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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., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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 PGF 2α 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., (Copyright © 2021 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2020 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to determine the association of beta-carotene concentration in plasma at the moment of artificial insemination (AI) on pregnancy/AI in lactating Holstein cows. A total of 399 events from 364 lactating Holstein cows were enrolled in the trial (143 primiparous and 221 multiparous). All cows were assigned to a timed AI protocol based on estradiol and progesterone. Blood samples were collected at the moment of AI and at 24 and 31d post-AI (samples on 31 d post-AI were collected only from cows that were diagnosed pregnant). The BCS were recorded at the time of AI. Plasma beta-carotene was quantified from blood samples taken at the time of AI using a single step denaturation and extraction into a solvent, followed by measurement using a portable spectrophotometer. Pregnancy-associated glycoproteins (PAG) were analyzed in blood samples taken at 24 and 31 d post-AI of pregnant cows. Milk production was collected for the entire experimental period. Pregnancy diagnosis was performed by ultrasound 31 and 60 d post-AI. Data was analyzed using the MIXED and GLIMMIX procedures of SAS. Cows classified as thin (<2.75) tended to have lower concentration of beta-carotene at AI when compared with those classified as Moderate (≥3.00; 3.8 ± 0.1 vs. 4.3 ± 0.1 μg/mL; P = 0.09). Concentration of beta-carotene were greater in multiparous compared with primiparous (P < 0.01). There was no correlation between concentration of beta-carotene and milk production (r = 0.04; P = 0.10). When plasma beta-carotene was categorized in quartiles, cows in the 1st quartile had lower pregnancy/AI and higher pregnancy losses when compared with cows that were in the 2nd, 3rd and 4th quartile (pregnancy/AI = 19.2 ± 4.5, 33.7 ± 4.7, 36.9 ± 5.0 and 39.8 ± 5.4%, respectively; P = 0.05; pregnancy losses = 41.9 ± 4.8, 20.4 ± 3.7, 22.1 ± 4.1, and 15.7 ± 4.2%, respectively; P < 0.05). There was no association between concentrations of beta-carotene at AI and PAG at 24 d post-AI (P = 0.60). Cows with greater concentrations of beta-carotene at AI were more likely to have greater concentrations of PAG at 31 d post-AI (P < 0.01). In conclusion, the concentration of beta-carotene at AI was affected by BCS and parity. Cows with higher concentrations of plasma beta-carotene at AI had greater pregnancy/AI, lower pregnancy losses and greater concentrations of PAG at d 31 post-AI, suggesting it may be associated with placental function in lactating dairy cows., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Expression of estrus (EST) near the time of fixed-time artificial insemination (FTAI) increases pregnancy success in beef females. This outcome has been associated with improved pregnancy establishment and maintenance, although research is still warranted to validate this theory. Hence, this experiment compared ovarian, uterine and conceptus factors associated with pregnancy establishment in Bos indicus beef cows according to estrous expression during a FTAI protocol. One hundred lactating multiparous Nelore cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device on day -11, a 12.5 mg injection of prostaglandin F2α on day -4, P4 device removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of equine chorionic gonadotropin on day -2, and FTAI on day 0. An estrous detection patch was attached to the tailhead of each cow on day -2, and estrous expression was defined as removal of >50% of the rub-off coating from the patch at FTAI. Overall, 39 cows expressed EST, 55 did not express EST (NOEST), and six cows lost their patch and were discarded from the experiment. Ovarian ultrasonography was performed at FTAI, and on days 7 and 15 of the experiment. Blood samples were also collected on days 7 and 15. Only cows without a corpus luteum (CL) on day 0, and with a CL on days 7 and 15 remained in the experiment (EST, n=36; NOEST, n=48). On day 15, cows were randomly selected within each group (EST, n=29; NOEST, n=30) for conceptus collection via transcervical flushing, followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Within cows not assigned to conceptus collection, blood samples were collected for whole blood RNA extraction (day 20) and pregnancy status was verified by transrectal ultrasonography (day 30). Diameter of dominant follicle on day 0 and plasma P4 concentrations on day 7 were greater (P⩽0.02) in EST v. NOEST cows. Conceptus length and messenger RNA (mRNA) expression of prostaglandin E synthase and interferon-tau were greater (P⩽0.04) in EST v. NOEST cows. Moreover, EST cows diagnosed as pregnant on day 30 had greater (P<0.01) blood mRNA expression of myxovirus resistance 2 on day 20 compared with NOEST. In summary, estrous expression near the time of FTAI enhanced pregnancy establishment factors in B. indicus cows, including conceptus development and mRNA expression of interferon-tau.

This experiment evaluated the impacts of estrus expression and intensity, estimated by physical activity during a timed-AI protocol, on reproductive performance of Bos indicus-influenced beef cows. A total of 290 lactating, primiparous, and multiparous nonpregnant Nelore × Angus cows received a 2 mg injection of estradiol benzoate and an intravaginal progesterone (P4) releasing device (CIDR) on d -11, a 12.5 mg injection of PGF2α on d -4, CIDR removal in addition to 0.6 mg injection of estradiol cypionate and 300 IU injection of eCG on d -2, and timed-AI on d 0. Cows were fitted with a pedometer behind their left shoulder on d -4. An estrus detection patch was attached to the tail-head of each cow on d -2. Pedometer results were recorded on d -2 and 0. Estrus expression was defined as removal of >50% of the rub-off coating from the patch on d 0. Net physical activity during estrus was calculated by subtracting total steps from d -4 to -2 (nonestrus basal activity) from total steps from d -2 to 0 (proestrus + estrus period) of each cow. Cows that did not express estrus were classified as NOESTR. Cows that expressed estrus were ranked by net physical activity; those above the median were classified as HIESTR and the remaining cows as LWESTR. Ovarian ultrasonography was performed on d 0 and 7. Blood was collected on d 0, 7, 20, and 30. Pregnancy status was verified by ultrasonography on d 30. Only data from cows responsive to the estrus synchronization protocol were utilized (NOESTR, n = 59; LWESTR, n = 100; HIESTR, n = 98). Diameter of dominant follicle on d 0, corpus luteum volume on d 7, and plasma P4 concentrations on d 7 were greater (P ≤ 0.05) in HIESTR vs. LWESTR and NOESTR and also greater (P ≤ 0.05) for LWESTR vs. NOESTR. Plasma P4 concentrations on d 0 were greater (P < 0.01) in NOESTR vs. HIESTR and LWESTR and similar (P = 0.93) between HIESTR and LWESTR. Whole blood mRNA expression of myxovirus resistance 2 on d 20 was greater (P ≤ 0.05) in HIESTR vs. LWESTR and NOESTR, and similar (P = 0.72) between LWESTR and NOESTR. Pregnancy rates were less (P ≤ 0.04) in NOESTR vs. HIESTR and LWESTR (52.4%, 68.9%, and 73.5%, SEM = 7.2), and similar (P = 0.57) between HIESTR and LWESTR. Hence, expression of estrus during a timed-AI protocol improved ovarian dynamics and pregnancy success, whereas estrus intensity modulated key biological markers associated with fertility but not pregnancy rates in B. indicus-influenced cows beef cows.

The objectives of this study were to explore Canadian dairy producers' attitudes toward reproductive performance and challenges they perceive to be related to reproduction and reproductive management practices. A survey in both English and French was developed, validated, and administered to Canadian dairy farmers between March and May 2014 to collect general farm, reproduction management, and reproductive performance data, as well as opinions and perceptions about different facets of reproduction. Associations between management practices and the perceived importance of reproduction were tested using a logistic regression model. Thematic network analysis was used to identify themes from the open-ended survey questions about challenges concerning reproduction. Finally, questions that were answered on a Likert scale were graphically represented using diverging stacked bar charts. A total of 832 questionnaires were completed online and by mail, which represents approximately 7% of all dairy farms in Canada. Respondents that ranked reproduction in lactating dairy cows as 1 of the 3 most important challenges faced on their farm (66%) were more likely to house their lactating cows in a tiestall and to have a lower herd annual 21-d pregnancy rate. Estrus detection and conception risk were 2 major themes raised and discussed by the respondents. Other concepts, including housing and milk production, were also perceived to affect estrus detection and conception risk. Whereas analysis of open-ended survey questions does not allow for quantification of the importance of different themes in the sample as a whole, it does show that respondents are aware of the multifactorial complexity of reproductive challenges on dairy farms. Improving performance was the main factor influencing decisions concerning reproduction for 80% of the respondents, and they adopted tools and technologies such as synchronization programs and automated activity monitoring systems to improve herd reproductive performance. More research is required to describe how this performance is defined and perceived by the respondents, and how it relates to the actual variability of performance (i.e., pregnancy rate) among farms., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Identifying cows in estrus remains a challenge on dairy cattle farms, and tools and technologies have been developed and used to complement or replace visual detection of estrus. Automated activity monitoring (AAM) systems and timed artificial insemination (TAI) are technologies available to dairy farmers, but many factors can influence their relative performance. The objective of the present study was to compare reproductive performance of cows managed with an AAM system combined with TAI, or with a TAI program (Double Ovsynch) for insemination before 88 DIM. From April 2014 to April 2015, 998 cows from 2 herds were randomly assigned either to be inseminated at 85 ± 3 DIM exclusively using the Double Ovsynch protocol for TAI, or to be inseminated based on estrus detection by AAM without hormonal intervention between 50 and 75 DIM; if no alarm was detected by 75 DIM, cows were inseminated following the single Ovsynch protocol (AAM + Ovsynch). The herds used different AAM systems. Parity, hyperketonemia at wk 1 and 2 postpartum (PP), purulent vaginal discharge at wk 5 PP, body condition score at wk 7 PP, and anovulation to wk 9 PP were recorded. These health indicators did not significantly differ between treatments, but did between herds. The effect of treatment on pregnancy at first insemination and by 88 DIM were assessed using logistic regression models. Time to pregnancy was assessed using survival analysis. Results are reported from intention-to-treat analyses. Treatment did not affect pregnancy at first insemination or pregnancy by 88 DIM, but we found significant interactions between treatment and herd for both outcomes. In herd 2, marginal mean pregnancy at first AI was greater with Double Ovsynch (38%) than AAM + Ovsynch (31%), but no difference was observed in herd 1 (Double Ovsynch = 31%; AAM + Ovsynch = 34%). By 88 DIM, a smaller proportion of cows in herd 1 were pregnant in Double Ovsynch (31%) than AAM + Ovsynch (49%), but there was no difference in herd 2 (Double Ovsynch = 38%; AAM + Ovsynch = 38%). We observed a treatment by herd interaction for median (95% confidence interval) time to pregnancy, which were, in herd 1, 110 (106 to 129) and 98 (88 to 113) d, and, in herd 2, 126 (113 to 139) and 116 (105 to 131) d for the Double Ovsynch and AAM + Ovsynch treatments, respectively. The relative performance of AAM-based reproductive management compared with TAI only is likely influenced by herd-specific variables, in particular related to insemination rate when estrus detection is employed., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this experiment was to compare hormonal, uterine, and conceptus factors associated with pregnancy establishment in beef cows supplemented or not with Ca salts of soybean oil (CSSO) for 21 d beginning after timed AI. One hundred lactating multiparous Nelore cows were allocated to 20 groups of 5 cows/group and timed inseminated on d 0 of the experiment. After AI, groups were randomly assigned to receive (as-fed basis) 100 g of protein-mineral mix + 100 g of ground corn per cow per day, in addition to 1) 100 g/cow daily of CSSO ( = 10) or 2) 100 g/cow daily of kaolin (CON; rumen-inert indigestible substance; = 10). Groups were maintained in 4 pastures (5 groups from the same treatment within each pasture) with ad libitum access to forage. Groups were segregated daily and individually offered treatments from d 0 to 21. Blood samples were collected and transrectal ultrasonography was performed to verify ovulation and corpus luteum (CL) volume immediately before AI (d 0) and on d 7 and 15. After ultrasonography on d 15, 60 cows (30 cows/treatment and 3 cows/group) diagnosed without the presence of a CL on d 0 but with a CL greater than 0.38 cm3 in volume on d 7 and 15 were assigned to conceptus collection via transcervical flushing with PBS followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Additional blood samples were collected for whole-blood RNA extraction (d 20), and pregnancy status was verified by transrectal ultrasonography (d 30) in cows not assigned to conceptus collection. Cows receiving CSSO had greater ( ≤ 0.04) mean plasma linoleic acid concentration, plasma linoleic:linolenic acid ratio, plasma progesterone (P4) concentration, and CL volume during the experiment compared with CON cows. Moreover, CSSO supplementation increased ( ≤ 0.04) length and mRNA expression of and by the conceptus as well as blood mRNA expression of interferon-stimulated genes on d 20 in gestating cows. No treatment differences were detected ( ≥ 0.30) for endometrial mRNA expression of and . In summary, post-AI CSSO supplementation to B. indicus beef cows increased plasma concentration of linoleic acid and enhanced pregnancy establishment factors, which included CL development and plasma P4 concentration, conceptus growth, and mRNA expression of as well as blood mRNA expression of interferon-stimulated genes.

The aim of this study was to test the effect of expression of estrus at artificial insemination (AI) on endometrium, conceptus, and CL gene expression of beef cows. Thirty-six multiparous nonlactating Nelore cows were enrolled on an estradiol- and progesterone (P4)-based timed AI protocol (AI = Day 0) and then slaughtered for the endometrium, CL, and conceptus collection on Day 19. The animals were retrospectively grouped on the basis of cows that (1) showed signs of estrus near AI (n = 19; estrus) and (2) did not show any signs of estrus (n = 17; nonestrus). Body condition score, blood sampling, and ultrasound examination were performed on Days 0, 7, and 18 of the experiment followed by messenger RNA extraction and quantitative reverse transcription polymerase chain reaction analysis of 58 target genes. Data were checked for normality and analyzed by ANOVA for repeated measures using proc GLM, MIXED, and UNIVARIATE of SAS. Only pregnant cows were included in the analyses (n = 12; nonestrus, n = 11). Estrous expression had no correlation with parameters such as body condition score, preovulatory follicle and CL diameter, P4 concentration in plasma on Days 7 and 18 after AI, and interferon-tau concentration in the uterine flushing (P > 0.15); however, a significant increase was observed in conceptus size from cows that expressed estrus (P = 0.02; 38.3 ± 2.8 vs. 28.2 ± 2.9 mm). The majority of transcripts affected by estrous expression in the endometrium belong to the immune system and adhesion molecule family (MX1, MX2, MYL12A, MMP19, CXCL10, IGLL1, and SLPI; P ≤ 0.05), as well as those related with prostaglandin synthesis (OTR and COX-2; P ≤ 0.05). Genes related to apoptosis, P4 synthesis, and prostaglandin receptor were downregulated (CYP11A, BAX, and FPr; P < 0.05) in the CL tissue of cows that expressed estrus. In addition, four genes were identified as differentially expressed in the 19-day-old conceptus from cows that expressed estrus (ISG15, PLAU, BMP15, and EEF1A1; P < 0.05). There was also a significant effect of Day 7 concentration of P4 mainly affecting the immune system, adhesion molecules, and wnt signaling pathway of the endometrium (IGLL1, MX2, SLPI, TRD, APC, WNT2, GLYCAM1, and MYL12A; P < 0.05). A significant interaction between estrous expression and P4 concentration on Day 7 was more pronounced in immune system genes (MX1, MX2, TRD, SLPI, and IGLL1; P < 0.05). This study reported that estrous expression at the time of AI favorably altered the gene expression profile in reproductive tissues during the preimplantation phase toward a more receptive state to the elongating conceptus. These effects seem to be more evident in the endometrium during the time of dynamic remodeling for embryo implantation., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objectives were to evaluate the effects of prepartum stocking density on innate and adaptive leukocyte responses, serum cortisol and haptoglobin concentrations and hair cortisol concentration of Jersey cows. The cows (254 ± 3d of gestation) were balanced for parity (nulliparous vs. parous) and previous lactation projected 305-d mature equivalent milk yield and assigned to one of two treatments: 80SD=80% stocking density (38 animals/48 headlocks) and 100SD=100% stocking density (48 animals/48 headlocks). Pens (n=4) were identical in size and design and each pen received each treatment a total of 2 times (4 replicates; 80SD: n=338; 100SD: n=418). A sub-group of cows (n=48/treatment per parity) was randomly selected on week 1 of each replicate from which blood was sampled weekly from d -14 to 14 (d 0=calving) to determine polymorphonuclear leukocyte (PMNL) phagocytosis, oxidative burst, and expression of CD18 and L-selectin, and hemogram. The same sub-group of cows was treated with chicken egg ovalbumin on d -21, -7, and 7 and had blood sampled weekly from d -21 to 21 for determination of serum IgG anti-ovalbumin concentration. Blood was sampled weekly from d -21 to 21 to determine glucose, cortisol, and haptoglobin concentrations in serum. Hair samples collected at enrollment and within 24h of calving were analyzed for cortisol concentration. The percentage of leukocytes classified as granulocyte and the granulocyte to the lymphocyte ratio were not affected by treatment. Treatment did not affect the percentage of PMNL positive for phagocytosis and oxidative burst or the intensity of phagocytosis and oxidative burst. Similarly, treatment did not affect the percentage of PMNL expressing CD18 and L-selectin or the intensity of expression of CD18 and L-selectin. Concentration of IgG anti-ovalbumin was not affected by treatment. Serum concentrations of haptoglobin and cortisol were not affected by treatment. Similarly, hair cortisol concentration at calving was not affected by treatment. According to the current experiment, a target stocking density of 80% did not improve leukocyte responses compared with 100% target stocking density., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The objective of this study was to determine risk-factors affecting increase in physical activity during estrus and pregnancy per artificial insemination (P/AI) in lactating dairy cows. Cows were monitored continuously by 2 automated activity monitors [a collar-mounted accelerometer (HT; Heatime, SCR Engineers, Netanya, Israel) and a leg-mounted pedometer (BO; Boumatic Heat-seeker-TX, Boumatic Dairy Equipment, Madison, WI)]. When an increase in activity was detected, body condition score (BCS) and blood samples were collected, ovaries were scanned by ultrasonography, and, if the cow was eligible for breeding, artificial insemination was performed. Milk production and health-related data were recorded throughout the experimental period. Pregnancy diagnosis was performed at 42 ± 7 d of gestation. Data were analyzed using Pearson correlation, ANOVA, and logistic regression. A total of 1,099 true events of estrus from 318 lactating Holstein cows were recorded, averaging 3.46 ± 1.1 events per cow. Positive predictive value for estrus episodes detected by the HT and BO systems were 89.6 and 85.5%, respectively. Mean peak activity at estrus (PA) recorded by the HT system was 71.6 ± 20.7 index-value, and 334.3 ± 155.7% relative increase by the BO system. Compared with primiparous, multiparous cows expressed estrus with lower PA (69.3 ± 0.8 vs. 75.9 ± 1.1 index for HT; 323.9 ± 6.0 vs. 354.8 ± 8.48% for BO) and shorter duration (DU; 10.7 ± 0.2 vs. 12.0 ± 0.3 h); DU was measured by HT only. Lower BCS was associated with decreased PA measured by both systems, estrus DU, and P/AI. Peak activity was weakly correlated with milk production on the day of artificial insemination (r = -0.20); however, when categorized into quartiles, the highest-yield cows had lower PA and DU. Follicle diameter was not correlated with PA or DU, but cows with greater concentrations of estradiol had higher PA. Cows with greater PA in both systems had greater P/AI than those with lower PA (36.5 vs. 24.6% for HT, 33.5 vs. 21.4% for BO). In conclusion, measurements of estrus events captured by automated activity monitors are correlated with BCS, parity, and secondary behavior signs related to estrus. Surprisingly, estrus intensity and duration were only weakly correlated with milk production, preovulatory follicle diameter, and concentrations of estradiol at estrus. Cows that had measurements of high-intensity estrus were significantly more fertile than low-intensity estrus., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Holstein heifers (n = 57) were monitored using accelerometers and video observations with the objective of better understanding the behavioral expression of estrus, the variation within and between the heifers, and the possible sources of variation. IceTags recorded walking activity from 7 to 13 months of age. Activity peaks (n = 282) were obtained from a rolling sum of steps within 24-hour periods and validated to be estrus by ovarian ultrasonography. Behavior around activity peak of one estrus for each of 12 heifers was described in detail from video recordings. Baseline behavior was monitored in a corresponding interval 1 week before. Estrus and baseline total steps and steps per hour, estrus relative increase in activity, duration, and interval between episodes were analyzed by descriptive statistics and Spearman rank correlations. Effects of category of baseline walking activity, estrus order (pubertal vs. second and greater episodes), season, hour of estrus onset, and number of heifers simultaneously in estrus were evaluated with proc MIXED. Behavioral changes from baseline to estrus were evaluated by a signed-rank test. Estrus total steps varied greatly (4743 ± 1740; range: 837-10,070), as well as the relative increase in activity (290 ± 160%; range: 30%-1190%). Duration of estrus was 14 ± 4 hours, ranging from 4 to 26 hours. The interval between episodes was the trait that varied the least. Pubertal estrus was shorter and had a smaller relative increase in activity than second and greater episodes (P < 0.05). The number of steps during estrus was greater for heifers of high baseline activity (P < 0.01). Estrus episodes occurring in the winter and starting between 4 PM and 3 AM had the greatest relative increase in activity (P < 0.05). The number of heifers simultaneously in estrus did not influence estrus expression (P > 0.05). The behaviors with greatest change from baseline to estrus were chin rest, sniff, back mount, crossover, accept chin rest, and follow, but variation was large. Overall, estrus was apparent in behavioral changes with large variation within and between the heifers. Estrus order, onset hour, season, and baseline walking activity are important factors affecting estrus activity. Therefore, estrus detection tools should account for potential sources of variation. The visual and automated measurements of estrus expression reported in this study reveal possibilities for improved on-farm estrus detection technologies and potential genetic selection for estrus expression., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Two activity monitoring systems-Heatime (SCR Engineers Ltd., Netanya, Israel) and IceTag (IceRobotics Ltd., Edinburgh, UK)-were compared on their ability to detect and quantify estrus expression. Holstein heifers (n=57) were fitted with Heatime (HT) and IceTag (IT) sensors from 12 mo of age until confirmation of pregnancy. Upon detection of high activity by HT, ovaries were scanned by ultrasound, a blood sample was collected for analysis of plasma estradiol, and signs of estrus (clear vaginal mucus, uterine muscle tone, visual mounting activity, standing to be mounted, or rump showing signs of repeated acceptance of mounts) were recorded. Because only estrus episodes detected by HT (n=111) were further evaluated, only the positive predictive value was measured. Heifers were housed in groups of 24 in a freestall pen. Data were analyzed using Proc CORR and GLM of SAS (SAS Institute Inc., Cary, NC). The positive predictive value was 84.7% (94/111) for HT and 98.7% (74/75) for IT. Estrus duration was recorded by HT as 14.3±4.1h [mean ± standard deviation (SD)] and by IT as 15.0±4.0h; duration measurements were correlated (r=0.60). The mean duration difference was 0.74±3.52h. Recordings of onset and end of estrus by IT were 3.5±4.3h and 2.9±4.9h earlier than those by HT. The overlap in duration was 9h. Measurements of estrus intensity were correlated (r=0.63). Peak activity was 77.3±19.5 index value (approximately 7.7 SD from basal activity) on HT. The relative increase in activity measured by IT was 360±170% baseline value. Measurements of intensity and duration from HT were correlated (r=0.64) but those from IT were not (r=0.13). Plasma estradiol concentration (11.2±4.6pg/mL) was not correlated with preovulatory follicle diameter or with duration or intensity of estrus. Diameter of preovulatory follicle (15.7±2.6mm) had no correlation with duration of estrus and was only weakly correlated with intensity measured by either system. Baseline steps/hour was negatively correlated with intensity from both sensors (r=-0.37 and -0.70 for HT and IT). Estrus episodes accompanied by 2 or 3 of the monitored signs of estrus had greater intensity and duration on HT but not on IT. Preovulatory follicle diameter and plasma estradiol concentration did not influence occurrence of estrus signs. Results indicate that both systems identified estrus precisely, with correlated characterization and similar timing. In contrast, relationships with plasma estradiol concentration and signs of estrus require further investigation., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this experiment was to compare fatty acid (FA) concentrations in plasma and reproductive tissues as well as hormones and expression of genes associated with pregnancy establishment in beef cows supplemented or not with Ca salts of soybean oil (CSSO) beginning after timed AI. Ninety nonlactating multiparous Nelore (Bos indicus) cows were timed inseminated on d 0 of the experiment and divided into 18 groups of 5 cows/group. Groups were randomly assigned to receive (as-fed basis) 100 g of a protein-mineral mix plus 100 g of ground corn per cow daily in addition to 1) 100 g/cow daily of CSSO (n = 9) or 2) 100 g/cow daily of kaolin (CON; rumen-inert indigestible substance; n = 9). All groups were maintained in a single Brachiaria brizanta pasture (24 ha) with ad libitum access to forage and water. However, groups were segregated daily and offered treatments individually at the working facility during the experimental period (d 0 to 18). Blood samples were collected and transrectal ultrasonography was performed to verify ovulation and estimate corpus luteum (CL) volume immediately before AI (d 0) and on d 7 and 18 of the experiment. On d 19, 36 cows (18 cows/treatment; 2 cows/group) diagnosed without the presence of a CL on d 0 but with a CL greater than 0.38 cm(3) in volume on d 7 and 18 were slaughtered for collection of conceptus, uterine luminal flushing, and tissue samples from the CL and endometrium. Cows receiving CSSO had greater concentrations of linoleic and other ω-6 FA in plasma (P < 0.01), endometrium (P ≤ 0.05), CL (P ≤ 0.05), and conceptus (P ≤ 0.08) compared to CON. On d 7 of the experiment, CSSO-supplemented cows had greater plasma progesterone concentrations (P < 0.01) and CL volume (P = 0.02) compared to CON, whereas no treatment effects were detected (P ≥ 0.15) for these parameters on d 18 (treatment × day interaction; P < 0.01). Cows receiving CSSO tended (P = 0.09) to have greater concentrations of interferon-tau in the uterine flushing media compared with CON. However, no treatment effects were detected for mRNA expression genes associated with pregnancy establishment in endometrial, CL, and conceptus samples (P ≥ 0.12). In summary, supplementing beef cows with 100 g of CSSO beginning after AI favored incorporation of ω-6 FA into their circulation, reproductive tissues, and conceptus, without impacting expression of genes associated with pregnancy establishment on d 19 of gestation.

Objectives were to determine effects of lactation and pregnancy on endometrial gene expression on d 17 of the estrous cycle and pregnancy. Heifers (n=33) were assigned randomly after parturition to lactating (L, n=17) or nonlactating (NL, n=16) groups. Cows were subjected to an ovulation synchronization program for a timed artificial insemination (TAI); 10 cows in L and 12 in NL were inseminated. Slaughter occurred 17 d after the day equivalent to TAI, and intercaruncular endometrial tissues were collected. Gene expression was determined by DNA microarray analysis for pregnant (L, n=8; NL, n=6) and noninseminated cyclic (L, n=7; NL, n=4) cows. Differentially expressed genes were selected with a P-value <0.01 and absolute expression >40. In addition, a fold effect >1.5 was used as a criterion for genes affected by pregnancy. In total, 210 genes were differentially regulated by lactation (136 downregulated and 74 upregulated), and 702 genes were differentially regulated by pregnancy (407 downregulated and 295 upregulated). The interaction effect of pregnancy and lactation affected 61 genes. Genes up- and downregulated in pregnant cows were associated with several gene ontology terms, such as defense response and interferon regulatory factor, cell adhesion, and extracellular matrix. The gene ontology analyses of up- and downregulated genes of lactating cows revealed terms related to immunoglobulin-like fold, immune response, COMM domain, and non-membrane-bounded organelle. Several genes upregulated by lactation, such as IGHG1, IGLL1, IGK, and TRD, were related to immune function, particularly for B cells and γδ T cells. Developmental genes related to limb and neural development and glucose homeostasis (e.g., DKK1, RELN, PDK4) were downregulated by lactation, whereas an interaction was also detected for RELN. The stated genes associated with immune function and developmental genes expressed in the endometrium affected by lactational state are possible candidate genes for interventions to improve fertility of lactating dairy cows., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objectives were to characterize postpartum metabolic and hormonal differences between nonlactating and lactating dairy cows, evaluate lactation and pregnancy effects on endometrium and conceptus expression of selected genes, and characterize associations between conceptus and endometrial expression of genes in early pregnancy (d 17). Pregnant heifers were assigned randomly after calving to a lactating group (L, n=17) and a nonlactating group (NL, n=16). The L cows were fed a total mixed ration [1.65 Mcal of net energy for lactation (NE(L))/kg, 16.5% crude protein (CP)] ad libitum, and the NL cows were fed a maintenance ration (1.45 Mcal of NE(L)/kg, 12.2% CP) once per day. All cows were presynchronized and enrolled in a timed artificial insemination (TAI) protocol; 10 cows in the L and 12 in the NL received TAI. On d 17 after GnRH and TAI, cows were slaughtered and endometrial and conceptus tissues collected. The Affymetrix Bovine Genome DNA Microarray (Affymetrix Inc., Santa Clara, CA) was used to assess conceptus and endometrial gene expression. The L cows had higher body temperature than the NL cows (38.4 vs. 38.2°C), and the NL cows cycled earlier than the L cows (26.3 vs. 34.7 d in milk). Cows in the L group had greater plasma concentrations of β-hydroxybutyrate (4.90 vs. 2.97 mg/dL) and blood urea N (11.6 vs. 6.5mg/dL) and lower concentrations of glucose (74.0 vs. 79.9 mg/dL) compared with NL cows. Insulin-like growth factor-1 was lower for L compared with NL (140.5 vs. 198.2 ng/mL) and was greater for cows subsequently classified pregnant compared with cyclic (191.0 vs. 147.6 ng/mL). The concentration of progesterone from GnRH or TAI (d 0) until d 17 was lower for L cows than for NL cows. Gene expression analyses indicated that all conceptuses (n=13) expressed pregnancy-associated glycoprotein (PAG) genes PAG2, PAG8, PAG11, and PAG12. The same PAG family genes were observed in the endometrium of some pregnant cows. Simple and standard partial correlation analyses detected associations of conceptus PAG11 with prostaglandin regulatory genes. In conclusion, lactation altered metabolic status, delayed initiation of cyclicity, and decreased concentrations of progesterone in pregnant cows. Early expression of PAG genes in the conceptus may contribute to successful development of early pregnancy and possibly alter mechanisms related with embryo survival such as prostaglandin synthesis., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objectives were to evaluate pregnancy per AI (P/AI) of dairy cows subjected to the 5-day timed AI protocol under various synchronization and luteolytic treatments. Cows were either presynchronized or received supplemental progesterone during the synchronization protocol, and received a double luteolytic dose of PGF2α, either as one or two injections. In Experiment 1, dairy cows (n=737; Holstein=250, Jersey=80, and crossbred=407) in two seasonal grazing dairy farms were randomly assigned to one of four treatments in a 2×2 factorial arrangement. The day of AI was considered study Day 0. Half of the cows were presynchronized (G6G: PGF2α on Day -16 and GnRH on Day -14) and received the 5-day timed AI protocol using 1 mg of cloprostenol, either as a single injection (G6G-S: GnRH on Day -8, PGF2α on Day -3, and GnRH+AI on Day 0) or divided into two injections of 0.5 mg each (G6G-T: GnRH on Day -8, PGF2α on Day -3 and -2, and GnRH+AI on Day 0). The remaining cows were not presynchronized and received a controlled internal drug-release (CIDR) insert containing progesterone from GnRH to the first PGF2α injection of the 5-day timed AI protocol, and 1 mg of cloprostenol either as a single injection on Day -3 (CIDR-S) or divided into two injections of 0.5 mg each on Days -3 and -2 (CIDR-T). Ovaries were examined by ultrasonography on Days -8 and -3 and plasma progesterone concentrations were determined on Days -3 and 0. In Experiment 2, 655 high-producing Holstein cows had their estrous cycle presynchronized with PGF2α at 46±3 and 60±3 days postpartum and were randomly assigned to receive 50 mg of dinoprost during the 5-day timed AI protocol, either as a single injection or divided into two injections of 25 mg each. Pregnancies per AI were determined on Days 35 and 64 after AI in both experiments. In Experiment 1, presynchronization with G6G increased the proportion of cows with a CL on Day -8 (80.6 vs. 58.8%), ovulation to the first GnRH of the protocol (64.2 vs. 50.2%), and the presence (95.6 vs. 88.4%) and number (1.79 vs. 1.30) of CL at PGF(2α) compared with CIDR cows. Luteolysis was greater for two injections compared to a single PGF2α injection (two PGF2α=95.9 vs. single PGF2α=72.2%), especially in presynchronized cows (G6G-T=96.2 vs. G6G-S=61.7%). For cows not presynchronized, two PGF2α injections had no effect on P/AI (CIDR-S=30.2 vs. CIDR-T=34.3%), whereas for presynchronized cows, it improved P/AI (G6G-S=28.7 vs. G6G-T=45.4%). In Experiment 2, the two-PGF2α injection increased P/AI on Days 35 (two PGF2α=44.5 vs. single PGF2α=36.4%) and 64 (two PGF2α=40.3% vs. single PGF2α=32.6%) after AI. Presynchronization and dividing the dose of PGF2α (either cloprostenol or dinoprost) into two injections increased P/AI in lactating dairy cows subjected to the 5-day timed AI protocol., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives were to compare the effect of presynchronization and resynchronization methods on fertility responses of grazing dairy cows at first and second artificial insemination (AI) and pregnancy rate during the entire breeding season. Lactating dairy cows (n = 1,263) in 2 seasonal grazing farms were blocked, within farm, by parity, breed and days in milk. Within each block, cows were randomly assigned to 1 of 4 treatments arranged as a 2 × 2 factorial with 2 presynchronization and 2 resynchronization treatments. Cows had their estrous cycles presynchronized with either a PGF(2α)-based program (Presynch) consisting of 2 injections of PGF(2α) administered 14 d apart and starting the timed AI protocol 11 d later, or with a PGF(2α)-GnRH-based presynchronization program (G6G) consisting of an injection of PGF(2α), followed 3 d later by an injection of GnRH and starting the timed AI protocol 6 d later. All cows received the first insemination on the same day, which was considered study d 0 and also d 0 of the breeding season. All cows received the 5-d timed AI protocol that consisted of GnRH on d -8, PGF(2α) on d -3 and -2, and GnRH+timed AI on d 0. Blood was sampled and analyzed for progesterone on d -8. On d 12, cows in each presynchronization treatment either remained as untreated controls (RCON) or received a controlled internal drug-release (CIDR) insert containing progesterone for 7 d (RCIDR). Estrus was observed daily starting on d 19 and cows in estrus were inseminated on the same day. On d 35, bulls were placed with the cows for an additional 65 d, completing a 100-d breeding season. Holstein cows were less likely to have progesterone ≥ 1 ng/mL on d -8, and had less expression of estrus and pregnancy per AI (P/AI), which resulted in a slower rate of pregnancy and a smaller proportion of pregnancy at the end of the study than did Jersey or crossbred cows. In addition, body condition, days in milk, and plasma progesterone concentration at the first GnRH injection of the timed AI protocol had marked effects on the reproductive performance of lactating grazing dairy cows. A greater proportion of G6G cows had progesterone ≥ 1 ng/mL at the first GnRH injection of the timed AI protocol compared with Presynch cows (82.0 vs. 74.3%). Presynchronization treatment did not influence P/AI, but cows in G6G had increased risk of pregnancy loss between d 30 and 65 after the first AI (12.9 vs. 8.1%). Nevertheless, an interaction between presynchronization and ovarian status was observed, and cows initiating the timed AI with progesterone ≥ 1 ng/mL had greater P/AI when previously treated with Presynch than G6G. On the other hand, G6G benefited P/AI of cows initiating the timed AI with progesterone < 1 ng/mL. Resynchronization with RCIDR altered the pattern of return to estrus, but it did not increase the rate of re-insemination and decreased the proportion of pregnant cows at the end of the 100-d breeding period (80.6 vs. 84.4%)., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Two experiments evaluated the influence of altering the concentrations of progesterone during the development of the ovulatory follicle on the composition of the follicular fluid, circulating LH and PGF(2α) metabolite (PGFM), and expression of endometrial progesterone receptor and estrogen receptor-α. In both experiments, the estrous cycles were presynchronized (GnRH and progesterone insert followed by insert removal and PGF(2α) 7 d later, and GnRH after 48 h) and cows were then enrolled in 1 of 2 treatments 7 d later (study d -16): high progesterone (HP) or low progesterone (LP). In experiment 1 (n=19), cows had their estrous cycle synchronized starting on study d -9 (GnRH and progesterone insert on d -9, and insert removal and PGF(2α) on d -2). In experiment 2 (n=25), cows were submitted to the same synchronization protocol as in experiment 1, but had ovulation induced with GnRH on study d 0. In experiment 1, plasma was sampled on d -4 and analyzed for concentrations of LH; the dominant follicle was aspirated on d 0 and the fluid analyzed for concentrations of progesterone, estradiol, and free and total IGF-1. In experiment 2, follicular development and concentrations of progesterone and estradiol in plasma were evaluated until study d 16. Uterine biopsies were collected on d 12 and 16 for progesterone receptor and estrogen receptor-α protein abundance. An estradiol/oxytocin challenge for PGFM measurements in plasma was performed on d 16. In experiments 1 and 2, LP cows had lower plasma concentrations of progesterone and greater concentrations of estradiol, and had larger ovulatory follicle diameter (20.4 vs. 17.2mm) at the end of the synchronization protocol than HP cows. Concentration of LH tended to be greater for LP than HP cows (0.98 vs. 0.84 ng/mL). The dominant follicle of LP cows had greater concentration of estradiol (387.5 vs. 330.9 ng/mL) and a lower concentration of total IGF-1 (40.9 vs. 51.7 ng/mL) than that of HP cows. In experiment 2, estradiol and progesterone concentrations did not differ between treatments from d 0 to 16; however, the proportion of cows with a short luteal phase tended to increase in LP than HP (25 vs. 0%). Concentrations of PGFM were greater for LP than HP. Uterine biopsies had a greater abundance of progesterone receptor, and tended to have less estrogen receptor-α abundance on d 12 compared with d 16. An interaction between treatment and day of collection was detected for estrogen receptor-α because of an earlier increase in protein abundance on d 12. Reduced concentrations of progesterone during the development of the ovulatory follicle altered follicular dynamics and follicular fluid composition, increased basal LH concentrations, and prematurely increased estrogen receptor-α abundance and exacerbated PGF(2α) release in the subsequent estrous cycle., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objectives were to evaluate the effects of differing progesterone concentrations during follicle development on follicular dynamics, fertilization, and embryo quality. Lactating Holstein cows (n=154) were assigned randomly to 1 of 2 treatments. Cows underwent a presynchronization of the estrous cycle composed of an injection of GnRH concurrently with the placement of a progesterone insert, an injection of PGF(2α) and insert removal 7 d later, and a second injection of GnRH 48 h later (study d -16). All cows were then submitted to a hormonal protocol identical to the presynchronization program starting on d 7 of the estrous cycle (study d -9). Cows enrolled in the high progesterone (HP) treatment received no further treatment. Cows in the low progesterone (LP) treatment received additional PGF(2α) injections on study d -14, -13.5, and -13 and again on study d -9, -7, -6.5, and -6. Ovaries were evaluated by ultrasonography, and blood was sampled for concentrations of progesterone and estradiol throughout the study. Uteri were flushed 6 d after artificial insemination (AI) and recovered oocytes-embryos were evaluated. Concentrations of progesterone were less for LP cows from study d -7 to -2; concentrations of estradiol at PGF(2α) and at the last GnRH of synchronization were greater for LP than HP. The proportion of cows in estrus at AI was greater for LP than for HP (38.0 vs. 5.3%). Ovulatory follicles of LP cows had larger diameters at the injections of PGF(2α) (17.2 vs. 14.6mm) and final GnRH (19.4 vs. 16.9%) of the synchronization, which resulted in a larger diameter of the corpus luteum 6 d after AI (24.3 vs. 22.6mm). Double ovulation after the last GnRH of the synchronization was increased in LP (18.6%) compared with HP (4.5%). Fertilization rate was similar and averaged 82.7%. The proportion of embryos and oocytes-embryos classified as grades 1 and 2, proportion of degenerated embryos, and unfertilized-degenerated oocytes-embryos were not different between LP and HP. Number of blastomeres did not differ between LP and HP, but the proportion of live blastomeres tended to be less for LP than HP (94.2 vs. 98.7%). Reducing progesterone concentrations during the synchronization program altered concentrations of estradiol and follicular dynamics, but resulted in similar fertilization and only minor changes in embryo quality., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objectives of this study were to evaluate the effect of administering 500 mg of recombinant bovine somatotropin (bST) every 10 d on ovulatory responses, estrous behavior, and fertility of lactating Holstein cows. Lactating dairy cows were assigned to 1 of 2 treatments: a control with no administration of bST (73 primiparous and 120 multiparous cows) or 6 consecutive administrations of 500 mg of bST (83 primiparous and 123 multiparous cows) given subcutaneously at 10-d intervals starting 61+/-3 d postpartum (study d 0), concurrent with the initiation of the timed artificial insemination (AI). Blood samples were collected thrice weekly from 61+/-3 to 124+/-3 d in milk (DIM), and plasma samples were analyzed for concentrations of estradiol, glucose, insulin, insulin-like growth factor 1, and progesterone. The estrous cycle of cows was presynchronized with 2 injections of PGF(2alpha) at 37+/-3 and 51+/-3 DIM, and the Ovsynch timed AI protocol was initiated at 61+/-3 DIM. Ovaries were scanned to determine ovulatory responses during the Ovsynch protocol. Pregnancy was diagnosed at 33 and 66 d after AI. Body condition was scored on study d 0, 10, 42, and 76. Sixty-four cows were fitted with a pressure mounting sensor with radiotelemetric transmitters to monitor estrous behavior. Treatment of lactating dairy cows with 500 mg of bST at 10-d intervals increased yields of milk and milk components in the first 2 mo after treatment. Body condition of bST-treated cows remained unaltered, whereas control cows gained BCS. Treatment with bST increased concentrations of insulin-like growth factor 1 chronically, but concentrations of insulin and glucose increased only transiently in the first 7 d after the first injection of bST. Concentrations of progesterone during and after the Ovsynch protocol remained unaltered after treatment with bST; likewise, ovulatory responses during the Ovsynch protocol were mostly unaltered by treatment. Concentration of estradiol tended to be greater for bST cows than for control cows immediately before induction of ovulation in the Ovsynch protocol. Similarly, the mean and the peak concentrations of estradiol were greater for bST cows than for control cows when monitored during spontaneous estrus. Nevertheless, duration of estrus and the median number of standing events were less for bST cows than for control cows. Pregnancies per AI after the first and second postpartum inseminations were not affected by bST treatment. Treatment of lactating dairy cows with 500 mg of bST every 10 d improved lactation performance, but it did not affect pregnancies per AI and it reduced expression of estrus., (Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objectives were to evaluate effects of feeding a calcium salt rich in linoleic and trans-octadecenoic acids (LTFA) on synthesis of prostaglandin F(2alpha) based on its metabolite (PGFM), uterine involution and pregnancy rates in lactating dairy cows. Five hundred and eleven Holstein cows were blocked according to parity, body condition score and milk yield in the previous lactation. Primiparous and multiparous cows were randomly assigned to one of the two treatments consisting of calcium salt (2% diet dry matter) of either palm oil (PO) or LTFA from 25 days prepartum to 80 days of lactation. Cows were time-inseminated at 70 +/- 3 days postpartum. Feeding LTFA tended (p = 0.08) to decrease the incidence of puerperal metritis (15.1% vs 8.8%). Primiparous cows supplemented with LTFA showed larger increase in plasma PGFM concentration at day 1 postpartum (17018 vs 6897 pm). Pregnancy rate after first insemination tended (p = 0.07) to be greater at 27 days after insemination (37.9% vs 28.6%), and was greater (p = 0.05) at 41 days after insemination (35.5% vs 25.8%) for cows fed LTFA compared with PO. These results indicate that unsaturated fatty acids fed in a rumen inert form have the potential to modulate reproductive events and improve pregnancy rates in lactating dairy cows.

Multiparous Holstein cows (n=717) from two dairy farms were blocked at calving by parity and previous lactation milk yield and, within each block, randomly assigned to one of two treatments: a diet containing no yeast culture (Control; n=359) or 30 g/d of a culture of Saccharomyces cerevisiae (YC; n=358) from 20 to 140 d postpartum. Only cows calving during months of heat stress, May-August were enrolled. Lameness score (1-5 scale) was evaluated at study enrollment and again at 100 d postpartum. The body condition score (BCS, 1-5 scale) was evaluated at calving, 28, 58 and 140 d postpartum. Cows received two injections of PGF(2alpha) at 37 and 51 d postpartum, and those observed in estrus were inseminated. Cows not in estrus were enrolled in a timed AI protocol at 65 d postpartum and inseminated at 75 d postpartum. Ovaries were examined by ultrasonography at 37 and 51 d postpartum to determine whether estrous cycling had been initiated by the presence of a corpus lutem (CL) in at least one of the two examinations. Pregnancy was diagnosed at 31, 38 and 66 d after the first AI and at 38 and 66 d after the second and third AI. Diet did not affect time of onset of estrous cycles postpartum, and 8.2% of the cows were anovular. Detection of estrus in the 7d after the second injection of PGF(2alpha) was similar for control and YC. For control and YC, conception rates 38 d after AI at first (30.8% and 31.4%), second (39.3% and 35.1%) and third (25.8% and 30.6%) inseminations, and pregnancy losses did not differ, which resulted in similar median days to pregnancy and proportion of pregnant cows at 140 d postpartum. Yeast culture did not affect incidence of lameness, but tended to reduce lameness score. Lame cows and anovular cows had lesser conception rates at first AI, and extended interval from calving to conception. A THI of 71 was identified as the critical point in which fertility was reduced in lactating dairy cows, although the sensitivity and specificity were minimal. Cows exposed to a THI>71 on the day of first AI had a 33% reduction in the rate of pregnancy resulting in extended interval to pregnancy. Feeding a yeast culture of S. cerevisiae had minor effects on lameness score, but no impact on reproduction of multiparous cows under heat stress.

Lactating Holstein cows (n=135) were randomly assigned to one of the two sources of supplemental selenium (Se), sodium selenite (SS) or Se yeast (SY), fed at 0.3mg/kg diet dry matter from 25 d before calving to 70 d in milk (DIM), in diets not suboptimal in basal Se concentrations. Cows were evaluated for health daily in the first 10 DIM, and uterine cytology of the previously gravid uterine horn was assessed at 30 DIM. The Ovsynch protocol was initiated at 42 DIM; ovarian responses to hormonal treatments were evaluated by ultrasonography. The uteri of cows were flushed 6d after timed AI for collection of embryos and oocytes. Plasma concentrations of Se and progesterone were measured throughout the postpartum period and during the reproductive protocol, respectively, and plasma glutathione peroxidase activity was determined 6d after AI. Concentrations of Se in pre- and postpartum diets ranged from 0.43 to 0.56 mg/kg of dry matter. Incidence of retained placenta, fever, ketosis, mastitis, acute puerperal metritis, clinical endometritis, and subclinical endometritis were not significantly different between treatments. There were no differences between groups in concentrations of Se and progesterone or glutathione peroxidase activity in plasma. Treatment did not influence ovarian responses to the synchronization protocol, fertilization rate, number of blastomeres and live blastomeres, or proportions of grades 1 and 2, degenerated, and degenerated-unfertilized embryos/oocytes. Odds of subclinical endometritis on Day 30 postpartum more than doubled in cows with fever of unknown origin or acute puerperal metritis in the first 10 DIM. Fertilization rate tended to be reduced in cows with subclinical endometritis. In summary, replacing SS with an organic source of Se in diets not suboptimal in basal Se concentrations did not improve Se status, uterine health, fertilization, or embryo quality in early lactation dairy cows.

The objectives were to evaluate the effects of source of fatty acids (FA) on embryo quality of dairy cows. A total of 154 Holstein cows were assigned randomly to 1 of 2 sources of FA supplemented at 2% of the dietary dry matter as calcium salts of either palm oil (PO) or linoleic and trans-octadecenoic acids (LTFA) from 25 d prepartum to 80 d in milk (DIM). Cows were presynchronized beginning at 30 +/- 3 DIM and then subjected to the Ovsynch protocol beginning on d 39 +/- 3 postpartum. Timed artificial insemination was performed 12 h after the final GnRH of the Ovsynch protocol with semen from a single sire of proven fertility. The uteri of cows were nonsurgically flushed at 5 d after artificial insemination for collection of embryos-oocytes. Ovaries were examined by ultrasonography throughout the synchronization protocol. Blood was sampled and plasma was analyzed for concentrations of metabolites and hormones. The body condition score and yields of milk and milk components were measured throughout the first 90 DIM. Treatment did not affect concentrations of nonesterified FA, beta-hydroxybutyrate, glucose, and progesterone in plasma. Body condition was similar between treatments. Milk production was similar between treatments, but concentrations of fat in milk and yields of fat and 3.5% fat-corrected milk decreased in cows fed LTFA, whereas concentration of true protein increased. Source of dietary FA did not influence ovulatory responses, diameter of the ovulatory follicle, and diameter of the corpus luteum during synchronization. Embryo-oocyte recovery relative to the number of corpora lutea did not differ between treatments. Fertilization tended to increase in cows fed LTFA compared with cows fed PO. Feeding LTFA improved the proportion of excellent-, good-, and fair-quality embryos, and embryos from cows fed LTFA had a greater number of blastomeres than embryos from cows fed PO. Feeding a more unsaturated source of FA improved fertilization and embryo development in lactating dairy cows, despite similar indicators of metabolic status.

Objectives were to evaluate progesterone concentrations after cows had initiated estrous cycles following calving and induction of estrous cycles in postpartum anovular high-producing Holstein dairy cows treated with controlled internal drug releasing (CIDR). In experiment 1 (EXP1), 62 cows that had initiated estrous cycles received a new CIDR (NCIDR) containing 1.38 g of progesterone or a 7-d used autoclaved CIDR (UCIDR) 48h after luteolysis for 7 d. Ovaries were examined by ultrasonography, and plasma analyzed for concentrations of progesterone. In experiment 2 (EXP2), 515 cows diagnosed as anestrus were randomly assigned to untreated control, NCIDR or UCIDR for 6d. Plasma was analyzed for concentration of progesterone 12 d after CIDR removal to determine ovulation. In EXP1, milk yield and body condition did not influence progesterone concentrations. Concentration of progesterone tended to increase faster (P=0.10) in cows receiving UCIDR than NCIDR, but both treatments reached a plateau at 90min. Cows receiving the NCIDR had greater (P=0.04) concentrations of progesterone during the 7-d treatment, but they were mostly subluteal (<1.0 ng/mL) after d 2. After removal, concentrations of progesterone were greater for NCIDR than UCIDR for the first 45 min, and were similar thereafter. Multiparous cows had lesser (P=0.004) concentrations than primiparous cows throughout the study. The pattern of ovarian follicular development was not affected by treatment. In EXP2, induction of onset of estrous cycles increased (P<0.01) with progesterone treatments, but was similar between NCIDR and UCIDR. Proportion of cows experiencing shorter than typical length estrous cycles after first AI tended to be greater (P=0.09) for control cows than those receiving the CIDR, and for cows remaining anestrous than those in which onset of estrous cycles was induced. Pregnancy per AI and pregnancy loss were similar among treatments. Cows that resumed estrous cyclicity prior to first AI had greater (P=0.01) pregnancy per AI. Treatment of high-producing Holstein cows that had previously initiated onset of estrous cycles with CIDR resulted in subluteal concentrations of progesterone, but in anestrous high-producing cows increased induction of estrous cycles with no effect on fertility at first insemination.

Nutrition of the donor cow can influence oocyte and embryo quality, which can affect the success of embryo transfer. Severe undernutrition compromised ovarian follicular development, with implications for superovulatory response and embryo quality. In postpartum lactating cows, undernutrition or inability to consume sufficient nutrients delayed resumption of ovulation, reduced the number of follicles, and compromised oocyte quality. Moderate undernutrition of nonlactating cows was unlikely to affect embryo quality; conversely, nonlactating animals on maintenance diets usually had better superovulatory responses and improved oocyte competence and embryo quality. The negative effects of overfeeding are thought to be mediated by alterations in endocrine cues, such as hyperinsulinemia and increased glucose and IGF-I, which may interfere with glucose transport in the embryo and increase apoptosis. Manipulating energy sources such as carbohydrates and fatty acids (FA) may influence embryo viability, but the effects of FA were not consistent in vitro; increasing concentrations of unsaturated FA in follicular and embryonic cells usually improved embryo viability and resistance to cryopreservation. Excess protein intake and increased urea and ammonia in body fluids can be toxic to embryos, impairing their development; these effects seemed to be associated with alterations in uterine pH and granulosa cell function. Likewise, toxins in feeds (e.g. gossypol), reduced embryo development and increased pregnancy losses. Diet of donor cows should be formulated to optimize the supply of nutrients to meet needs; however, manipulating energy intake, source of FA and protein content of donor diets, particularly moderate underfeeding in nonlactating cows, may further optimize responses.

The objectives of this study were to evaluate the effect of 3 methods of resynchronization of estrus and ovulation for lactating dairy cows of unknown pregnancy status on conception rate and time to pregnancy. Holstein cows (n = 495) were randomly assigned to 1 of 3 treatments: 1) control (n = 167), resynchronization with a timed AI protocol upon diagnosis of nonpregnancy on d 31 after preenrollment AI (PAI); 2) CIDR-G (n = 159), use of an intravaginal progesterone insert from d 14 to 21 after AI, with AI at estrus from d 21 to 24 and initiation of a timed AI protocol on d 24 after AI in cows not reinseminated; 3) CIDR-G + ECP (n = 169), the same treatment as CIDR-G but with an injection of 1 mg of estradiol cypionate at the time of progesterone insert removal. Cows were continuously reenrolled in the same treatment until diagnosed as pregnant, which resulted in a total of 1,148 AI (495 PAI and 653 resynchronized AI; RAI). Blood was collected from 1,001 cows on d 14, 21, and 24 after each AI for analysis of progesterone, and ovaries were scanned on d 21, 24, and 31 after AI. The presence of an active corpus luteum was presumed based on progesterone > or = 1 ng/mL. Pregnancy was diagnosed by ultrasonography on d 31 and 61 after AI. The presence of an active corpus luteum and the incidence of luteolysis were similar for all treatments from d 14 to 24; however, luteolysis increased in the CIDR-G + ECP treatment from d 21 to 24. Conception rates for the PAI and all AI were similar on d 31 and 61 after insemination. Conception rates at 31 and 61 d after the RAI were also similar among treatments. Overall pregnancy loss for the PAI, RAI, and all AI were similar for all treatments. The accuracy of estrous detection, based on progesterone concentration within 2 d of detection of estrus, was similar for all treatments for the RAI and averaged 95.3%. The estrus-detection rate (EDR) decreased for the CIDR-G and CIDR-G + ECP treatments from d 14 to 21, but increased from d 21 to 24 compared with control cows; however, the EDR was smaller for cows in the CIDR-G treatment during the entire resynchronization period compared with those in the CIDR-G + ECP and control groups. The reinsemination interval was reduced in cows receiving the CIDR-G + ECP treatment compared with control cows because of increased EDR after removal of the intravaginal insert; however, the interval from study enrollment to pregnancy was not different among treatments. These results indicate that the reproductive performance of dairy cows did not differ among the 3 resynchronization treatments evaluated.

A total of 799 Holstein cows from 3 herds were randomly assigned at 37 +/- 3 d in milk (DIM) to timed artificial insemination (AI) or insemination at detected estrus. Cows were presynchronized with injections of PGF(2alpha) at 37 and 51 DIM. At 65 DIM, cows received an injection of GnRH, followed 7 d later by PGF(2alpha). Cows in the estrus-detected group were inseminated after being observed in estrus during the 7 d after the last PGF(2alpha). Cows in the timed AI group received an injection of 1 mg of estradiol cypionate (ECP) 24 h after the last PGF(2alpha). If detected in estrus or=1 ng/mL; L = <1 ng/mL), resulting in 8 combinations (LLL, LHL, LLH, LHH, HHH, HHL, HLH, and HLL). Conception rates and pregnancy rates were higher for cows in the timed AI group than in the estrus-detected group at 30, 44, and 58 d (e.g., at 58 d, pregnancy rates were 42.2% for multiparous cows or 34.4% for primiparous cows in the group receiving ECP and timed AI compared with only 20.8 or 18.8% for respective parity subgroups for the treatment group inseminated only at detected estrus). Pregnancy losses were 11.5% from 30 to 58 d and did not differ between treatments. Cyclic cows within both treatments had higher estrous responses, conception rates, and pregnancy rates. Cows that responded to presynchronization and to luteolysis (HHL) had the highest conception and pregnancy rates, followed by cows classified as LHL. Use of 1 mg of ECP to induce ovulation as part of a synchrony regimen improved reproduction at first postpartum insemination in dairy cows.

The objective was to determine the effects of bovine somatotropin (bST) and two artificial insemination (AI) protocols on reproductive performance of Holstein cows. Lactating cows (n = 840) were assigned at 37 d in milk (DIM) to one of four treatments in a 2 x 2 factorial arrangement. Treatments consisted of either bST (500 mg/14 d) starting at 63 +/- 3 DIM or no bST (control), with cows either submitted for timed AI following a synchronized ovulation (Ovsynch) protocol or assigned to receive AI based on estrus detection (ED). Two injections of PGF2, at 37 +/- 3 and 51 +/- 3 DIM were used to presynchronize estrous cycles. Cows then received an injection of GnRH at 63 +/- 3 DIM, followed 7.5 d later by PGF2. Cows assigned to ED treatments were inseminated after observed estrus during a 7-d period. Cows in Ovsynch treatments received a second GnRH injection 48 h after the last PGF2alpha and received timed AI 16 to 18 h later. Pregnancy was diagnosed by ultrasound at 31 d after AI and confirmed 14 d later. Frequency of anovulation (18.5%) at 63 DIM was similar across treatments, but proportions of anovulatory cows decreased quadratically as body condition at 70 DIM increased from 2.25 to 3.75. Estrus detection rate after PGF2alpha tended to be lower in multiparous cows receiving bST, and bST reduced returns to estrus in nonpregnant cows. Conception rates were higher in cows receiving AI after ED and bST improved conception rates to first AI in cyclic cows by reducing embryonic mortality. Pregnancy loss was similar for cows inseminated following ED or the Ovsynch protocol. There was a positive impact of bST on fertility of cyclic cows inseminated at fixed time or at detected estrus, but effective resynchronization protocols are needed to optimize reinsemination of non-pregnant bST-treated cows.

This study examined the influence of a GnRH agonist containing either 450 or 750 microg of deslorelin in an implant form or a gonadorelin injection (control) to induce ovulation in the Ovsynch protocol on pregnancy rates (PR), embryonic loss, and ovarian function in 593 lactating Holstein cows. Cows were given two injections of PGF2alpha 14 days apart, followed 14 days later by the Ovsynch protocol, and were timed artificially inseminated (TAI) at 68 +/- 3 days postpartum. Blood samples for determination of plasma progesterone concentrations were collected at 24 and 10 days prior to and 11 days after TAI. Pregnancy was diagnosed on Day 27 and reconfirmed on Day 41 after TAI. Non-pregnant, not re-inseminated cows at Day 27 had their ovaries examined by ultrasonography, and the number and size of follicles and presence of luteal tissue were determined. Simultaneously, these cows were re-synchronized with the Ovsynch protocol. Pregnancy during the re-synchronization period was determined between 35 and 41 days after insemination. On Day 27, PR were higher for control (39.0%) and deslorelin 450 microg (DESLORELIN 450) implant (41.3%) than for those receiving the deslorelin 750 microg (DESLORELIN 750) implant (27.5%; P<0.05). Pregnancy losses tended to decrease for DESLORELIN 450 compared with control (5.0% versus 12.7%; P<0.13). Plasma progesterone concentrations did not differ significantly among treatments. Deslorelin suppressed ovarian activity and decreased PR during the re-synchronization period compared with control. The percentage of non-pregnant animals that were re-inseminated by Day 27 was less for deslorelin compared with control. In conclusion, incorporation of an implant of the GnRH agonist deslorelin to induce ovulation in the Ovsynch protocol has the potential to reduce pregnancy losses, but the response was dependent upon implant concentration. Evaluation of lower doses to minimize the negative effects on subsequent fertility is warranted.

Objectives of this study were to determine the influence of timing of first clinical mastitis case occurrence on lactational and reproductive performance in high producing lactating dairy cows during the first 320 days in milk (DIM). Holstein cows, 1001, from two commercial dairy farms in California were retrospectively divided into four treatment groups according to timing of first clinical mastitis case caused by environmental pathogens: control with no recorded clinical cases of mastitis (C; n=501); first clinical mastitis prior to first postpartum AI (MG1; n=250); first clinical mastitis between first postpartum AI and pregnancy diagnosis (MG2; n=147); and first clinical mastitis after diagnosed pregnant (MG3; n=103). Clinical cases of mastitis were identified at every milking by the herd personnel based on abnormal milk or swelling of the mammary gland. A fore sample of milk was aseptically collected from every clinical case for microbiological culture. Mastitis decreased yields of milk, 3.5% fat-corrected milk, and milk components, but the effect was only observed for MG1 and MG2. Cows in the control group had lower linear somatic cell count (SCC) score throughout the lactation. Culling was increased by mastitis, and cows in the mastitis groups left the study earlier than controls. Conception rate at first postpartum AI and pregnancy rate at the end of the study were both decreased by mastitis prior to or after first AI, and MG1 and MG2 cows had extended days open. Furthermore, cows experiencing mastitis during lactation had a higher incidence of abortions. The negative effects of mastitis on reproduction were observed regardless of clinical case being caused by either Gram positive or negative bacteria. Mastitis either prior to or after first postpartum AI impairs lactation performance, increases culling, and decreases reproductive efficiency in high producing Holstein dairy cows.

Objectives were to determine the effect of source of supplemental Se on postpartum health, immune responses and Se status of periparturient Holstein cows. Treatments were sodium selenite (SS, n=291) or Se yeast (SY, Sel-Plex®, n=286) supplemented at 0.3 ppm from d 25 prior to calving to 80 d in milk. Health of dairy cows was monitored daily in the first 80 d in milk, and rectal temperature was taken for the first 10 d postpartum. A subset of 15 primiparous and 24 multiparous in the SS group and 10 primiparous and 26 multiparous in the SeY group was used to evaluate cellular and humoral immune responses. A colostrum sample at the first milking after calving was analyzed for total IgG concentrations. Concentrations of Se in plasma were determined at -45, 0, 21, 42 and 60 d relative to calving. Glutathione peroxidase activity in plasma, neutrophil phagocytic activity and its oxidative metabolism were determined on days 0 and 42 postpartum. Each animal received an i.m. injection of 1 mg of ovalbumin at -45, -25 and 0 d relative to calving. Anti-ovalbumin IgG concentrations in serum were analyzed at every injection and at 21 and 42 d postpartum. Concentration of Se in plasma was similar (P = 0.38) for SY and SS throughout the study (0.107 vs 0.101 μg/mL). Glutathione peroxidase activity in plasma was not affected (P = 0.70) by source of Se. Concentration of IgG in colostrum was similar (P = 0.24) for SY and SS (60.9 vs 71.0 g/dL). Phagocytic and killing activities of neutrophils were influenced (P <0.01) by days postpartum, but not (P > 0.10) by source of Se. Similarly, the ability of neutrophils to reduce nitroblue tetrazolium was not (P > 0.10) influenced by source of Se in stimulated and nonstimulated neutrophils. Incidence of retained placenta (SY=9.4 vs SS=8.6%), fever (SY=47.0 vs SS=44.7%), clinical ketosis (SY=22.1 vs SS=22.3%), displacement of abomasum (SY=2.5 vs SS=3.8%), and mastitis (SY=27.3 vs SS=25.1%) were not affected (P > 0.10) by source of supplemental Se, but a greater (P < 0.01) proportion of cows fed SY experienced acute metritis (21.3 vs 13.4%). Source of Se did not influence health or immunological status of periparturient dairy cows. [ABSTRACT FROM AUTHOR]