OXTR to pre-existing cellular machinery to synthesize PGF2α and cause luteal regression.

A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep.

Brazilian Santa Inês (SI) sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecG(E) (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecG(E) allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.