Association between genomic daughter pregnancy rate and expected milk production on the resumption of estrus behavior in Holstein cattle.

The objective of this observational study was to evaluate estrous expression at the first estrus occurring between 7 to 30 d in milk (DIM), as detected by an automated activity monitor (AAM), and its association with genomic daughter pregnancy rate (GDPR) and genomic expected milk production (GEM) in lactating dairy cows. A total of 4,119 lactations from 2,602 Holstein cows were included. Cows were enrolled as first lactation (n = 1,168), second lactation (n = 1,525) and third and greater lactation (n = 1,426). Hair samples were collected from the tail switch, and cows were genotyped using an SNP platform (Clarifide, Zoetis, São Paulo, SP, Brazil). Postpartum cows were examined daily by the farm personnel from calving until 10 DIM. Calving was classified as assisted (forced calf extraction) and unassisted (normal calving). Retained fetal membranes (RFM), hyperketonemia (KET), and left displaced abomasum (LDA) were also recorded. Mean GDPR (± SD) was -0.29 ± 1.4, and the intensity and duration of the first estrus event was 15.9 ± 13.1 x-factor (intensity unit measurement) and 11.1 ± 3.8 h, respectively. Cows that had greater GDPR had greater intensity and longer duration of estrus at the alert, independent of parity. Overall resumption of estrous expression, between 7 to 30 DIM, was 41.2% (1,695/4,119), where 58.8% (2,424/4,119) did not have an estrus event, 31.0% (1,274/4,119) of cows had one event of estrus, and 10.2% (421/4,119) of cows had 2 or more events of estrus early postpartum. Mean DIM (± SD) at first estrus event, detected by the AAM, was 19.4 ± 4.4 d. Days in milk at first event for cows with one event was 20.7 ± 1.6 d and 15.9 ± 3.1 d for cows with 2 or more events of estrus. First lactation cows were more likely to have an estrus event early postpartum when compared with second and third and greater lactation cows (45.2 ± 1.4% [530/1168] vs. 41.6 ± 1.3% [636/1525] vs. 37.2 ± 1.3% [529/1426], respectively). There was an interaction of parity and GDPR on the proportion of cows demonstrating an early postpartum estrus. There was no difference in the proportion of cows with an early postpartum estrus between those with assisted or unassisted calving, RFM, or LDA. However, cows that had KET were less likely to have an alert early postpartum when compared with cows that did not have KET. Mean genomic expected milk production (± SD) was 256.8 ± 600.1 kg. There was no interaction between GEM and parity on estrous expression (i.e., intensity and duration). There was no interaction between GEM and GDPR on the proportion of estrus early postpartum.

Is prolonged luteal phase a problem in lactating Holstein cows?

In this study, the main objective was to assess if long luteal phases could have causes other than pregnancy loss. We enrolled Holstein dairy cows ≥50 DIM from a commercial herd in Brazil from October 2016 to August 2017. All cows received an estradiol-based synchronization protocol, and, on the day of insemination (d 0), were randomly assigned either an AI or a placebo insemination (PBO) in a 3:1 ratio. An ultrasound was used to assess the presence of a corpus luteum (CL) on d 17, 24, and 31, which, combined to the information from patches for the detection of estrus, was used to determine the length of the luteal phase following AI or PBO. Pregnancy was assessed by ultrasound on d 31 and cows that were pregnant were excluded from the analyses. The length of the estrous cycles was categorized as short (<17 d), normal (17-23 d), long (24-30 d), and very long (≥31 d). We compared the proportion of cows in each category between the AI and PBO groups using a cumulative ordinal mixed model. We define prolonged luteal phase as estrous cycles ≥24 d and tested its association with potential risk factors (parity, season, DIM, uterine size and position score, milk production, BCS, and the presence of a CL at enrollment to the synchronization protocol) using mixed logistic regression models. Results are presented as odds ratio (OR) and 95% Bayesian credible intervals (BCI). Data from 876 inseminations (AI: n = 616, PBO: n = 260) was collected. Overall, 12% of estrous cycles were short, 31% were normal, 19% were long, and 38% were very long. There was no difference in the odds of being in longer estrous cycle categories for the AI compared with the PBO group (OR = 0.92; 95% BCI = 0.76-1.10). Season and presence of a CL at enrollment were associated with prolonged luteal phase. In the AI group, there was a possible effect of early pregnancy losses on the lifespan of the CL, but not the PBO group, which led us to conclude that long and very long estrous cycles were not all caused by the embryonic loss. In fact, the high prevalence of cows with an extended CL lifespan in the present study suggests this could be an under- or miss-reported characteristic of high-producing lactating Holstein cows. This finding may have important repercussions in the understanding of the CL function physiology of lactating Holstein cows.

Effects of additional gonadotropin-releasing hormone and prostaglandin F2α treatment to an estradiol/progesterone-based embryo transfer protocol for recipient lactating dairy cows.

This study was designed to evaluate whether the utilization of a second PGF2α treatment at the end of an estradiol/progesterone (E2/P4)-based protocol with or without GnRH at the beginning of the protocol would improve pregnancy rates of lactating Holstein cows assigned to timed embryo transfer. A total of 501 lactating Holstein cows in 5 farms were enrolled in the experiment. Within farm, cows were blocked by parity and, within block, were assigned randomly to (1) insertion of an intravaginal P4 device (controlled internal drug-releasing device; CIDR) and estradiol benzoate on d -11, PGF2α on d -4, CIDR withdrawal and an injection of estradiol cypionate on d -2, and timed embryo transfer on d 7 (1-PGF; n = 164); (2) the same treatments as 1-PGF, but with PGF2α administered on d -4 and -2 (2-PGF; n = 171); and (3) 2-PGF with the addition of a GnRH treatment on d -11 (GnRH+2-PGF; n = 166). Ovaries were scanned by transrectal ultrasonography on d -11, -4, and 7, and blood samples were collected on d -11, -4, 0, and 7 for P4 determination. Treatment comparisons were performed using contrasts. The proportion of cows with a new corpus luteum on d -4 was greater in GnRH+2-PGF cows. Cows in 1-PGF had a greater P4 concentration on d 0 but lesser P4 on d 7 compared with cows in the other groups. Cows assigned to receive 2-PGF (2-PGF and GnRH+2-PGF) had greater estrus expression, and a greater proportion of cows ovulated to estradiol cypionate. No further contrast effects were observed for follicle diameter, double ovulation rate, pregnancy per embryo transfer (P/ET) on d 32 and 60, or pregnancy loss. As P4 concentration on d -4 increased, P/ET on d 60 tended to increase. Cows with P4 ≥3.66 ng/mL on d -4 had greater P/ET on d 32 and 60 than those with P4 below that threshold. Regardless of treatment, cows with P4 concentration ≥3.66 ng/mL also had a greater pregnancy per synchronized protocol (P/SP) on d 60. Also, a P4 concentration on d -4 (low or high) × follicle diameter (continuous) interaction tendency was observed when evaluating P/ET. Although P/ET did not differ among cows with different follicles sizes with reduced P4 concentration on d -4 (<3.66 ng/mL), it increased in cows with larger follicles exposed to increased P4 concentration (≥3.66 ng/mL). When P4 on d 0 was evaluated, P/ET on d 32 and 60 was greater for cows with low (≤0.09 ng/mL) versus high (>0.21 ng/mL) P4; as P4 concentration on d 0 increased, P/ET linearly decreased. In summary, cows with increased P4 concentrations during growth of the ovulatory follicular wave had improved P/ET. Administering a second PGF2α dose reduced P4 concentration on d 0 and increased ovulatory response to the protocol, but no benefits were observed on P/ET or P/SP.

Association between genomic daughter pregnancy rates and reproductive parameters in Holstein dairy cattle.

The objective of this observational study was to evaluate the relationship between genomic daughter pregnancy rate (GDPR) with reproduction parameters such as pregnancy at first artificial insemination (AI), pregnancy per AI, and pregnancy losses (PL). A total of 12,949 events from 3,499 Holstein cows were included. Cows were enrolled as nulliparous (n = 1,220), primiparous (n = 1,314), or multiparous (n = 965). Cows were bred either after a timed AI protocol, timed embryo transfer (ET), or spontaneous estrus. Most lactating cows were bred following a timed AI protocol based on estradiol and progesterone, and most nulliparous were artificially inseminated following estrus detection. Hair samples were collected from the tail switch and cows were genotyped using a SNP platform (Clarifide, Zoetis). Cows that were bred by timed AI were evaluated for estrous behavior using tail chalk. Tail chalk was applied on the head of the tail 2 d before timed AI and the chalk was evaluated at AI (no estrus: 100% of chalk remaining or ≥50% of chalk remaining; Estrus: <50% of chalk remaining). Pregnancy diagnosis was performed at d 32 and 60 after AI using ultrasonography, and the presence of a heartbeat was considered a positive diagnosis. Pregnancy loss was defined as a pregnant cow on d 32 that was nonpregnant on d 60. As GDPR increased, the odds of pregnancy at first AI increased [odds ratio (OR) = 1.28, 95% CI = 1.20-1.35], the odds of pregnancy per AI increased (OR = 1.31, 95% CI = 1.25-1.36), and the odds of PL decreased (OR = 0.66, 95% CI = 0.60-0.72). Most cows that were bred on the day of the timed AI demonstrated estrus (n = 6,075; 92.9%). The odds of demonstrating estrus on the day of timed AI increased as GDPR increased (OR = 1.31, 95% CI = 1.17-1.48). There was no interaction between GDPR and parity or breeding management for pregnancy at first AI, pregnancy per AI, and PL. In conclusion, the odds of pregnancy at first AI and pregnancy per AI increased as GDPR increased. Moreover, the odds of PL increased as GDPR decreased. Greater GDPR was also associated with greater occurrence of estrus on the day of timed AI. These results suggest that selecting for higher GDPR could result in better reproductive performance, but this would need to be assessed with additional research.

Occurrence and greater intensity of estrus in recipient lactating dairy cows improve pregnancy per embryo transfer.

The aim of this study was to determine the association between occurrence and intensity of estrous expression with pregnancy success in recipient lactating dairy cows subjected to embryo transfer (ET). Two observational studies were conducted. Holstein cows were synchronized using the same timed ET protocol, based on estradiol and progesterone in both experiments. At 9 d after the end of the timed ET protocol only animals that had ovulated were implanted with a 7-d embryo [experiment 1 (Exp. 1); n = 1,401 ET events from 1,045 cows, and experiment 2 (Exp. 2); n = 1,147 ET events from 657 cows]. Embryos were produced in vivo (Exp. 1 and Exp. 2) and in vitro (only Exp. 2), then transferred to recipient cows as fresh or frozen-thawed. Pregnancy was confirmed at 29 and 58 d after the end of timed ET protocol. In Exp. 1, animals had their estrous expression monitored through a tail chalk applied on the tail head of the cows and evaluated daily for chalk removal (no estrus: 100% of chalk remaining; estrus: <50% of chalk remaining). In Exp. 2, cows were continuously monitored by a leg-mounted automated activity monitor. Estrous expression was quantified using the relative increase in physical activity at estrus in relation to the days before estrus. Estrous expression was classified as no estrus [<100% relative increase in activity (RI)], weak intensity (100-299% RI), and strong intensity (≥300% RI). Data were analyzed by analysis of variance using mixed linear regression models (GLIMMIX) in SAS (SAS Institute Inc.). A total of 65.2% (914/1,401) and 89.2% (1,019/1,142) of cows from Exp. 1 and Exp. 2, respectively, displayed estrus at the end of the ovulation synchronization protocol. In Exp. 1, cows expressing estrus before to ET had greater pregnancy per ET than those that did not [41.0 ± 2.3% (381/914) vs. 31.5 ± 2.9% (151/487), respectively]. Similarly, in Exp. 2, cows classified in the strong intensity group had greater pregnancy per ET compared with cows in the weak intensity and no estrus groups [41.3 ± 2.2% (213/571) vs. 32.7 ± 2.7% (115/353) vs. 11.3 ± 3.5% (26/218), respectively]. There was no effect of ET type on pregnancy per ET in Exp. 1. However, in Exp. 2, cows that received an in vivo-produced embryo, either fresh or frozen, had greater pregnancy per ET compared with cows that received in vitro-produced embryo. Cows receiving embryos in the early blastocyst and blastocyst stage had greater fertility compared with cows receiving embryos in the morula stage. There was an interaction between the occurrence of estrus and the stage of embryo development on pregnancy per ET, cows which displayed estrus and received a morula or early blastocyst had greater pregnancy per ET than cows that did not display estrus. In conclusion, the occurrence and the intensity of estrous expression improved pregnancy per ET in recipient lactating dairy cows and thus could be used as a tool to assist in the decision making of reproduction strategies in dairy farms.

Increasing the length of an estradiol with progesterone timed artificial insemination protocol with 2 controlled internal drug release devices improves pregnancy per artificial insemination in lactating dairy cows.

The objective of this study was to compare the effects of different lengths of ovulation synchronization protocols using 2 controlled internal drug release (CIDR) devices on ovarian dynamics and pregnancy outcomes in lactating dairy cows. Lactating Holstein cows (n = 1,979) were randomly assigned to receive timed artificial insemination (TAI; d 0) following 1 of 2 treatments: (1) 9-d protocol (n = 988; 9D) with 2 intravaginal devices containing 1.9 g of progesterone (CIDR) and 2.0 mg of estradiol benzoate on day -11; 25 mg (i.m.) of dinoprost tromethamine (PG) and withdrawal of 1 CIDR on d -4; 1.0 mg (i.m.) of estradiol cypionate, second CIDR withdrawal, and PG on d -2; and TAI on d 0 and (2) 10-d protocol (n = 991; 10D) with 2 CIDR and 2.0 mg of estradiol benzoate on d -12; 25 mg of PG and withdrawal of 1 CIDR on d -4; 1.0 mg of estradiol, second CIDR withdrawal, and PG on d -2; and TAI on d 0. There was no effect of protocol on estrus detection, whereas a greater percentage of cows from 10D had ovulated close to TAI [no corpus luteum (CL) at AI and a CL at d 7] versus cows assigned to 9D protocol. A protocol × heat stress (average cow temperature ≥39.1°C on day of AI and d 7) interaction was observed in a manner that pregnancy per AI (P/AI) was greater in non-heat-stressed 10D versus 9D cows, whereas P/AI did not differ when cows were under heat stress. Furthermore, 10D protocol did not increase P/AI when all cows that received AI were included in the analysis or in cows that ovulated near TAI. However, animals assigned to 9D without any event of heat stress had a reduced P/AI when compared with cows assigned to 10D without heat stress. A protocol × CL presence at the beginning of the protocol interaction was observed and cows with a CL at the beginning of the protocol had a greater P/AI in 10D versus 9D; meanwhile, in cows without a CL, no differences on P/AI were observed. The protocol × CL presence at the beginning of the protocol interaction on P/AI was also observed for cows that ovulated near TAI. A greater percentage of cows assigned to 9D had follicles of medium size (13-15.9 mm), and greater percentage of cows assigned to 10D had larger follicles (>16 mm). Increasing the length of an estradiol with progesterone-based ovulation synchronization protocol (10D vs. 9D) increased the proportion of cows with larger follicles (>16 mm) and increased P/AI in cows without heat stress and in cows with a CL at beginning of the protocol. Moreover, the 10D protocol increased the proportion of cows with ovulation near TAI, demonstrating the effectiveness of this protocol in improving the reproductive performance of lactating Holstein cows.

Symposium review: Linking activity-sensor data and physiology to improve dairy cow fertility.

Several studies have demonstrated that the intensity of estrous expression is associated with ovulation, ovarian and uterine function, and fertility, and is dependent on social hierarchy and the housing system used. Data from recent studies involving spontaneous and induced estrus have shown that a greater relative increase and longer estrus (captured by different automated activity monitors; AAM) are both associated with improved pregnancy per artificial insemination (AI; around 10 to 14% increase) and decreased pregnancy losses. Intensity and duration of estrus were surprisingly weakly associated with preovulatory follicle diameter and concentrations of plasma estradiol at estrus, whereas ovulation failure was associated with low estrus intensity. Studies have also shown that the display of estrous behavior near AI was associated with the modification of expression of genes related to the immune system, adhesion molecules, and prostaglandin synthesis in the endometrium. Transcripts in leukocytes and in the conceptus tissue associated with maternal recognition of pregnancy as well as conceptus elongation were all associated with differences in the intensity of estrous expression. Most recently, studies from the United States and Canada have demonstrated that reproductive programs emphasizing detection of estrus using AAM can be successful and comparable to intensive timed AI protocol-based programs that incorporate GnRH and PGF2α treatments. Further, one study concluded that the administration of GnRH at AI for spontaneous estrus events greatly improved pregnancy per AI, but only for cows with reduced intensity of estrous expression, showing the potential to use AAM data as a tool in targeted reproductive programs. Quantitative information from estrus events could be used to improve estrus detection and develop decision-making strategies at the farm level. Future studies in this field should aim to better understand ovarian, conceptus, and endometrial mechanisms associated with either the expression or the intensity of estrus, and to refine the identification of phenotypes related to estrus (relative increase, absolute increase, baseline levels, duration, and repeatability within cow) to improve data usage, estrus detection, and possibly genetic selection.

Technical note: Validation of an in-house bovine serum enzyme immunoassay for progesterone measurement.

Measuring circulating progesterone (P4) of dairy cows is a key component of many research studies dealing with basic and applied reproduction physiology. The gold standard in dairy cows for the measurement of P4 in serum is radioimmunoassay (RIA), but it generates radioactive waste and requires licensed facilities. The purpose of this study was to develop and validate an in-house competitive enzyme immunoassay (EIA) to measure the P4 concentration in serum of dairy cattle. The secondary objective was to validate a commercial EIA. In the present study, a competitive EIA was developed using commercially available antibodies and conjugates. Ninety-six well microtiter plates were coated with the secondary antibody and incubated overnight. Following a washing step, the wells were blocked using the primary antibody. Serum samples were prepared by first extracting P4 using petroleum ether, then diluted in working conjugate solution. Samples were pipetted into the coated and blocked plates, then the matching HRP conjugate label (P4-3-HRP, East Coast Bio, North Berwick, ME) was added. The plates were incubated for 2 h, then washed. The substrate solution was added, and the plate was incubated up to 1 h at room temperature in the dark until a blue color had developed. A stop solution was added, and the optical density measured on a microplate reader was set at 450 nm. The binding proportion was calculated by a visible spectrum absorbance reader, and the amount of P4 was calculated using a log-logit regression line. The commercial EIA was executed as suggested by the manufacturer. The validation of the in-house EIA was done by calculating the inter- and intraassay coefficients of variation (CV) and evaluating the parallelism of diluted samples. The results from the in-house and commercial EIA were also compared with the ones from the RIA graphically (scatterplots and Bland-Altman plots) and statistically, using the Spearman correlation coefficient (r) and the Cohen's kappa statistics using a threshold of 1.0 ng/mL (κ). For the in-house EIA, the intraassay CV were all <10%, but the interassay for samples with small and large P4 concentration had CV of 12.5 and 11.0%, respectively. The correlations between the results from the EIA and the RIA were strong (in-house: r = 0.90; commercial: r = 0.83). At small concentrations (<1.0 ng/mL), however, the correlation with the gold standard was weak (in-house: r = 0.27; commercial: r = 0.14). This was likely due to the lack of accuracy at small concentrations, also shown by the absence of parallelism in samples ≤0.4 ng/mL. In conclusion, results from both the in-house and commercial EIA strongly correlated with the gold standard, but less so at smaller concentrations. The in-house EIA offers good accuracy to measure P4 in samples with a concentration >0.4 ng/mL, and a perfect agreement with RIA using a threshold of 1.0 ng/mL.

North African dust intrusions and increased risk of respiratory diseases in Southern Portugal.

The study of dust intrusions in Portugal is still a subject on which little investigation has been made, especially in terms of their effects. Thus, this work aims to achieve two goals: firstly, to characterize the dust intrusions in the study area; and secondly, to evaluate the possible statistical association between the dust intrusion days and hospital admissions due to respiratory diseases. Dust intrusions in Portugal are prevalent during the summer season. During this season, the dust plumes tend to cover broader areas than in the other seasons and they have origin in the North African countries. In the study area for the period between 2005 and 2015, the relative risk of urgent hospitalizations due to respiratory diseases was 12.6% higher during dust intrusion days. In order to obtain this statistical association, a Distributed Lag Nonlinear Model was developed. With this work, we expect to help the development of further studies regarding North African dust intrusions in Portugal, more precisely their effects on human health.

Inflammatory diseases in dairy cows: Risk factors and associations with pregnancy after embryo transfer.

The objectives of the present prospective cohort study were to identify risk factors for inflammatory diseases in Holstein-Gyr crossbred dairy cows and characterize the associations of those diseases with pregnancy per embryo transfer (ET). Diseases were diagnosed in the first 60 d postpartum in 252 primiparous and 481 multiparous cows. Uterine diseases (UTD) included retained placenta, metritis, clinical endometritis, and subclinical endometritis. Nonuterine diseases (NUTD) included mastitis, lameness, pneumonia, and displaced abomasum. Blood was sampled on d 0, 1, and 2 postpartum and analyzed for concentrations of haptoglobin, fatty acids, total Ca (tCa), P, and Mg, and again on d 8 postpartum and analyzed for concentration of ß-hydroxybutyrate. The association between concentrations of metabolites in serum and inflammatory diseases was determined. Cows received a timed ET program starting 28 ± 3 d postpartum with first ET at 46 ± 3 d postpartum using fresh in vitro-produced embryos. Pregnancy was diagnosed on d 31 and 59 of presumptive gestation. Overall, 63.3% of the cows were diagnosed with UTD and 20.6% with NUTD. The risk factors for UTD included season of calving, parity group, calving problems, days with subclinical hypocalcemia, and serum concentrations of haptoglobin and Mg, whereas the risk factors for NUTD were parity group and serum Mg concentration. Cows that developed UTD had increased concentrations of haptoglobin on d 2 and fatty acids on d 1 and 2, and reduced concentrations of tCa on d 1 and 2 and of P and Mg on d 2 postpartum compared with cows without UTD. Cows that developed NUTD had increased concentrations of fatty acids on d 0 to 2 postpartum, and decreased concentrations of tCa and P on d 0 and 1, and of Mg on d 1 and 2 postpartum compared with cows without NUTD. Cows that developed NUTD had a 340-kg reduction in milk yield in the first 60 d postpartum. Inflammatory diseases were associated with lesser body condition score and increased loss of body condition in the first 70 d postpartum. Maintenance of pregnancy after ET was reduced in UTD cows following the first (41.7 vs. 25.4%) or all ET (46.4 vs. 36.2%), whereas maintenance of pregnancy was reduced in NUTD cows only at the second ET (39.0 vs 25.9%). The reduced pregnancy maintenance in UTD cows combined with a reduced 21-d service rate (61.9 vs. 54.8%) decreased the 21-d cycle pregnancy rate (28.6 vs. 19.9%) and the hazard of pregnancy to 300 d postpartum by 35%, resulting in an extra 32 d open. In conclusion, inflammatory diseases depressed fertility in dairy cows receiving ET, with the greatest impact observed in UTD cows. This suggests that local inflammation of the uterus impairs maintenance of pregnancy in dairy cows following ET.

Short communication: Greater intensity of estrous expression is associated with improved embryo viability from superovulated Holstein heifers.

The aim of this study was to determine the association between estrous expression, measured using a breeding indicator and an automated activity monitor (AAM), and the success of embryo collection after superovulation. Holstein heifers (n = 51; 10.5 to 14.5 mo, and 325.0 ± 21.1 kg of body weight) were superovulated (n = 69 events) for the collection of embryos using a protocol based on sequential administration of FSH for follicle superstimulation and GnRH to induce ovulation. Artificial insemination (AI) was performed twice, once at the moment of GnRH administration and again 12 h later, using thawed, sexed semen. Ovaries were scanned via ultrasonography on the day of the first AI to count the total number of preovulatory follicles and 7 d later for the total number of corpora lutea present. Embryos were collected 7 d post-AI, counted, and assessed for viability. A breeding indicator (Estrotect, Rockway Inc., Spring Valley, WI) and a collar-mounted AAM (CowScout Activity Monitoring System, GEA, Dusseldorf, Germany) were used to measure standing mounts and an algorithmic estimate of estrous expression, respectively. A score for the breeding indicator was given as follows: score 1 = 100% of the indicator was intact; score 2 = 50% of the indicator was rubbed off; score 3 = greater than 50% of the indicator was rubbed off. Estrous expression detected by the AAM was quantified through the relative increase in physical activity and duration of time spent above a set threshold. Data were analyzed by ANOVA using the MIXED procedures of SAS (SAS Institute Inc., Cary, NC). The number of follicles present at AI was not affected by estrous expression. The mean (± SD) ovulatory response was 67.5 ± 26.3%. We found an effect of estrous expression as detected by the breeding indicator on the ovulatory response (42.1 ± 8.0, vs. 78.2 ± 9.0, vs. 74.0 ± 4.9%, for scores 1, 2, and 3, respectively) but not from the AAM. Heifers that had a score of 3 (versus those with scores of 1 and 2) on the breeding indicator had a greater number of embryos (4.1 ± 0.5, vs. 1.2 ± 1.0, vs. 1.8 ± 1.0 embryos), and a greater percentage of these embryos were viable (43.1 ± 0.05, vs. 35.5 ± 0.1, vs. 34.3 ± 0.1%). Similarly, heifers that showed a greater intensity of activity (as measured by the AAM) had a greater number of embryos collected (10.2 ± 1.2 vs. 6.0 ± 1.3 embryos), and a greater percentage of those embryos were viable (53.1 ± 5.0 vs. 23.4 ± 5.1%). Longer-duration estrus episodes were associated with a higher percentage of viable embryos (51.2 ± 5.2 vs. 25.3 ± 5.3%). In conclusion, stronger estrous intensity was associated with a greater number of total embryos collected and a greater percentage of viable embryos. These results suggest that monitoring the intensity of estrus could be used to predict superovulatory response as well as embryo quality in Holstein heifers.

Mitochondrial DNA haplogroups and age at onset of Machado-Joseph disease/spinocerebellar ataxia type 3: a study in patients from multiple populations.

BACKGROUND AND PURPOSE: Mitochondrial dysfunction has been implicated in the pathogenesis of several neurodegenerative disorders, including Machado-Joseph disease (MJD), an autosomal dominant late-onset polyglutamine ataxia that results from an unstable expansion of a CAG tract in the ATXN3 gene. The size of the CAG tract only partially explains age at onset (AO), highlighting the existence of disease modifiers. Mitochondrial DNA (mtDNA) haplogroups have been associated with clinical presentation in other polyglutamine disorders, constituting potential modifiers of MJD phenotype. METHODS: A cross-sectional study, using 235 unrelated patients from Portugal, Brazil, India and Japan, was performed to investigate if mtDNA haplogroups contribute to AO of MJD. mtDNA haplogroups were obtained after sequencing the mtDNA hypervariable region I. Patients were classified in 15 phylogenetically related haplogroup clusters. RESULTS: The AO was significantly different among populations, implying the existence of other non-CAG factors, which seem to be population specific. In the Portuguese population, patients classified as belonging to haplogroup JT presented the earliest onset (estimated onset 34.6 years of age). Haplogroups W and X seem to have a protective effect, causing a delay in onset (estimated onset 47 years of age). No significant association between haplogroup clusters and AO was detected in the other populations or when all patients were pooled. Although haplogroup JT has already been implicated in other neurodegenerative disorders, no previous reports of an association between haplogroups W and X and disease were found. CONCLUSIONS: These findings suggest that haplogroups JT, W and X modify AO in MJD. Replication studies should be performed in European populations, where the frequency of the candidate modifiers is similar.

Age at onset prediction in spinocerebellar ataxia type 3 changes according to population of origin.

BACKGROUND AND PURPOSE: In spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD), the length of CAG repeat expansions in ATXN3 shows an inverse correlation with age at onset (AO). Recently, a formula for predicting AO based on CAG expansion was developed for European carriers. We tested this formula in SCA3/MJD carriers from distinct origins and developed population-specific models to predict AO. METHODS: This was a parametric survival modelling study. RESULTS: The European formula (EF) was tested in 739 independent SCA3/MJD carriers from South Brazil, Taiwan and the Portuguese Azorean islands, and it largely underestimated AO in South Brazilian and Taiwanese test cohorts. This finding challenged the universal use of the EF, leading us to develop and validate population-specific models for AO prediction. Using validation cohorts, we showed that Brazilian and Taiwanese formulas largely outperformed the EF in a population-specific manner. Inversely, the EF was more accurate at predicting AO among Portuguese Azorean patients. Hence, specific prediction models were required for each SCA3/MJD ethnic group. CONCLUSIONS: Our data strongly support the existence of as yet unknown factors that modulate AO in SCA3/MJD in a population-dependent manner, independent of CAG expansion length. The generated models are made available to the scientific community as they can be useful for future studies on SCA3/MJD carriers from distinct geographical origins.

Natural infection by Culex flavivirus in Culex quinquefasciatus mosquitoes captured in Cuiabá, Mato Grosso Mid-Western Brazil.

New species of insect-specific viruses (ISV) have been reported worldwide. In the present study, the complete genome of Culex flavivirus (CxFV) and partial sequences of other ISVs in Culex quinquefasciatus Say 1823 females (n = 3425) sampled in 200 urban areas census tracts of Cuiaba, state of Mato Grosso, were identified via reverse transcriptase-polymerase chain reaction for a NS5 region of flaviviruses, nucleotide and high-throughput sequencing, and viral isolation in C6/36 cells. CxFV was detected in 16 of 403 mosquito pools; sequences found in the study presented a high similarity with isolates from São Paulo, Brazil and other countries in Latin American that belong to genotype II, supporting the geographical influence on CxFV evolution. The monthly maximum likelihood estimation for CxFV ranged from 1.81 to 9.94 per 1000 mosquitoes. In addition to the CxFV complete genome, one pool contained an ORF1 sequence (756 bp) that belongs to a novel Negevirus from the Sandewavirus supergroup most similar to the Santana virus (77.1%) and another pool presented an RNA-dependent RNA polymerase sequence (1081 bp) of a novel Rhabdovirus most similar to Wuhan mosquito virus 9 (44%). After three passages in C6/36 cells, only CxFV was isolated from these co-infected pools. The importance of ISVs relies on their possible ability to interfere with arbovirus replication in competent vectors.

Intensity of estrus following an estradiol-progesterone-based ovulation synchronization protocol influences fertility outcomes.

The objective of this study was to examine the association between increased physical activity at the moment of timed artificial insemination (AI), detected by an automated activity monitor (AAM), and fertility outcomes. This paper also investigated factors affecting estrous expression in general. A total of 1,411 AI events from 1,040 lactating Holstein cows were recorded, averaging 1.3 ± 0.6 (±standard deviation) events per cow. Activity (measured as steps/h) was monitored continuously by a leg-mounted AAM located on the rear leg of the cow. Ovulation was synchronized by a timed AI protocol based on estradiol and progesterone. Ovarian ultrasonography was performed in all cows on d -11 (AI = d 0) and in a subset of cows on d 0 (n = 588) and d 7 (n = 819) to determine the presence of a corpus luteum and follicles. The body condition score (1 to 5 scale) was assessed on d 0 and a blood sample was collected for progesterone measurement on d 7. Using the AAM, an estrus event was determined when the relative increase (RI) in physical activity of the cow exceeded 100% of the baseline activity. The physical activity was classified as strong RI (≥300% RI), moderate RI (100-300% RI), or no estrus (<100% RI). Milk production was measured daily and averaged between d -11 and 0. Pregnancy was diagnosed at 32 and 60 d post-AI and pregnancy losses were calculated. The mean RI at estrus was 328.3 ± 132.1%. Cows with strong RI had greater pregnancy per AI than those with moderate RI and those that did not express estrus (35.1 vs. 27.3 vs. 6.2%). When including only cows that successfully ovulated after timed AI, those that displayed strong intensity RI still had greater pregnancy per AI than those with moderate intensity RI or those that did not express estrus (45.1 vs. 34.8 vs. 6.2%). Cows expressing strong RI at timed AI had greater ovulation rates compared with moderate RI and cows that did not express estrus (94.9 vs. 88.2 vs. 49.5%). Furthermore, pregnancy losses were reduced in cows with strong RI compared with cows expressing moderate RI (13.9 vs. 21.7%). Cows with a strong RI at estrus were more likely to have a corpus luteum at the beginning of the protocol and had greater concentration of progesterone 7 d post-AI. Multiparous cows expressed lower RI compared with primiparous cows. Cows with lower body condition score tended to have decreased RI at estrus. No correlation between estrous expression and pre-ovulatory follicle diameter was observed. Also, no correlation was observed between milk production at AI and RI. In conclusion, strong intensity RI of estrus events at timed AI was associated with improved ovulation rates and pregnancy per AI, and reduced pregnancy losses. These results provide further evidence that measurements of estrous expression can be used to predict fertility at the time of AI and possibly be used as a tool to assist decision making strategies of reproduction programs.

Reducing prepartum urine pH by supplementing anionic feed ingredients: Effects on physiological and productive responses of Holstein × Gir cows.

This study compared physiological and productive parameters in 3/4 Holstein × 1/4 Gir dairy cows receiving a prepartum concentrate containing ammonium chloride to reduce urine pH near 7.0 (CON; n = 17), or a commercial anionic supplement to reduce urine pH near 6.0 (SUPP; n = 17). Nonlactating, multiparous, pregnant cows were assigned to receive SUPP or CON beginning 21 d before expected date of calving. Cows were maintained in a single drylot pen with ad libitum access to corn silage, and individually received their prepartum concentrate once daily (0800 h) before calving. Cows from both treatments completely consumed their concentrate allocation within 30 min after feeding. Cow body weight and body condition score were recorded once weekly, urine pH measured every 3 d, and blood samples collected on d -21, -14, -9, -6, and -3 relative to expected calving date. After calving (d 0), cows were moved to an adjacent drylot pen with ad libitum access to water and a total mixed ration, and were milked twice daily (0600 and 1700 h). Cow body weight and body condition score were recorded once weekly and individual milk production was recorded daily until 30 d in milk (DIM). Blood samples were collected before each milking during the first 5 DIM, as well as at 6, 9, 16, 23, and 30 DIM before the morning milking. Based on actual calving dates, cows received SUPP or CON for (mean ± standard error) 19.2 ± 1.2 and 19.0 ± 0.9 d before calving, respectively. Urine pH was less in SUPP versus CON cows during the last 15 d of gestation (6.12 vs. 7.15, respectively). Milk yield during the first 5 DIM and throughout the experimental period was greater in SUPP versus CON cows (by 20 and 14%, respectively), whereas serum Ca concentrations did not differ between treatments during the first 5 DIM. Serum concentrations of fatty acids were greater in SUPP versus CON cows 3 d before and at calving (by 52 and 22%, respectively), whereas SUPP cows had lower serum glucose and cortisol concentration at calving (by 23 and 27%, respectively). Hence, the SUPP treatment decreased prepartum urine pH near 6.0 in Holstein × Gir dairy cows without depressing concentrate intake compared with CON, although total dry matter intake was not evaluated to fully investigate feed intake responses. Moreover, the SUPP treatment transiently affected serum glucose, fatty acids, and cortisol concentrations near the time of calving, and resulted in greater milk yield during the initial 30 DIM compared with CON.

Effects of supplemental calcium salts of palm oil and chromium-propionate on insulin sensitivity and productive and reproductive traits of mid- to late-lactating Holstein × Gir dairy cows consuming excessive energy.

This experiment compared insulin sensitivity, milk production, and reproductive outcomes in dairy cows consuming excessive energy during mid to late lactation and receiving in a 2 × 2 factorial design (1) concentrate based on ground corn (CRN; n = 20) or including 8% (DM basis) of Ca salts of palm oil (CSPO; n = 20), and (2) supplemented (n = 20) or not (n = 20) with 2.5 g/d of Cr-propionate. During the experiment (d 0-203), 40 multiparous, nonpregnant, lactating 3/4 Holstein × 1/4 Gir cows (initial days in milk = 81 ± 2; mean ± SE) were offered corn silage for ad libitum consumption, and individually received concentrate formulated to allow diets to provide 160% of their daily net energy for lactation requirements. From d -15 to 203, milk production was recorded daily, blood samples collected weekly, and cow body weight (BW) and body condition score (BCS) recorded on d 0 and 203. For dry matter intake evaluation, cows from both treatments were randomly divided in 5 groups of 8 cows each, and allocated to 8 individual feeding stations for 3 d. Intake was evaluated 6 times/group. Glucose tolerance tests (GTT; 0.5 g of glucose/kg of BW) were performed on d -3, 100, and 200. Follicle aspiration for in vitro embryo production was performed via transvaginal ovum pick-up on d -1, 98, and 198. Mean DMI, net energy for lactation intake, as well as BW and BCS change were similar across treatments. On average, cows gained 40 kg of BW and 0.49 BCS during the experiment. Within weekly blood samples, CRN cows had lower serum concentrations of glucose, insulin, fatty acids, and insulin-to-glucose ratio compared with CSPO cows, suggesting increased insulin sensitivity in CRN cows. During the GTT, insulin-sensitivity traits were also greater in CRN versus CSPO cows. Supplemental Cr-propionate resulted in lower serum insulin concentrations and insulin-to-glucose ratio within CRN cows only, indicating that Cr-propionate improved basal insulin sensitivity in CRN but not in CSPO cows. During the GTT, however, Cr-propionate supplementation reduced hyperinsulinemia and insulin-to-glucose ratio across CSPO and CRN cows. Milk production, as well as number of viable oocytes collected and embryos produced within each aspiration, were not affected by treatments. Hence, replacing corn by Ca salts of palm oil in the concentrate did not improve insulin sensitivity in Holstein × Gir dairy cows consuming excessive energy during mid to late lactation, whereas Cr-supplementation was effective in improving basal insulin sensitivity in cows not receiving Ca salts of palm oil.

Comparison of 2 protocols to increase circulating progesterone concentration before timed artificial insemination in lactating dairy cows with or without elevated body temperature.

Two treatments designed to increase circulating progesterone concentration (P4) during preovulatory follicle development were compared. One treatment used 2 intravaginal P4 implants (controlled internal drug-releasing inserts; CIDR) and the other used a GnRH treatment at beginning of the protocol. Lactating Holstein cows that had been diagnosed as nonpregnant were randomly assigned to receive timed artificial insemination (TAI) following 1 of 2 treatments (n = 1,638 breedings): (1) GnRH: CIDR+ 2 mg of estradiol (E2) benzoate + 100 µg of GnRH on d -11, PGF2α on d -4, CIDR withdrawal + 1.0 mg of E2-cypionate + PGF2α) on d -2, and TAI on d 0; or (2) 2CIDR: 2 CIDR + 2 mg of E2-benzoate on d -11, 1 CIDR withdrawn + PGF2α on d -4, second CIDR withdrawn + 1.0 mg of E2-cypionate + PGF2α on d -2, and TAI on d 0. Milk yield was measured daily between d 0 and d 7. Rectal temperature was measured using a digital thermometer at d 0 and 7, and elevated body temperature was defined as an average rectal temperature ≥39.1°C. Pregnancy diagnoses were performed on d 32 and 60 after TAI. We detected no effect of treatments on pregnancy per AI or pregnancy loss regardless of elevated body temperature, body condition score, parity, milk yield, or presence or absence of a corpus luteum (CL) on d -11 or d -4. Pregnancy per AI at 60 d was reduced [elevated body temperature = 22.8% (162/709), no elevated body temperature 34.1% (279/817)] and pregnancy loss tended to increase [elevated body temperature = 20.2% (41/203), no elevated body temperature 14.4% (47/326)] in cows with elevated body temperature. Various physiological measurements associated with greater fertility were also reduced in cows with elevated body temperature, such as percentage of cows with a CL at PGF2α (decreased 7.9%), ovulatory follicle diameter (decreased 0.51 mm), expression of estrus (decreased 5.1%), and ovulation near TAI (decreased 2.8%) compared with cows without elevated body temperature. A greater proportion of cows (30.2%) had a CL at PGF2α in the GnRH treatment [74.1% (570/763)] than in the 2CIDR treatment [56.9% (434/763)]; however, circulating P4 concentration was greater at the time of PGF2α treatment (d -4) for cows 2CIDR (4.26 ± 0.13 ng/mL) than in cows in GnRH (3.99 ± 0.14 ng/mL). Thus, these 2 protocols yield similar fertility results that might be due to somewhat different physiological alterations. Treatment with GnRH increased the proportion of cows with a CL at PGF2α; however, the 2CIDR protocol increased circulating P4 under all circumstances.

Daily lying behavior of lactating Holstein cows during an estrus synchronization protocol and its associations with fertility.

Objectives were to quantify lying behavior (LB) during an estradiol and progesterone-based synchronization protocol, to assess risk factors for ovulation, pregnancy per AI (P/AI), and degree of behavioral change at estrus, and to investigate the associations between estrus LB and walking activity. Holstein cows (43.6 ± 11.0 kg of milk/d) were fitted with leg-mounted accelerometers. Total lying time/d (L_time), bout frequency (bout_N), average lying bout duration, and relative increase in walking activity (ACT%) were evaluated for 1,411 timed artificial insemination events. The day with lowest L_time or bout_N among d -2, -1, and 0 (day of timed artificial insemination) determined the day of behavioral estrus. The variables L_time% and bout_N% represent relative ratios between lowest L_time and baseline (d -7), L_time, and lowest bout_N, and baseline (d -7) bout_N, respectively [e.g., (lowest L_time/baseline L_time) × 100]. Correlation coefficients between L_time% and bout_N% and ACT% were -0.38 and -0.31, respectively. Estrus LB change was considered large if <75% of baseline and small if ≥75% of baseline for both L_time% and bout_N%; average lying bout duration did not change with estrus. Lowest L_time% and bout_N% corresponded to, respectively, 65 ± 21% (mean ± standard deviation; 447 ± 157 min/d) and 65 ± 24% (8.5 ± 4.0 bouts/d) of baseline. The change in L_time% at estrus was smaller when cows had milk yield above average; the change in bout_N% was smaller among multiparous cows and for estrus occurring in the colder season. Likelihood of ovulation was greater when there was larger change in L_time% [odds ratio = 4.9; ovulation rate = 93 (large change) and 76% (small change)], as well as when a corpus luteum was present at start of protocol (odds ratio = 3.6; in the model with L_time%). Likelihood of pregnancy at d 32 was 1.6 times greater for estrus with large change in LB [L_time% or bout_N%; P/AI = 34% (large change in L_time%) and 26% (small change in L_time%)]. Among estrus events with ACT% ≥300% (high intensity), classification by small or large L_time% did not influence P/AI at 32 d. The magnitude of LB change at estrus and its association with fertility suggest potential application toward improved use of activity monitors (e.g., increased estrus detection, fertility prediction). The contribution of LB to accuracy of estrus detection when physical activity is known remains to be addressed. The relationship between intensity of estrus expression and fertility requires further investigations of its physiological rationale and on-farm applications.

Supplementing an immunomodulatory feed ingredient to modulate thermoregulation, physiologic, and production responses in lactating dairy cows under heat stress conditions.

This study compared vaginal temperature, physiologic, and productive parameters in lactating dairy cows supplemented or not with Omnigen-AF (Phibro Animal Health, Teaneck, NJ) during the summer months in a tropical environment. Thirty-two lactating, primiparous (n = 16) and multiparous (n = 16) pregnant Holstein × Gir cows were ranked by parity, days in milk, body weight, and body condition score (BCS), and assigned to receive (SUPP; n = 16) or not (CON; n = 16) Omnigen-AF (Phibro Animal Health, Teaneck, NJ) at 56 g/cow daily (as-fed basis). During the experimental period (d -6 to 56), cows were maintained in a single drylot pen with ad libitum access to water and a total mixed ration, and milked twice daily. Cows received Omnigen-AF mixed with 200 g of corn (as-fed basis) after the daily morning milking through self-locking head gates, whereas CON cows concurrently received 56 g of kaolin mixed with 200 g of corn. For feed intake evaluation, cows from both treatments were randomly divided in 4 groups of 8 cows each, and allocated to 8 individual feeding stations for 3 d. Intake was evaluated 4 times per group from d 1 to 56. From d -6 to 0, d 15 to 28, and d 43 to 56, cow vaginal temperature was recorded hourly. Environmental temperature-humidity index (THI) was also recorded hourly from d 15 to 28 and d 43 to 56. Cows were evaluated for body weight and BCS on d -6 and 56, individual milk production was recorded daily from d -6 to 56, and milk samples were collected on d -6, 0, 7, 14, 21, 28, 35, 42, 49, and 56 for analyses of somatic cell count and milk components. Blood samples were collected on d -6, -3, 0, 9, 15, 18, 21, 24, 27, 36, 45, 48, 51, 54, and 56. Results from samples or observations collected from d -6 to 0 were included as an independent covariate in each respective analysis. Environmental THI was 74.2 ± 0.5 and cows were exposed to THI >68 for 633 h within a total of 672 h of evaluation. Cows assigned to CON had greater vaginal temperature on d 28, 43, 45, and from d 48 to 55 (by 0.38 to 0.52%), as well as greater mean somatic cell count (by 97%) and serum haptoglobin concentrations (by 89%) compared with SUPP cows. Cows assigned to SUPP had greater mean dry matter intake (by 7%), BCS on d 56 (by 11%), and mean serum insulin concentrations (by 35%) compared with CON cows. Hence, SUPP ameliorated hyperthermia, improved nutritional status, and modulated systemic and mammary gland immune parameters in lactating dairy cows exposed to heat stress conditions.