Estimates of intra- and interclass correlation coefficients for rump touches and the number of steps during estrus in postpartum cows.

Estrus traits have economic value in dairy production systems and could be incorporated into genetic selection indices. In an effort to further understand selection responses, 2 studies were performed to estimate the intra- and interclass correlation coefficients for estrus traits. Holstein-Friesian cows (n = 1,197; study 1) across 5 pasture-based grazing dairy herds were fitted with a capacitive touch sensing (CTS) device on the rump (FlashMate, Farmshed Labs Limited, Hamilton, New Zealand). The daily number of rump touches were subjected to a peak detection program to objectively identify periods of increased rump touches above baseline (indicative of estrus). The number of times touched and the sum of the touch duration were used to compare farms and estimate the intraclass correlation (repeatability). For study 2, postpartum Holstein (n = 85) and Guernsey (n = 5) cows in a confinement-style dairy were used. Cows were fitted with an IceQube accelerometer (IceRobotics Ltd., Edinburgh, United Kingdom) to measure steps taken per hour and a CTS device was applied to both rumps. The interclass correlation for the number of rump touches and number of steps taken during estrus was calculated. Data collected from 5 herds (study 1) demonstrated a 2- to 3-fold difference between herds in the number of rump touches and total touch time during estrus. The intraclass correlation (repeatability; estimates of maximum heritability) for rump touches during estrus was 0.22. For study 2, the number of steps and the number of rump touches during estrus increased in a synchronous manner. The intraclass correlation (repeatability) for number of steps during estrus was 0.26. The interclass correlation (r) for the number of rump touches and the number of steps was 0.46 (R2 = 0.21). Based on the R2, at least 20% of the variation in the number of steps during estrus was explained by the number of touches to the rump of the cow. Selecting cows for the number of steps taken during estrus could increase the number of rump touches (mounts, chin rests, and so on, received from other cows) if a genetic correlation exists for the phenotypic correlation that we observed.

Short communication: Simultaneous measurements of estrus behavior and plasma concentrations of estradiol during estrus in lactating and nonlactating dairy cows.

Estrus is an important behavior that can potentially be subjected to genomic selection. Circulating estradiol concentrations at estrus may be a useful phenotype if the absolute concentrations of estradiol are associated with overt phenotypes for estrus (activity, rump touches, or both; e.g., mounts, chinrests) that can be easily observed. The objective was to measure plasma estradiol concentrations at estrus and associate these measurements with the increase in activity (steps per hour) and rump touches received at estrus. We also tested the effect of lactation on the estrus traits that we measured. Cows (n = 11 lactating and n = 9 nonlactating) were treated with PGF2α to synchronize estrus. A jugular vein was cannulated to collect blood every 2 h for plasma estradiol measurement. Plasma LH was measured during the periestrual period to determine the time of the LH surge. Cows were fitted with an accelerometer to measure activity (steps per hour) and a capacitive touch sensing device to measure the number of rump touches and total touch time. Plasma estradiol concentrations were poorly correlated with overt signs of estrus during the period leading up to maximum estrus activity. After peak estrus activity (when cows were going out of estrus and plasma estradiol concentrations were decreasing), a stronger correlation was detected between overt signs of estrus and plasma estradiol concentrations. Effective selection for improved estrus expression based on plasma estradiol concentrations will depend on whether the cow is coming into or going out of estrus at the time of blood sampling. An association existed between lactation and fewer number of hours in estrus when estrus was defined by an increase in activity (steps per hour). Lactating cows had a shorter interval from the onset of estrus to the LH surge, and the shorter interval to the LH surge may have reduced the period of elevated estradiol during estrus in the lactating cows. Understanding mechanisms that control the sensitivity of the cow to estradiol and making appropriate selection decisions based on these mechanisms will likely increase overt signs of estrus in dairy cows.

Role of Anions and Mixtures of Anions on the Thermochromism, Vapochromism, and Polymorph Formation of Luminescent Crystals of a Single Cation, [(C6H11NC)2Au].

Noncoordinating anions, which generally play a subordinate role in coordination chemistry, alter the structure, the luminescence, as well as the thermochromic and vapochromic behaviors of salts of the two-coordinate cation, [(C6H11NC)2Au]+. Thus whereas the yellow polymorphs of [(C6H11NC)2Au](PF6) and [(C6H11NC)2Au](AsF6) contain single chains of cations and are vapochromic, yellow [(C6H11NC)2Au](SbF6) does not form the same polymorph and is not vapochromic but contains two distinct chains of cations connected through aurophilic interactions. Mixed crystals such as [(C6H11NC)2Au](PF6)0.50(AsF6)0.50 have been prepared by adding diethyl ether to a dichloromethane solution containing equimolar amounts of [(C6H11NC)2Au](PF6) and [(C6H11NC)2Au](AsF6). The initial (kinetic) product for the three combinations of anions ((PF6)-/(AsF6)-, (PF6)-/(SbF6)-, and (AsF6)-/(SbF6)-) was a precipitate of fine yellow needles with a green emission, which were gradually transformed at rates that depended on the anions present into colorless crystals with a blue emission. Whereas neither polymorph of [(C6H11NC)2Au](PF6) nor [(C6H11NC)2Au](SbF6) is thermochromic, the colorless mixed crystal [(C6H11NC)2Au](PF6)0.50(SbF6)0.50 is thermochromic and converts from blue-emitting to green-emitting at 87-95 °C. The temperature required to transform a crystal of the type [(C6H11NC)2Au](PF6)n(AsF6)1-n from colorless (blue-emitting) to yellow (green-emitting) increases as the fraction of hexafluorophosphate ion in the crystal increases. The yellow crystals of [(C6H11NC)2Au](PF6)0.75(AsF6)0.25, [(C6H11NC)2Au](PF6)0.50(AsF6)0.50, and [(C6H11NC)2Au](PF6)0.25(AsF6)0.75 are vapochromic, whereas the yellow crystals of [(C6H11NC)2Au](PF6)0.50(SbF6)0.50 and [(C6H11NC)2Au](AsF6)0.50(SbF6)0.50 are not.

Unsymmetrical Coordination of Bipyridine in Three-Coordinate Gold(I) Complexes.

The unsymmetrical coordination of gold(I) by 2,2'-bipyridine (bipy) in some planar, three-coordinate cations has been examined by crystallographic and computational studies. The salts [(Ph3P)Au(bipy)]XF6 (X = P, As, Sb) form an isomorphic series in which the differences in Au-N distances range from 0.241(2) to 0.146(2) Å. A second polymorph of [(Ph3P)Au(bipy)]AsF6 has also been found. Both polymorphs exhibit similar structures. The salts [(Et3P)Au(bipy)]XF6 (X = P, As, Sb) form a second isostructural series. In this series the unsymmetrical coordination of the bipy ligand is maintained, but the gold ions are disordered over two unequally populated positions that produce very similar overall structures for the cations. Although many planar, three-coordinate gold(I) complexes are strongly luminescent, the salts [(R3P)Au(bipy)]XF6 (R = Ph or Et; X = P, As, Sb) are not luminescent as solids or in solution. Computational studies revealed that a fully symmetrical structure for [(Et3P)Au(bipy)]+ is 7 kJ/mol higher in energy than the observed unsymmetrical structure and is best described as a transition state between the two limiting unsymmetrical geometries. The Au-N bonding has been examined by natural resonance theory (NRT) calculations using the "12 electron rule". The dominant Lewis structure is one with five lone pairs on Au and one bond to the P atom, which results in a saturated (12 electron) gold center and thereby inhibits the formation of any classical, 2 e- bonds between the gold and either of the bipy nitrogen atoms. The nitrogen atoms may instead donate a lone pair into an empty Au-P antibonding orbital, resulting in a three-center, four-electron (3c/4e) P-Au-N bond. The binuclear complex, [µ2-bipy(AuPPh3)2](PF6)2, has also been prepared and shown to have an aurophillic interaction between the two gold ions, which are separated by 3.0747(3) Å. Despite the aurophillic interaction, this binuclear complex is not luminescent.

Ovarian function and the establishment and maintenance of pregnancy in dairy cows with and without evidence of postpartum uterine disease.

Uterine disease early postpartum reduces fertility during the breeding period. One potential mechanism involves the reduced functional capacity of the uterus to support pregnancy. A second potential mechanism involves damage to ovarian follicles associated with systemic inflammation. We categorized lactating Holstein cows into healthy (n = 63) and diseased (n = 39) uterus groups based on the percentage of polymorphonuclear neutrophils in the uterine lumen during the second and third month postpartum and evaluated the functionality of their ovaries and their capacity to establish and maintain pregnancy. Cows were enrolled in a timed artificial insemination protocol (Presynch Ovsynch) so that the first artificial insemination was approximately 75 d postpartum. Ovarian follicles and corpora lutea were counted and measured using transrectal ultrasound, ovulatory responses were assessed, and luteal phase progesterone concentrations were measured. Pregnancy was detected on d 18, 20, 22, 25, 32, and 45 through chemical (d 18 to 25) or ultrasonographic methods (d 32 and 45). The percentage of cows ovulating during the Presynch period; the number, diameter, and ovulatory capacity of follicles during the Ovsynch period; and plasma progesterone concentrations following ovulation were similar for healthy and diseased cows. The initial period of pregnancy establishment (d 18 to 22) appeared to be unaffected by disease because a similar percentage of healthy and diseased cows were pregnant during this period. Embryonic loss occurred in both healthy and diseased cows after d 22. Based on a relatively small number of pregnancies (n = 30 healthy and n = 17 diseased), the cumulative embryonic loss after d 22 was greater in diseased compared with healthy cows. In short, uterine disease as defined in this study did not affect cyclicity, ovarian follicular growth, or plasma progesterone concentrations. Percentages of healthy and diseased cows that were pregnant were similar from d 18 to 22 after artificial insemination. Greater embryonic loss was observed after d 22 in diseased compared with healthy cows, but this observation was based on a small number of pregnancies and should be studied further in larger trials with greater statistical power.

Scanning electron microscopy of the surface epithelium of the bovine endometrium.

The surface epithelium of the bovine endometrium comprises at least 2 cell types (ciliated cells and secretory cells with microvilli), but their distribution and morphological changes over the estrous cycle are poorly understood. The objective was to quantify the number of ciliated cells and assess morphological changes in secretory cells on the uterine surface epithelium during the estrous cycle. Caruncular endometrium (CAR) and intercaruncular endometrium (ICAR) samples were collected from the uterine body, the horn ipsilateral to the corpus luteum or dominant follicle (H-CL/DF), and the horn contralateral to the corpus luteum or dominant follicle (H-NCL/NDF) from heifers following slaughter on d 0 (estrus; n = 5) or d 14 (mid-luteal phase; n = 5) of the estrous cycle. Samples were prepared for scanning electron microscopy at 1,000× magnification. Four to 10 fields (256 × 225 µm) for each sample were examined (n = 567 images). The number of ciliated cells was counted and the surface was scored for the morphology of the secretory cells (0 = absence of microvilli on surface; 3 = 100% of surface covered with microvilli). Ciliated cells were present in both the CAR and ICAR regions. The number of ciliated cells per field increased from d 0 to 14 in CAR and decreased from d 0 to14 in ICAR. The scanning electron microscopy revealed a general lack of uniformity in the lawn of microvilli on the surface of the endometrium. Based on the scores, approximately 25% of the fields had a surface that was <50% covered by microvilli. Depletion of microvilli may be explained by a normal process where apical protrusions are formed and either regress back into the cell surface or break to release their contents into the uterine lumen. These studies support the hypothesis that the surface of the luminal epithelium changes during the estrous cycle through a process that involves remodeling of the apical surface. The morphology of the apical surface may have a key role in governing pregnancy establishment.

Vapoluminescent Behavior and the Single-Crystal-to-Single-Crystal Transformations of Chloroform Solvates of [Au2 (µ-1,2-bis(diphenylarsino)ethane)2 ](AsF6 )2.

The mono- and di-chloroform solvates of [Au2 (µ-1,2-bis(diphenylarsino)ethane)2 ](AsF6 )2 undergo single-crystal-to-single-crystal transformations that result in gain (over 12 hours) or slow loss (over five years) of only one chloroform molecule. The change in solvation results in changes in the structure and luminescence of the digold cation. The cation consists of a pair of slightly bent As-Au-As units that are connected through the two bridging dpae ligands and by aurophilic interactions with Au⋅⋅⋅Au contacts of 3.05152(15) Šin the disolvate or 2.9570(5) Šin the monosolvate.

Symposium review: Selection for fertility in the modern dairy cow-Current status and future direction for genetic selection.

The establishment of pregnancy following insemination is the primary definition of fertility in most dairy systems. Highly fertile cows establish pregnancy sooner after calving and require fewer inseminations than lower-fertility cows. Pregnancy occurs through a series of individual events in sequence. In postpartum cows, for example, the uterus involutes, estrous cycles are re-established, estrus is expressed and detected, sperm are deposited in the reproductive tract and capacitate, ovulation occurs and is followed by fertilization, and the corpus luteum forms and produces sufficient progesterone to maintain pregnancy. The oviduct supports early cleavage and the uterus establishes a receptive environment for the developing pregnancy. Each individual event is theoretically heritable and these events collectively contribute to the phenotype of pregnancy after insemination. Across most dairy systems, genetic selection for fertility in cows is primarily based on reduced days from calving to pregnancy (i.e., days open). Dairy systems differ with respect to reproductive management applied to cows, which may affect the relative importance of individual components to the overall fertility of the cow. In some systems, cows are inseminated after detected estrus with minimal intervention. In these systems, days open effectively captures the summation of the individual components of fertility. More intensive systems use hormonal treatments (e.g., PGF2α, GnRH) followed by timed artificial insemination (AI). Timed AI does not invalidate days open but the individual components that contribute to days open may be more or less important. Selection of cows for days open within populations that are managed differently may place different pressures on the individual components of fertility. Ensuring uniform performance of future cows across a variety of reproductive management systems may require a greater understanding of the underlying genetics of the individual components of fertility.

Strategic application of convective cooling to maximize the thermal gradient and reduce heat stress response in dairy cows.

This study determined the effectiveness of convective cooling at different times of day when air temperature (Ta) was cycled from day to night. Mid-lactation Holstein cows (n = 12) were placed in 3 environmental chambers (4 cows per chamber) and acclimated to Ta 19.9°C (thermoneutral; TN) for 7 d followed by an incremental increase over 3 d to a heat stress (HS) condition. Conditions were maintained for 11 d at high and low daily Ta of 33 and 23°C, respectively. To determine adaptive HS response, the HS period was divided into early (E: d 11 to 14) and late (L: d 17 to 20) periods. During HS, cows were exposed to continuous fan (convective) cooling (CC), 8-h day fan cooling (1100 to 1900 h; DC), or 8-h night fan cooling (2300 to 0700 h; NC). Compared with DC, the NC treatment maximized the thermal gradient during the convective cooling. Each animal received all treatments within 3 trials using a repeated 3 × 3 Latin square design. Cows were fed a total mixed ration and milked twice daily. Thermal status was assessed by using thermal conductance and average daily values for mean, minimum, and maximum rectal temperature (Tre), skin temperatures, and respiration rate. Percent reduction in dry matter intake from TN to HS was less for CC than DC and NC, with no change from E to L periods. The DC group exhibited the greatest trend for a percent reduction in total milk yield below CC due to the significantly lower morning milk production. All values for total daily milk production decreased from E to L periods, with E to L reductions in both morning and afternoon milk production. Minimum Tre for CC and NC cows was 0.4°C below DC. In contrast, maximum Tre was similar for NC and DC groups, at 0.5 to 0.6°C above the CC group. Skin temperature for CC cows was always less than DC cows. Skin temperature for NC cows was equal to CC for minimum skin temperature, but exceeded both CC and DC cows for maximum skin temperature. Average skin temperature decreased from E to L, which suggested heat adaptation. The thermal advantage of night (lowest Ta and greatest thermal gradient) versus day cooling (greatest Ta and lowest thermal gradient) was increased heat transfer via thermal conductance with NC. The higher thermal strain of DC cows caused a larger percent decrease in morning milk yield than for NC cows. In contrast, use of convective cooling at night in the absence of elevated humidity could sufficiently reduce heat strain beyond DC to maintain milk production at a level that is closer to that of CC cows.

The effect of exogenous glucose infusion on early embryonic development in lactating dairy cows.

The objective of this study was to examine the effect of intravenous infusion of glucose on early embryonic development in lactating dairy cows. Nonpregnant, lactating dairy cows (n = 12) were enrolled in the study (276 ± 17 d in milk). On d 7 after a synchronized estrus, cows were randomly assigned to receive an intravenous infusion of either 750 g/d of exogenous glucose (GLUC; 78 mL/h of 40% glucose wt/vol) or saline (CTRL; 78 mL/h of 0.9% saline solution). The infusion period lasted 7 d and cows were confined to metabolism stalls for the duration of the study. Coincident with the commencement of the infusion on d 7 after estrus, 15 in vitro-produced grade 1 blastocysts were transferred into the uterine horn ipsilateral to the corpus luteum. All animals were slaughtered on d 14 to recover conceptuses, uterine fluid, and endometrial tissue. Glucose infusion increased circulating glucose concentrations (4.70 ± 0.12 vs. 4.15 ± 0.12 mmol/L) but did not affect milk production or dry matter intake. Circulating ß-hydroxybutyrate concentrations were decreased (0.51 ± 0.01 vs. 0.70 ± 0.01 mmol/L for GLUC vs. CTRL, respectively) but plasma fatty acids, progesterone, and insulin concentrations were unaffected by treatment. Treatment did not affect either uterine lumen fluid glucose concentration or the mRNA abundance of specific glucose transporters in the endometrium. Mean conceptus length, width, and area on d 14 were reduced in the GLUC treatment compared with the CTRL treatment. A greater proportion of embryos in the CTRL group had elongated to all length cut-off measurements between 11 and 20 mm (measured in 1-mm increments) compared with the GLUC treatment. In conclusion, infusion of glucose into lactating dairy cows from d 7 to d 14 post-estrus during the critical period of conceptus elongation had an adverse impact on early embryonic development.

Technical note: Evaluation of fine needle aspiration cytology for the diagnosis of fatty liver in dairy cattle.

Fatty liver is a common condition affecting dairy cattle during the periparturient period, characterized by a pathological accumulation of triglycerides (TG) in the hepatocytes. The objective of this study was to evaluate the diagnostic potential of fine needle aspiration cytology in fresh liver specimens using liver TG concentrations as a gold standard. Fifty-seven liver samples from Holstein cows were collected during processing at a slaughterhouse. Tissue and fine needle aspirate samples were obtained from the parietal upper portion of the caudate lobe. Two samples of liver tissue were collected with a 16 gauge × 15 cm biopsy needle for histological and TG concentration assessment. A third sample was collected for cytology using an 18 gauge × 5.08 cm needle. The contents of the needle were transferred to a glass slide, spread, and air-dried. Liver samples were assayed by colorimetry/fluorimetry to determine TG concentrations. Concentrations of TG <2% were considered normal. Histological and cytological evaluations were conducted by 2 different pathologists blind to the visual classification. Sensitivity (Se) and specificity (Sp) were calculated. Cytology had a Se and Sp of 73 and 85%, respectively. Histopathology had a Se and Sp of 45.9 and 100%, respectively. The likelihood of having higher scores for histopathology and cytology increased as a function of liver TG content (mg/g).

Hot topic: 16S rRNA gene sequencing reveals the microbiome of the virgin and pregnant bovine uterus.

We tested the hypothesis that the uterus of virgin heifers and pregnant cows possessed a resident microbiome by 16S rRNA gene sequencing of the virgin and pregnant bovine uterus. The endometrium of 10 virgin heifers in estrus and the amniotic fluid, placentome, intercotyledonary placenta, cervical lumen, and external cervix surface (control) of 5 pregnant cows were sampled using aseptic techniques. The DNA was extracted, the V4 hypervariable region of the 16S rRNA gene was amplified, and amplicons were sequenced using Illumina MiSeq technology (Illumina Inc., San Diego, CA). Operational taxonomic units (OTU) were generated from the sequences using Qiime v1.8 software, and taxonomy was assigned using the Greengenes database. The effect of tissue on the microbial composition within the pregnant uterus was tested using univariate (mixed model) and multivariate (permutational multivariate ANOVA) procedures. Amplicons of 16S rRNA gene were generated in all samples, supporting the contention that the uterus of virgin heifers and pregnant cows contained a microbiome. On average, 53, 199, 380, 382, 525, and 13,589 reads annotated as 16, 35, 43, 63, 48, and 176 OTU in the placentome, virgin endometrium, amniotic fluid, cervical lumen, intercotyledonary placenta, and external surface of the cervix, respectively, were generated. The 3 most abundant phyla in the uterus of the virgin heifers and pregnant cows were Firmicutes, Bacteroidetes, and Proteobacteria, and they accounted for approximately 40, 35, and 10% of the sequences, respectively. Phyla abundance was similar between the tissues of the pregnant uterus. Principal component analysis, one-way PERMANOVA analysis of the Bray-Curtis similarity index, and mixed model analysis of the Shannon diversity index and Chao1 index demonstrated that the microbiome of the control tissue (external surface of the cervix) was significantly different from that of the amniotic fluid, intercotyledonary placenta, and placentome tissues. Interestingly, many bacterial species associated with postpartum uterine disease (i.e., Trueperella spp., Acinetobacter spp., Fusobacteria spp., Proteus spp., Prevotella spp., and Peptostreptococcus spp.) were also present in the uterus of virgin heifers and of pregnant cows. The presence of 16S rRNA gene sequence reads in the samples from the current study suggests that the uterine microbiome is established by the time a female reaches reproductive maturity, and that pregnancies are established and maintained in the presence of a uterine microbiome.

Technical note: Validation of a chemical pregnancy test in dairy cows that uses whole blood, shortened incubation times, and visual readout.

Chemical pregnancy testing is an alternative to traditional methods of pregnancy diagnosis (either manual palpation or ultrasound) in postpartum dairy cows and heifers. The objective was to validate a chemical pregnancy test that confers the advantages of using whole blood, rapid incubation times, and visual readout. Blood and milk samples were collected from Holstein dairy cows [n=320; 162±62 (mean ± SD) d in milk] on a confinement farm in northeast Missouri at 28 d after artificial insemination (AI). The samples were assayed for pregnancy-associated glycoproteins (PAG) by using a rapid visual test as well as traditional plasma- and milk-based tests. Transrectal ultrasonography diagnosis for pregnancy at 35 to 38 d after AI was the reference (gold) standard for all PAG tests. One hundred fifty-nine cows were diagnosed as pregnant by the reference standard (pregnancies per AI=49.7%). The tests were ELISA and either optical density (OD; measured with a microtiter plate reader; plasma, milk, and rapid visual tests) or visual readout (rapid visual test) were used to diagnose pregnancy. When OD was used, the percentage of pregnant cows classified correctly (sensitivity) for the plasma, milk, and rapid visual tests were 97±1, 96±2, and 95±1% (±SE), respectively. The sensitivity of the rapid visual test when assessed visually was 98±1%. The specificity (proportion of nonpregnant cows classified correctly) for the plasma, milk, and rapid visual was 94±2%, 94±2%, and 93±2% when an OD was used. When read visually, the specificity of the rapid visual test was lesser (85±3%) because some cows with faint visual signals yielded false positive diagnosis. The overall accuracy (proportion of pregnant and nonpregnant cows diagnosed correctly) was similar for all tests (plasma, milk, rapid visual OD, and rapid visual; 96±1, 95±1, 94±1, and 92±2%, respectively). In a second experiment, lactating Holstein cows (n=291) from 4 commercial confinement dairy farms in western Kentucky were tested 25 to 95 d after AI using the rapid visual test. The OD of the rapid visual test followed the known profile for PAG in circulation during the first trimester of pregnancy. The conclusion is that the rapid visual test has equal sensitivity and accuracy as existing PAG tests. A slightly lower specificity was found when the rapid visual test was read visually.

Short communication: Test for nonpregnancy in dairy cows based on plasma progesterone concentrations before and after timed artificial insemination.

Timed artificial insemination (AI) programs have increased reproductive efficiency in dairy herds. A low timed AI pregnancy per AI is partially explained by cows that fail to respond optimally to the series of treatments that are designed to synchronize ovulation for AI. We hypothesized that testing cows for plasma progesterone concentrations during a timed AI protocol could be used as an early diagnostic test for nonpregnancy. Lactating Holstein cows (n=160) in 2 confinement-style dairies were used. Cows were treated with Presynch Ovsynch 56 for timed AI. Concentrations of progesterone in plasma were measured at -3, 0, 7, and 25 d relative to timed AI. Progesterone data were analyzed and receiver operating characteristic curves were generated by using logistic regression. The area under the receiver operating curves for a progesterone test for nonpregnancy on d -3 (PGF2α), 0 (AI), 7, and 25 d relative to timed AI were 0.68, 0.52, 0.55, and 0.89, respectively. The cutpoints and sensitivity (respectively) for the progesterone test were 0.51ng/mL (lower=nonpregnant) and 28.2% for the day of PGF2α, 0.43ng/mL (greater=nonpregnant) and 17.9% for the day of AI, 1.82ng/mL (lower=nonpregnant) and 23.1% for 7 d after AI, and 2.67ng/mL (lower=nonpregnant) and 76.0% for 25 d after AI. The false positive rate was less than 5% for all tests. Analysis of a second data set from a published study gave approximately the same cutpoints and sensitivity. When both studies were combined, approximately 20% of nonpregnant cows could be identified with a single test that was done before or shortly after AI with a false positive rate of less than 5%. When 2 and 3 tests were applied sequentially, the sensitivity for identifying nonpregnant cows increased from 38.4 to 50.5%. The pregnancy per AI for those cows that met the established progesterone criteria was approximately 3 to 4 times greater than those that failed to meet the criteria. The conclusions were that cows destined to be nonpregnant after timed AI can be identified before or shortly after AI. Testing for nonpregnancy before or shortly after AI may have utility with respect to eliminating a nonproductive AI (cows identified before AI) or shortening the time to reinsemination (cows identified by 1 wk after AI).

Effect of manipulating progesterone before timed artificial insemination on reproductive and endocrine parameters in seasonal-calving, pasture-based Holstein-Friesian cows.

Fertility to timed AI (TAI) is profoundly affected by progesterone (P4) levels during hormonal synchronization protocols. Holstein-Friesian dairy cows managed in a seasonal-calving, pasture-based production system were randomly assigned to 2 treatments to manipulate P4 before TAI during growth of the preovulatory follicle. Cows in the first treatment (High P4; n=30) were submitted to a Double-Ovsynch protocol {Pre-Ovsynch [GnRH; 7 d, PGF2α; 3 d, GnRH] followed 7 d later by Breeding-Ovsynch [GnRH (G1); 7 d PGF2α; 24 h, PGF2α; 32 h, GnRH (G2); 16 h, TAI]}. Cows in the second treatment (n=30; Low P4) received the same Double-Ovsynch protocol but with an additional PGF2α treatment 24 h after G1. Overall, synchronization rate did not differ between treatments and was 92% (55/60). Unexpectedly, 37% of Low P4 cows were detected in estrus ~24 h before scheduled TAI and were inseminated ~16 h before scheduled TAI. Overall, P4 did not differ between treatments at G1, whereas High P4 cows had greater P4 concentrations at PGF2α and G2 than Low P4 cows. High P4 cows had the smallest mean follicle diameter at G2, whereas Low P4 cows with no estrus before TAI had intermediate mean follicle diameter at G2, and Low P4 cows with estrus before TAI had the largest mean follicle diameter. Low P4 cows with estrus before TAI had larger corpora lutea 15 d after TAI than Low P4 cows without estrus before TAI or High P4 cows. In accordance with corpus luteum size on d 15, High P4 cows and Low P4 cows without estrus before TAI had lower P4 from 4 to 46 d after TAI than Low P4 cows with estrus before TAI. Relative mRNA levels of the interferon-stimulated genes ISG15, MX1, MX2, and OAS1 were greater for Low P4 than for High P4 cows, whereas relative mRNA levels of RTP4 were greater for High P4 than for Low P4 cows 18 d after TAI. Treatment did not affect plasma pregnancy-associated glycoprotein concentrations after TAI; however, pregnancy-associated glycoprotein concentrations were affected by pregnancy status and parity. Treatment did not affect pregnancy per artificial insemination at 29, 39, or 60 d after TAI, and no pregnancy losses were observed from 39 to 60 d after TAI. We concluded that (1) Low P4 cows were more likely to express estrus than High P4 cows; (2) the subpopulation of Low P4 cows that expressed estrus had larger preovulatory follicles and greater P4 concentrations after TAI; and (3) regardless of estrus before TAI, all Low P4 cows had greater mRNA expression for 5 of 6 interferon-stimulated genes than High P4 cows 18 d after TAI.

Gene expression in liver and adipose tissue is altered during and after temporary changes to postpartum milking frequency.

Short-term changes to milking frequency can alter the metabolic status of dairy cows depending on the duration, magnitude, and stage of lactation at which the milking frequency changes occur. Additionally, effects of altered milking frequency that are subsequent to cows returning to a normal twice-daily (2×) milking regimen are not well established. This study tested the hypothesis that plasma concentrations of key hormones and metabolites and transcription of genes involved in the somatotropic axis and lipid metabolism would be altered in liver and subcutaneous adipose tissue from cows milked with different frequencies. Multiparous Holstein-Friesian dairy cows were allocated to 2× milking for the whole lactation, or once-(1×) or 3 times-(3×) daily milking for 3 or 6 wk, immediately postpartum, and then 2× milking for the remainder of the lactation. Liver and subcutaneous fat were biopsied at wk 1 (liver only), 3, 6, and 9 postpartum, and transcription of genes involved in the somatotropic axis and lipid metabolism were measured. At wk 3, cows milked 3× had lower hepatic expression of growth hormone receptor (GHR1A) compared with cows milked 2× or 1×, and lower IGF1 expression compared with cows milked 1×, indicating greater uncoupling of the somatotropic axis. At wk 6, reduced transcription of total GHR and GHR1B occurred in the adipose tissue of cows milked 3×. Cows milked 1× had greater transcription in adipose tissue of lipogenesis genes at wk 3 and 6, and lipolysis genes at wk 6, compared with cows milked 2×, indicating a period of increased fatty acid storage, followed by increased fatty acid reesterification. At wk 9, cows previously milked 3× for 6 wk maintained lower transcription of genes involved in lipogenesis, lipolysis, and ketolysis in adipose tissue compared with cows milked 2×, indicating that the effects of 3× milking persist for at least 3 wk after switching to 2× milking. Results indicate that alterations to milking frequency affect the transcription of genes involved in lipid mobilization and storage, enabling the animal to manage the energy demands associated with the change in milk production. Some of these gene transcription changes were maintained in cows previously milked 3×, indicating that the adipose tissue gene expression changes were still required even after 3 wk of the less-demanding 2× milking regimen.

Follicular populations and luteal function in dairy heifers treated with a controlled internal drug release insert for 14 days as a method to synchronize the estrous cycle before prostaglandin F2α treatment and artificial insemination.

Progesterone-containing controlled internal drug release (CIDR) inserts are used to synchronize the estrous cycle before PGF2α is administered for timed AI (14dCIDR-PGF2α program). The program, initially designed for beef cattle, was recently shown to be efficacious in dairy heifers. We hypothesized that the 14-d CIDR treatment would synchronize the estrous cycle in dairy heifers and result in a uniformly sized corpus luteum (CL) and largest follicle (LF) at the time of PGF2α treatment. Holstein (n=110) or Holstein × Guernsey (n=4) dairy heifers were assigned to 2 treatments: (1) 14dCIDR-PGF2α [CIDR in for 14 d, CIDR out for 16d, PGF2α and AI after observed estrus (n=57)] or (2) control [PGF2α and AI after observed estrus (n=57)]. Regardless of treatment, additional PGF2α injections were administered at 14-d intervals to heifers that were not seen in estrus. Ovarian ultrasonography and blood sampling were done on d 0 (CIDR administered), 14 (day CIDR removed), 19 (5d after CIDR removed), 30 (PGF2α administered), and 44 (second PGF2α dose administered to heifers that were not detected in estrus after the first PGF2α). Compared with control (untreated), more CIDR-treated heifers were categorized as having a small CL (≤ 9.9 mm) and large LF (15.0-19.9 mm) on d 14 (CIDR removal) and, as expected, a greater percentage of CIDR-treated heifers were in estrus during the 5d after the CIDR removal compared with control heifers (75.4 vs. 22.8%, respectively). On d 19, the CIDR-treated heifers had apparently ovulated based on disappearance of LF and appearance of small CL. On d 30 (PGF2α administration), 89% of 14dCIDR-PGF2α heifers had CL that were ≥ 20 mm in diameter compared with 55% for control. Presence of larger CL on d 30 was associated with greater concentrations of plasma progesterone in 14dCIDR-PGF2α compared with control (10.5 ± 0.5 vs. 5.0 ± 0.6 ng/mL, respectively). The percentages of heifers with LF in the smallest category (≤ 9.9 mm) tended to be less (5.3 vs. 16.6%) and the percentage of heifers with LF in the medium-size category (10.0 to 14.9 mm) tended to be greater (84.2 vs. 69.1%) for 14dCIDR-PGF2α versus control, respectively, on d 30. More heifers were detected in estrus within 5d after the first PGF2α (86.0 vs. 56.1%) and conception rate to AI using sexed semen tended to be greater (61.2% vs. 40.6%) for 14dCIDR-PGF2α compared with control (respectively). Treating dairy heifers with a CIDR for 14 d was an effective method to synchronize an estrous cycle before PGF2α was administered.

Short communication: Glucose infusion into early postpartum cows defines an upper physiological set point for blood glucose and causes rapid and reversible changes in blood hormones and metabolites.

Low blood glucose concentrations after calving are associated with infertility in postpartum dairy cows perhaps because glucose is a master regulator of hormones and metabolites that control reproductive processes. The hypothesis was that low blood glucose postpartum is caused by inadequate glucose entry rate relative to whole-body demand as opposed to the alternative possibility that postpartum cows have a lower regulatory set point for blood glucose. Eight early postpartum (10 to 25 d) dairy cows (5 Holstein and 3 Guernsey) were jugular catheterized. During the first 24 h, cows were infused with physiological saline at 83.3 mL/h. After 24 h, the infusion solution was switched to 50% dextrose that was infused at a rate of 41.7 mL/h (total daily glucose dose=500 g). On d 3 and d 4, the rate of glucose infusion was increased to 83.3 mL/h (daily dose=1,000 g) and 125 mL/h (daily dose=1,500 g), respectively. On d 5, physiological saline was infused at 83.3 mL/h. Blood was sampled hourly through a second jugular catheter (contralateral side) and analyzed for glucose, nonesterified fatty acids, ß-hydroxybutyrate, insulin-like growth factor 1, and insulin. Blood glucose concentrations on d 1 (saline infusion) averaged 53.4±1.7 mg/dL. Blood glucose concentrations increased on d 2 when cows were infused with 500 g/d and increased further on d 3 when cows were infused with 1,000g of glucose/d. Increasing the infusion rate to 1,500 g/d on d 4 did not cause a further increase in blood glucose concentrations. Based on a segmented regression analysis, the upper physiological set point for blood glucose was 72.1 mg/dL. Both insulin and insulin-like growth factor 1 concentrations increased in response to glucose infusion and decreased when cows were infused with saline on d 5. Serum nonesterified fatty acids and ß-hydroxybutyrate concentrations decreased in response to glucose infusion and rebounded upward on d 5 (saline infusion). In conclusion, early postpartum cows had circulating blood glucose concentrations that were well below the upper set point defined in this study (72.1 mg/dL). Infusing approximately 1,000 g of glucose daily increased blood glucose to the physiological set point and rapidly changed the hormonal and metabolic profile that typifies postpartum cows. The inability of the early postpartum cow to achieve an adequate entry rate for glucose relative to whole-body demand is a possible mechanism that links postpartum physiology and nutrition to reproduction in dairy cows.

Reproduction in grazing dairy cows treated with 14-day controlled internal drug release for presynchronization before timed artificial insemination compared with artificial insemination after observed estrus.

Progesterone-releasing (controlled internal drug release, CIDR) devices inserted for 14 d are used to presynchronize the estrous cycle for timed artificial insemination (TAI) in beef heifers (14-d CIDR-PGF(2α) program). The objective was to test a similar program in dairy cows by measuring first-service conception rates (FSCR), pregnancy rates after 2 AI, and time to pregnancy compared with a control (AI after observed estrus). Postpartum cows (Holstein, Jersey, or crossbred; n=1,363) from 4 grazing dairy farms were assigned to 1 of 2 programs: 14dCIDR_TAI [CIDR in for 14 d, CIDR out, PGF(2α) injection at 19 d after CIDR removal, GnRH injection 56 h later, and then TAI 16 h later; n=737] or control [AI after observed estrus; reproductive program with PGF(2α) (cycling cows) and CIDR (noncycling cows) to synchronize estrus with the start of the breeding season; n=626]. Body condition was scored (1 to 5; thin to fat) at the start of the trial. The interval from the start of the breeding period (final PGF(2α) injection of either program) to first AI was shorter for 14dCIDR_TAI compared with the control (3.0±0.2 vs. 5.3±0.2 d; mean ± SEM) but 14dCIDR_TAI cows had lesser FSCR than controls (48 vs. 61%). Farm affected FSCR (50, 51, 67, and 58% for farms 1 to 4). The BCS affected FSCR (50, 55, and 62% for BCS=2, 2.5, and 3, respectively). Cows that either calved the year before (carryover) or that calved early in the calving season had greater FSCR than cows that calved later in the calving season (55, 61, and 42%, respectively). The percentage of cows pregnant to AI (first and second inseminations within 31-d breeding season) was similar for 14dCIDR_TAI and control (64 vs. 70%) cows, but farm (64, 62, 80, and 69%) and time of calving (70, 76, and 56%: carryover, early, and late, respectively) affected the percentage. Survival analyses showed an initial advantage for 14dCIDR_TAI (more cows inseminated and more pregnancies achieved early in the breeding season) that was not maintained over time. Conclusions were that the 14dCIDR_TAI program achieved acceptable FSCR (48%) and overall AI pregnancy rates (64%), but did not surpass a control program that used AI after observed estrus (61 and 70%, respectively).

Comparison of innate immune responses and somatotropic axis components of Holstein and Montbéliarde-sired crossbred dairy cows during the transition period.

Objectives were to compare parameters related to innate immune responses and somatotropic axis of Holstein (HO) and Montbéliarde (MO)-sired crossbred cows during the transition from late gestation to early lactation. Cows (40 HO and 47 MO-sired crossbred) were enrolled in the study 45d before expected calving date (study d 0=calving). Polymorphonuclear leukocytes (PMNL) isolated from blood samples collected weekly from study d -7 to 21 and on study d 42 were used for determination of percentage of PMNL positive for phagocytosis (PA+) and oxidative burst (OB+), intensity of PA and OB, percentage of PMNL expressing CD18 (CD18+) and L-selectin (LS+), and intensity of CD18 and LS expression. Blood was sampled weekly from study d -7 to 14 and on study d 28, 42, and 56 for determination of insulin, growth hormone (GH), leptin, and insulin-like growth factor (IGF)-1 concentrations. Blood sampled weekly from study d -14 to 21 and on study d 42 was used to determine cortisol concentration. Liver biopsies were performed on study d -14, 7, 14, and 28 for determination of mRNA expression for insulin receptor B (IRB), total GH receptor (GHRtot), GHR variant 1A (GHR1A), and IGF-1. Data were analyzed by ANOVA for repeated measures or by ANOVA using the GLM procedure of SAS (SAS Institute Inc., Cary, NC). Intensity of CD18 expression was greater in PMNL from crossbred cows compared with PMNL from HO cows [1,482.1 ± 82.3 vs. 1,286.6 ± 69.8 geometric mean fluorescence intensity (GMFI)]. Furthermore, among HO cows, the percentage of PA+ PMNL on study d -7 (64.4 ± 5.2%) tended to be greater than on study d 0 (57.1 ± 5.1%), but no differences in percentage of PA+ PMNL between study d -7 and 0 were observed in crossbred cows. Similarly, intensity of PA in PA+ PMNL from HO cows decreased from study d -7 to 0 (4,750.6 ± 1,217.0 vs. 1,964.7 ± 1,227.9 GMFI), but no changes in intensity of PA in PA+ PMNL from crossbred cows were observed. On study d 0, intensity of PA tended to be reduced in PA+ PMNL from HO cows compared with PA+ PMNL from crossbred cows (1,964.7 ± 1,227.9 vs. 4,688.1 ± 1,271.8 GMFI). Concentrations of GH (7.4 ± 0.4 vs. 5.1 ± 0.4 ng/mL) and cortisol (9.5 ± 0.8 vs. 7.1 ± 0.8 ng/mL) were greater for HO than for crossbred cows. Crossbred cows had improved innate immune responses compared with HO cows, as determined by a lack of decrease in intensity of PA on the day of calving, which may result in improved health. Furthermore, HO cows appeared to be less sensitive to the negative feedback of IGF-1 on GH secretion because cows from both breeds had similar IGF-1 concentrations but MO-sired crossbred cows had greater GH concentrations.