Short communication: Validation of a novel milk progesterone-based tool to monitor luteolysis in dairy cows using cost-effective,on-farm measured data

The progesterone (P4) monitoring algorithm using synergistic control (PMASC) uses luteal dynamics to identify fertility events in dairy cows. This algorithm employs a combination of mathematical functions describing the increasing and decreasing P4 concentrations during the development and regression of the corpus luteum and a statistical control chart that allows identification of luteolysis. The mathematical model combines sigmoidal functions from which the cycle characteristics can be calculated. Both the moment at which luteolysis is detected and confirmed by PMASC, as well as the model features themselves, can be used to inform the farmer on the fertility status of the cows.     

Atypical progesterone profiles and fertility in Swedish dairy cows

The incidence of normal and atypical progesterone profiles in Swedish dairy cows was studied. Data were collected from an experimental herd over 15 yr, and included 1,049 postpartum periods from 183 Swedish Holstein and 326 Swedish Red and White dairy cows. Milk progesterone samples were taken twice weekly until initiation of cyclical ovarian activity and less frequently thereafter. Progesterone profiles were 1) normal profile: first rise in milk progesterone above the threshold value before d 56 postpartum, followed by regular cyclical ovarian activity (70.4%); 2) delayed onset of cyclical ovarian activity: low milk progesterone the first 56 d postpartum (15.6%); 3) cessation of cyclical ovarian activity: ovarian activity resumed within 56 d postpartum, but ceased for a period of 14 d or more (6.6%); and 4) prolonged luteal phase: ovarian activity resumed within 56 d postpartum, but milk progesterone remained elevated in the nonpregnant cow for a period of 20 d or more (7.3%). Swedish Holsteins had 1.5 times higher risk of atypical profile than Swedish Red and Whites. Risk of atypical profiles was 0.5 and 0.7 times lower for older cows compared with first-parity cows; 2.3 times higher for cows in tie-stalls compared with those in loose housing; 2.6 times higher for cows calving during winter compared with summer; 0.5 times lower for cows in earlier (1994-1999) calving-year groups compared with the most recent (2000-2002); 2.5 times higher for cows with planned extended calving interval compared with conventional calving interval; and 2.2 times higher for an atypical profile in previous lactation compared with a normal profile. Cows with atypical profiles had a 15-d increase in interval from calving to first artificial insemination and an 18-d increase in interval from calving to conception. Progesterone samples taken within the first 60 d postpartum were used to calculate the percentage of samples above the threshold value of luteal activity. This measure had a significantly different mean in profiles and can be used to separate delayed onset of cyclical ovarian activity profiles and prolonged luteal phase profiles from normal. Thereby, it may be a more effective tool than measurements based only on the onset of ovarian cyclical activity in genetic evaluation of early postpartum fertility in dairy cows.     

Short communication: Effects of the progesterone receptor variants on fertility traits in cattle

The progesterone receptor (PGR) gene is a key factor in the initiation and maintenance of pregnancy and in embryo development. Currently, it is unknown what variants of the PGR gene are related to fertility traits in cattle. Identification of such variants would allow the implementation of marker-assisted selection in breeding schemes. The objective of this study was to investigate the association of single nucleotide polymorphisms (SNP) of PGR with fertility traits in Holstein dairy cattle. An in vitro fertilization system was used to maximize the efficiency of the identification of genetic factors affecting fertility. This in vitro fertilization system would allow the assessment of fertilization and embryonic survival rates independently of influences from the uterine environment. A total of 5,566 fertilization attempts were performed, and a total of 3,679 embryos were produced using oocytes from 324 Holstein cows and semen from 10 Holstein bulls. Sequencing of pooled DNA samples from ovaries revealed an SNP (G/C) in intron 3 of PGR. A generalized linear model was used to analyze the association of this SNP with fertilization and embryonic survival rates for each ovary. Oocytes obtained from CC ovaries showed a 61% fertilization rate, compared with 68 and 69% for GC and GG ovaries, respectively. The survival rate of embryos produced from GG ovaries was 5 and 6% higher than that of GC and CC ovaries. These results indicate that the PGR SNP could be used in marker-assisted selection breeding programs in Holstein dairy cattle.     

Characterization of progesterone profiles in fall-calving Norwegian Red cows

Progesterone profiles in Norwegian Red cows were categorized, and associations between the occurrence of irregularities in the profiles and the commencement of luteal activity were investigated. The cows were managed in 3 feeding trials from 1994 to 2001 and from 2005 to 2008 at the Norwegian University of Life Sciences. The cows were followed from calving, and the milk samples collected represented 502 lactations from 302 cows. Milk samples for progesterone analysis were taken 3 times weekly from 1994 throughout 1998 and from 2005 to 2008 and 2 times weekly from 1999 to 2001. Commencement of luteal activity was defined as the first day of 2 consecutive measurements of progesterone concentration ≥3 ng/mL not earlier than 10 d after calving. Delayed ovulation type I was defined as consistently low progesterone concentration, <3 ng/mL for ≥50 d postpartum. Delayed ovulation type II was defined as prolonged interluteal interval with milk progesterone measurements <3 ng/mL for ≥12 d between 2 luteal phases. Persistent corpus luteum (PCL) type I was defined as delayed luteolysis with milk progesterone ≥3 ng/mL for ≥19 d during the first estrous cycle postpartum. Persistent corpus luteum type II was defined as delayed luteolysis with milk progesterone ≥3 ng/mL for ≥19 d during subsequent estrous cycles before first artificial insemination. Delayed ovulation type I was present in 14.7%, delayed ovulation type II in 2.8%, PCL type I in 6.7%, and PCL type II in 3.3% of the profiles. Commencement of luteal activity was related to milk yield, parity, PCL type I, and the summated occurrence of PCL type I and II. The least squares means for the interval to commencement of luteal activity were 24.2 d when PCL type I and II were present and 29.5 d when PCL type I and II were absent. The likelihood of pregnancy to first service was not affected in cows with a history of PCL when artificial insemination was carried out at progesterone concentrations <3 ng/mL (i.e., during estrus); however, cows that had experienced PCL were more likely to be inseminated during a luteal phase. The occurrence of delayed ovulation and PCL in Norwegian Red cows was less than that reported in most other dairy populations.     

Relationships among body condition score,milk constituents,and postpartum luteal function in Norwegian dairy cows

Relationships among body condition score (BCS), milk constituents, and resumption of postpartum luteal function were studied in 162 lactations of first- and second-parity Norwegian dairy cows. Milk components included acetone, lactose, fat, protein, urea, and ratios of fat to protein and fat to lactose. Milk progesterone concentrations were used to determine intervals from calving to first luteal response (> 5 ng/ml). Intervals to first luteal response were divided into categories of early (< or = 24 d) or late (> 24 d) responses. Higher BCS were observed during wk 4 and 5 postpartum among both first- and second-parity cows with early compared with delayed luteal responses. Second-parity cows with early onset of luteal function also had higher BCS from wk 6 through 12, whereas first-parity cows with early onset of luteal function had higher BCS from wk 13 through 15. Higher acetone levels from wk 2 through 4 postpartum were associated with late luteal response in second-parity cows. Greater milk lactose content during wk 1, 2, 3, 6, 7, and 8 postpartum and higher fat fractions during wk 4 postpartum were related to early luteal response in second-parity cows. Relationships between milk constituents and onset of luteal function were less evident and occurred later postpartum among first-parity cows than among second-parity cows. Measures of weekly milk composites obtained during the early postpartum period and BCS were closely associated with postpartum resumption of luteal function. Acetone and lactose values in milk from the first 4 wk postpartum predicted postpartum luteal function in second-parity cows at a sensitivity of 0.84 and specificity of 0.86.     

Concentrations of progesterone in milk of cows administered an intravaginal progesterone insert

Milk from pregnant cows contains concentrations of progesterone (P4) considered safe for human consumption. The objective of this study was to determine if concentrations of P4 in milk during administration of an intravaginal progesterone insert (CIDR insert) are less than concentrations of P4 in milk associated with pregnancy. Results have implications for human use of milk from cows receiving CIDR inserts. Holstein cows (N = 64; > 40 and < 150 d after calving) were administered 25 mg of PGF2alpha i.m. (study d 0) and 20 cows detected in estrus from 2 to 4 d later were assigned randomly to either control (N = 10; no further treatment) or CIDR insert (N = 10; 1.38 g of P4) inserted on study d 17 (14 +/- 1 d after estrus) and removed 7 d later. Composite milk samples were collected contemporaneously from each of the 20 estrous cycling cows and from 10 pregnant cows (> or = 60 and < or = 220 d of gestation) twice daily from study d 17 to 27. Concentrations of P4 in defatted milk samples were quantified using a validated radioimmunoassay. Mean logs of areas under the curve of concentrations of P4 from the afternoon on study d 17 through the afternoon on study d 27 were 3.05 ng day/ml for control, 3.33 ng day/ml for CIDR insert, and 3.81 ng day/ml for pregnant cows. Therefore, increased P4 due to pregnancy was 0.76 ng day/ml (3.81-3.05), whereas the increase in P4 due to CIDR insert was only 0.28 ng day/ml (3.33-3.05). Applying a 95% confidence interval to 0.28 ng day/ml provided an upper value of 0.70 ng day/ml, lower than the increase due to pregnancy. Because milk from pregnant cows is considered safe for human consumption, it follows that milk from cows administered CIDR inserts should also be considered safe, based on concentrations of P4.     

Genetic analysis of atypical progesterone profiles in Holstein-Friesian cows from experimental research herds

The objective of this study was to quantify the genetic variation in normal and atypical progesterone profiles and investigate if this information could be useful in an improved genetic evaluation for fertility for dairy cows. The phenotypes derived from normal profiles included cycle length traits, including commencement of luteal activity (C-LA), interluteal interval, luteal phase length. and interovulatory interval. In total, 44,977 progesterone test-day records were available from 1,612 lactations on 1,122 primiparous and multiparous Holstein-Friesian cows from Ireland, the Netherlands, Sweden, and the United Kingdom. The atypical progesterone profiles studied were delayed cyclicity, prolonged luteal phase, and cessation of cyclicity. Variance components for the atypical progesterone profiles were estimated using a sire linear mixed model, whereas an animal linear mixed model was used to estimate variance components for the cycle length traits. Heritability was moderate for delayed cyclicity (0.24 ± 0.05) and C-LA (0.18 ± 0.04) but low for prolonged luteal phase (0.02 ± 0.04), luteal phase length (0.08 ± 0.05), interluteal interval (0.08 ± 0.14), and interovulatory interval (0.03 ± 0.04). No genetic variation was detected for cessation of cyclicity. Commencement of luteal activity, luteal phase length, and interovulatory interval were moderately to strongly genetically correlated with days from calving to first service (0.35 ± 0.12, 0.25 ± 0.14, and 0.76 ± 0.24, respectively). Delayed cyclicity and C-LA are traits that can be important in both genetic evaluations and management of fertility to detect (earlier) cows at risk of compromised fertility. Delayed cyclicity and C-LA were both strongly genetically correlated with milk yield in early lactation (0.57 ± 0.14 and 0.45 ± 0.09, respectively), which may imply deterioration in these traits with selection for greater milk yield without cognizance of other traits.     

Estimating probability of insemination success using milk progesterone measurements

The aim of this study was to quantify the effects of progesterone profile features and other cow-level factors on insemination success to provide a real-time predictor equation of probability of insemination success. Progesterone profiles from 26 dairy herds were analyzed and the effects of profile features (progesterone slope, cycle length, and cycle height) and cow traits (milk yield, parity, insemination during the previous estrus) on likelihood of artificial insemination success were estimated. The equation was fitted on a training data set containing data from 16 herds (6,246 estrous cycles from 3,404 lactations). The equation was tested on a testing data set containing data from 10 herds (8,105 estrous cycles from 3,038 lactations). Predictors were selected to be implemented in the final equation if adding them to a base model correcting for timing of insemination and parity decreased the overall likelihood distance of the model. Selected variables (cycle length, milk yield, cycle height, and insemination during the previous estrus) were used to build the final model using a stepwise approach. Predictors were added 1 by 1 in different order, and the model that had the smallest likelihood distance was selected. The final equation included the variables timing of insemination, parity, milk yield, cycle length, cycle height, and insemination during the previous estrus, respectively. The final model was applied to the testing data set and area under the curve (AUC) was calculated. On the testing data set, the final model had an AUC of 58%. When the farm effect was taken into account, the AUC increased to 63%. This equation can be implemented on farms that monitor progesterone and can support the farmer in deciding when to inseminate a cow. This can be the first step in moving the focus away from the current paradigm associated with poorer estrus detection, where each detected estrus is automatically inseminated, to near perfect estrus detection, where the question is which estrous cycle is worth inseminating?     

The effect of presynchronization with prostaglandin F2α and gonadotropin-releasing hormone simultaneously, 7 d before Ovsynch,compared with Presynch-10/Ovsynch on luteal function and first-service pregnancies per artificial insemination

Pregnancy per artificial insemination (P/AI) following Ovsynch is optimized when cows ovulate to the first GnRH of Ovsynch. Fertility programs are designed to presynchronize cows to d 6 or 7 of the estrous cycle to increase the chances of ovulation of a first-wave dominant follicle to the first GnRH of Ovsynch. The hypothesis of this experiment was that simplification of a presynchronization program through the combination of PGF_2α and GnRH on the same day, 7 d before Ovsynch, would allow for similar P/AI compared with Presynch-10. Lactating dairy cows (n = 432) 41 to 47 d in milk (DIM) were randomly assigned to 2 treatments within parities for first service. Control cows received Presynch-10/Ovsynch consisting of the following: PGF_2α–14 d–PGF_2α–10 d–GnRH–7 d–PGF_2α–56 h–GnRH–16 h–AI. Treated cows received PGF_2α and GnRH–7 d–GnRH–7 d–PGF_2α–56 h–GnRH–16 h–AI. All cows received a supplemental injection of PGF_2α 24 h after the PGF_2α of Ovsynch to enhance complete luteolysis. All cows received timed AI between 75 and 81 DIM. Blood was collected to assess circulating concentrations of progesterone (P4), and the number and size of corpora lutea (CL) were recorded using ultrasonography on day of PGF_2α of Ovsynch. The administration of PGF_2α simultaneously with GnRH and 7 d before Ovsynch (PG+G) had similar P/AI at 28 (46 vs. 48%), 35 (43 vs. 43%), 49 (39 vs. 39%), and 77 d post-AI (38 vs. 39%) compared with Presynch-10. No differences were observed in P/AI in primiparous versus multiparous cows at 28 (52 vs. 45%), 35 (48 vs. 41%), 49 (45 vs. 37%), and 77 d post-AI (43 vs. 36%). No difference existed between treatments in percentage of cows with functional CL at PGF_2α of Ovsynch, total luteal area (mm²), or serum concentrations of P4 at time of PGF_2α of Ovsynch, regardless of parity. Number of CL had a tendency to be greater for multiparous PG+G vs. Presynch-10 cows (2.34 ± 0.09 vs. 2.15 ± 0.08) but not in primiparous cows (1.95 ± 0.10 vs. 1.98 ± 0.11). In summary, administering both PGF_2α and GnRH on the same day, 7 d before the start of Ovsynch, appears to be a simple and effective alternative to Presynch-10 Ovsynch.     

Infrequent milk progesterone measurements in daughters enable bull selection for cow fertility

The interval from calving to first luteal activity (CLA) has been suggested as an unbiased and, therefore, preferable measure for selection on female fertility in dairy cattle. However, measurement of this interval for individual cows is not feasible for reasons of cost and labor associated with the necessary frequent (milk) progesterone measurements. The objective of this study was to test the hypothesis that mean sire progesterone profiles based on individual progesterone measurements of daughters at 3- to 6-wk intervals have prospects as a measure for female fertility when selecting sires in a progeny testing scheme. In this study, progesterone concentrations were measured in milk samples collected at routinely performed milk recordings during the first 100 d of lactation of daughters of 20 test bulls. It is demonstrated that a) mean progesterone profiles can be used to calculate the earliest stage of lactation at which at least 50% of the daughters of a test bull has a milk progesterone level >3 ng/mL (indicating luteal activity) and that b) this stage, at which 50% of the daughters of a bull have an active corpus luteum (CLA50%), varies largely between test bulls. We conclude that selecting sires based on daughter CLA50% may improve female fertility.     

Timed artificial insemination programs during the summer in lactating dairy cows: Comparison of the 5-d Cosynch protocol with an estrogen/progesterone-based protocol

The objective of this study was to compare a GnRH-based to an estrogen/progesterone (E2/P4)-based protocol for estrous cycle synchronization and fixed timed artificial insemination (TAI), both designed for synchronization of ovulation and to reduce the period from follicular emergence until ovulation in cows with a synchronized follicular wave. A total of 1,190 lactating Holstein cows (primiparous: n = 685 and multiparous: n = 505) yielding 26.5 ± 0.30 kg of milk/d at 177 ± 5.02 d in milk were randomly assigned to receive one of the following programs: 5-d Cosynch protocol [d −8: controlled internal drug release (CIDR) + GnRH; d −3: CIDR removal + PGF_2α; d −2: PGF_2α; d 0: TAI + GnRH] or E2/P4 protocol (d −10: CIDR + estradiol benzoate; d −3: PGF_2α; d −2: CIDR removal + estradiol cypionate; d 0: TAI). Rectal temperature and circulating progesterone (P4) were measured on d −3, −2, 0 (TAI), and 7. The estrous cycle was considered to be synchronized when P4 was ≥1.0 ng/mL on d 7 in cows that had luteolysis (P4 ≤0.4 ng/mL on d 0). To evaluate the effects of heat stress, cows were classified by number of heat stress events: 0, 1, and 2-or-more measurements of elevated body temperature (≥39.1°C). Pregnancy success (pregnancy per artificial insemination, P/AI) was determined at d 32 and 60 after TAI. The cows in the 5-d Cosynch protocol had increased circulating P4 at the time of PGF_2α injection (2.66 ± 0.13 vs. 1.66 ± 0.13 ng/mL). The cows in the E2/P4 protocol were more likely to be detected in estrus (62.8 vs. 43.4%) compared with the cows in the 5-d Cosynch protocol, and expression of estrus improved P/AI in both treatments. The cows in the 5-d Cosynch protocol had greater percentage of synchronized estrous cycle (78.2%), compared with cows in the E2/P4 protocol (70.7%). On d 60, the E2/P4 protocol tended to improve P/AI (20.7 vs. 16.7%) and reduced pregnancy loss from 32 to 60 d (11.0 vs. 19.6%), compared with the 5-d Cosynch protocol. In cows withtheir estrous cycle synchronized, the E2/P4 protocol had greater P/AI (25.6 vs. 17.7%) on d 60 and lower pregnancy loss from 32 to 60 d (6.7 vs. 21.7%) compared with cows in the 5-d Cosynch protocol. Follicle diameter affected pregnancy loss from 32 to 60 d only in the cows in the 5-d Cosynch protocol, with smaller follicles resulting in greater pregnancy loss. Pregnancy per AI at d 60 was different between protocols in the cows with 2 or more measurements of heat stress (5-d Cosynch = 12.2% vs. E2/P4 = 22.8%), but not in the cows without or with 1 heat stress measurement. In conclusion, the 5-d Cosynch protocol apparently produced better estrous cycle synchronization than the E2/P4 protocol but did not improve P/AI. The potential explanation for these results is that increased E2 concentrations during the periovulatory period can improve pregnancy success and pregnancy maintenance, and this effect appears to be greatest in heat-stressed cows when circulating E2 may be reduced.     

Genetic analysis of new progesterone-based fertility traits in dairy cows measured on-farm

The objectives of this study were (1) to analyze the agreement of a standard laboratory ELISA for progesterone (P4) with an automated on-farm ELISA kit operated under commercial conditions in 1,297 milk samples from 50 dairy cows; (2) to study the influence of the method of detection of luteal activity on genetic parameters of fertility traits based on P4 measured with an automated on-farm ELISA once weekly from wk 3 to 9 postpartum in the milk of 1,304 cows; and (3) to study the influence of sampling frequency (once or twice weekly from wk 3 to 9) on the same traits from 296 cows. Luteal activity can be detected when there is an active corpus luteum in the ovary producing P4 and indicating the onset of reproductive cyclicity after calving. The on-farm ELISA overestimated P4 contents by a mean square error of prediction of 2.76 ng/mL and had an intermediate Spearman correlation with the laboratory kit (0.54). For the second objective, the postpartum interval to the commencement of luteal activity (C-LA), proportion of luteal activity between d 15 and 63 postpartum (P-LA), calculated as the number of samples above the threshold for high P4 values divided by the number of all samples, and delay of first ovulation (DOV1), defined as C-LA occurring later than d 45 postpartum, were derived from the P4 profiles. Both C-LA and DOV1 were determined by (a) thorough qualitative visual inspection of the profile, (b) the profile's mean as threshold for the first increase in P4 postpartum, indicating commencement of luteal activity, and (c) 3 ng/mL as threshold for the first increase in P4, a value that has been used by many other studies. Similarly, P-LA was determined by using methods (b) and (c). Estimates of heritability were 0.04 to 0.13 for C-LA, 0.12 to 0.23 for P-LA, and 0.03 to 0.07 for DOV1. Genetic correlation of P-LA with C-LA and with the profile's mean P4 was ?1.00. The profile's mean had a higher estimate of heritability (0.11–0.12) than C-LA or DOV1. It can be calculated as the arithmetic mean of all P4 values of a profile, whereas C-LA, P-LA, and DOV1 need a definition of a threshold for high P4 values. We therefore suggest the profile's mean as a promising candidate for further research. For the third objective, once-weekly sampling was mimicked by neglecting every second sample, and C-LA and DOV1 shifted toward a later onset of cyclicity. Thus, a common standard for sampling regimen and detection algorithm is essential to avoid incompatibility between studies.     

Diurnal variations of progesterone,testosterone, and androsta-1,4-diene-3,17-dione in the rumen and in vitro progesterone transformation by mixed rumen microorganisms of lactating dairy cows

Five Holstein lactating dairy cows fed 5 total mixed rations (TMR) with different forage combinations were used in a 5 × 5 Latin square design to investigate diurnal variations of progesterone (P4), testosterone, and androsta-1,4-diene-3,17-dione (ADD) concentrations in the rumen. Meanwhile, different P4 inclusion levels [0 (control), 2, 20, 40, 80, and 100 ng/mL in culture fluids] were incubated in vitro for 6, 12, 24, 36, 48, and 72 h together with rumen mixed microorganisms grown on a maize-rich feed mixture (maize meal:Chinese ryegrass hay = :1) with an aim to determine microbial P4 transformation into testosterone and ADD. Ruminal P4, testosterone, and ADD concentrations of lactating dairy cows were greater in the TMR with forage combination of corn silage plus alfalfa hay or Chinese wild ryegrass hay than the TMR with the corn stover-based forage combination. The diurnal fluctuation pattern showed that P4, testosterone, and ADD concentrations in the rumen were greater at nighttime than daytime and peaked 6 h after feeding in the morning or afternoon. The in vitro batch cultures showed that the P4 elimination rate was highest at the P4 addition of 20 ng/mL and declined with the further increased addition of P4. The treatments after dosing P4 exhibited a shorter time than the control group until half of the initial P4 inclusion was eliminated (i.e., half time), and the lowest half time (1.46 h) occurred at the P4 addition of 20 ng/mL. In summary, the ruminal steroids concentration was affected by forage type and quality, and the rumen microorganisms exhibited great ability to transform P4 into testosterone and ADD, depending on incubation time and initial P4 addition level, suggesting that the host might affect the metabolism of its rumen microorganisms via the endogenous steroids.     

Genome-wide association study for age at puberty and resumption of cyclicity in a crossbred dairy cattle population

Fertility is of primary economic importance in dairy cattle and the most common reason for involuntary culling. However, standard fertility traits have very low heritability that renders genetic selection slow and difficult. In this study, we explored fertility from an endocrine standpoint. A total of 1,163 crossbred Holstein-Normande females in a 3-generation familial design were studied for progesterone level measured every 10 d to determine age at puberty (PUB) and commencement of postpartum luteal activity (CPLA). Genetic parameters were estimated using REML with WOMBAT software. The heritability estimates were 0.38 ± 0.10 and 0.16 ± 0.07 for PUB and CPLA, respectively. Moreover, the 2 traits were genetically correlated (0.45 ± 0.23), suggesting a partially common determinism. Because of the family structure, a linkage disequilibrium and linkage analysis approach was preferred over standard genome-wide association study to map genomic regions associated with these traits. Ten quantitative trait loci (QTL) were detected for PUB on chromosomes 1, 3, 11, 13, 14, 21, and 29, whereas 3 QTL were associated with CPLA on chromosomes 21 and 26. Only the QTL on chromosome 21 was common to both traits. Four functional candidate genes (NCOA2, GAS2, OVOL1, and FOSL1) were identified in the detected regions. These findings will contribute to a clearer understanding of fertility determinism and enhance the value of introducing endocrinological data in fertility studies.     

The creation and evaluation of a model predicting the probability of conception in seasonal-calving,pasture-based dairy cows

Reproductive performance in pasture-based production systems has a fundamentally important effect on economic efficiency. The individual factors affecting the probability of submission and conception are multifaceted and have been extensively researched. The present study analyzed some of these factors in relation to service-level probability of conception in seasonal-calving pasture-based dairy cows to develop a predictive model of conception. Data relating to 2,966 services from 737 cows on 2 research farms were used for model development and data from 9 commercial dairy farms were used for model testing, comprising 4,212 services from 1,471 cows. The data spanned a 15-yr period and originated from seasonal-calving pasture-based dairy herds in Ireland. The calving season for the study herds extended from January to June, with peak calving in February and March. A base mixed-effects logistic regression model was created using a stepwise model-building strategy and incorporated parity, days in milk, interservice interval, calving difficulty, and predicted transmitting abilities for calving interval and milk production traits. To attempt to further improve the predictive capability of the model, the addition of effects that were not statistically significant was considered, resulting in a final model composed of the base model with the inclusion of BCS at service. The models' predictions were evaluated using discrimination to measure their ability to correctly classify positive and negative cases. Precision, recall, F-score, and area under the receiver operating characteristic curve (AUC) were calculated. Calibration tests measured the accuracy of the predicted probabilities. These included tests of overall goodness-of-fit, bias, and calibration error. Both models performed better than using the population average probability of conception. Neither of the models showed high levels of discrimination (base model AUC 0.61, final model AUC 0.62), possibly because of the narrow central range of conception rates in the study herds. The final model was found to reliably predict the probability of conception without bias when evaluated against the full external data set, with a mean absolute calibration error of 2.4%. The chosen model could be used to support a farmer's decision-making and in stochastic simulation of fertility in seasonal-calving pasture-based dairy cows.     

A novel system for on-farm fertility monitoring based on milk progesterone

Timely identification of a cow's reproduction status is essential to minimize fertility-related losses on dairy farms. This includes optimal estrus detection, pregnancy diagnosis, and the timely recognition of early embryonic death and ovarian problems. On-farm milk progesterone (P4) analysis can indicate all of these fertility events simultaneously. However, milk P4 measurements are subject to a large variability both in terms of measurement errors and absolute values between cycles. The objective of this paper is to present a newly developed methodology for detecting luteolysis preceding estrus and give an indication of its on-farm use. The innovative monitoring system presented is based on milk P4 using the principles of synergistic control. Instead of using filtering techniques and fixed thresholds, the present system employs an individually on-line updated model to describe the P4 profile, combined with a statistical process control chart to identify the cow's fertility status. The inputs for the latter are the residuals of the on-line updated model, corrected for the concentration-dependent variability that is typical for milk P4 measurements. To show its possible use, the system was validated on the P4 profiles of 38 dairy cows. The positive predictive value for luteolysis followed by estrus was 100%, meaning that the monitoring system picked up all estrous periods identified by the experts. Pregnancy or embryonic mortality was characterized by the absence or detection of luteolysis following an insemination, respectively. For 13 cows, no luteolysis was detected by the system within the 25 to 32 d after insemination, indicating pregnancy, which was confirmed later by rectal palpation. It was also shown that the system is able to cope with deviating P4 profiles having prolonged follicular or luteal phases, which may suggest the occurrence of cysts. Future research is recommended for optimizing sampling frequency, predicting the optimal insemination window, and establishing rules to detect problems based on deviating P4 patterns.     

Short communication: Validation of a test-day milk test for β-hydroxybutyrate for identifying cows with hyperketonemia

Subclinical ketosis is a common metabolic disorder affecting dairy cattle that results in a greater risk for the development of subsequent metabolic and infectious disease. Canwest Dairy Herd Improvement (DHI; Guelph, ON, Canada) has begun to use an infrared test (MilkoScan FT600, Foss Analytical A/S, Hillerød, Denmark) applied to metered composite milk samples to detect β-hydroxybutyrate (BHB) levels as a herd surveillance test for hyperketonemia. However, the test has not been compared with the gold standard, serum BHB as determined in a reference diagnostic laboratory. The objective of this cross-sectional diagnostic accuracy study was to validate the DHI milk BHB test to identify cows with hyperketonemia as determined by quantification of BHB in serum. A total of 316 cows from 17 dairy herds in southwestern Ontario had a milk and blood sample taken. Milk was collected at a routine DHI test, and blood from the same cow was sampled within 24 h of the milk test. The BHB concentration in milk was determined using the DHI milk BHB test, and serum was sent to the Animal Health Laboratory at the University of Guelph (Guelph, ON, Canada). A nonparametric receiver operating characteristic curve was generated to compare DHI milk BHB concentrations with serum BHB concentrations. Overall, a total of 34 cows (11%) had a level of serum BHB ≥1.2 mmol/L. The concentration of DHI milk BHB was moderately correlated with the concentration of serum BHB, yielding a coefficient of determination value of 0.61. The optimal cut point for determining hyperketonemia (≥1.2 mmol/L) on the DHI milk BHB test was ≥0.14 mmol/L, yielding a sensitivity of 81% and specificity of 92%. The performance of the DHI milk BHB test varied depending on the days in milk (DIM) of the cows tested, with a higher specificity being found in cows that were ≤25 DIM compared with cows tested >25 DIM. If the herd-level prevalence of hyperketonemia was ≥14%, the DHI milk BHB test had an improved sensitivity when compared with a herd-level prevalence of <14%. This study demonstrates that the DHI milk BHB test is a reliable measure for evaluating hyperketonemia using routine DHI milk samples and could be used as a herd-level monitoring tool for ketosis when evaluating nutritional management or preventative medicine strategies.     

Technical note: Direct enzyme immunoassay of progesterone in bovine milk whey

A simple extraction-free or direct quantitative ELISA for progesterone in bovine milk whey was developed. Whey samples are easy to collect, transport, and store. This method also allows for monitoring progesterone levels in cattle, which is important in reproductive management. The assay was designed to cover the concentration range 0.05 to 2 ng/mL, and the sensitivity of the method was 1.5 pg/mL. The intra- and interassay coefficients of variation were 8 and 12%, respectively. A high correlation (r = 0.90) between ELISA and radioimmunoassay measurements of progesterone in the same milk whey samples was obtained. The method can be easily applied in practice because samples can be stored at room temperature (22 to 26°C) for 4 d. Moreover, because analysis requires milk coagulation, that process can be initiated during transport by standard mail services to the laboratory. Upon arrival at the laboratory, whey can be kept refrigerated for 1 wk before analysis. This tool is useful for monitoring luteal activity of dairy cows.     

Concentration of progesterone during the development of the ovulatory follicle: II. Ovarian and uterine responses

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. 17.2 mm) 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.     

Resynchronization strategies to improve fertility in lactating dairy cows utilizing a presynchronization injection of GnRH or supplemental progesterone: I. Pregnancy rates and ovarian responses

Objectives were to evaluate 3 resynchronization protocols for lactating dairy cows. At 32 ± 3 d after pre-enrollment artificial insemination (AI; study d −7), 1 wk before pregnancy diagnosis, cows from 2 farms were enrolled and randomly assigned to 1 of 3 resynchronization protocols after balancing for parity, days in milk, and number of previous AI. All cows were examined for pregnancy at 39 ± 3 d after pre-enrollment AI (study d 0). Cows enrolled as controls (n = 386) diagnosed not pregnant were submitted to a resynchronization protocol (d 0-GnRH, d 7-PGF_2α, and d 10-GnRH and AI) on the same day. Cows enrolled in the GGPG (GnRH-GnRH-PGF_2α-GnRH) treatment (n = 357) received a GnRH injection at enrollment (d −7) and if diagnosed not pregnant were submitted to the resynchronization protocol for control cows on d 0. Cows enrolled in CIDR treatment (n = 316) diagnosed not pregnant received the resynchronization protocol described for control cows with addition of a controlled internal drug release (CIDR) insert containing progesterone (P₄) from d 0 to 7. In a subgroup of cows, ovaries were scanned and blood was sampled for P₄ concentration on d 0 and 7. After resynchronized AI, cows were diagnosed for pregnancy at 39 ± 3 and 67 ± 3 d (California herds) or 120 ± 3 d (Arizona herds). Cows in the GGPG treatment had more corpora lutea than CIDR and control cows on d 0 (1.30 ± 0.11, 1.05 ± 0.11, and 1.05 ± 0.11, respectively) and d 7 (1.41 ± 0.14, 0.97 ± 0.13, and 1.03 ± 0.14, respectively). A greater percentage of GGPG cows ovulated to GnRH given on d 0 compared with CIDR and control cows (48.4, 29.6, and 36.6%, respectively), but CIDR and control did not differ. At 39 ± 3 d after resynchronized AI, pregnancy per AI (P/AI) was increased in GGPG (33.6%) and CIDR (31.3%) cows compared with control (24.6%) cows. At 67 or 120 ± 3 d after resynchronized AI, P/AI of GGPG and CIDR cows was increased compared with control cows (31.2, 29.5, and 22.1%, respectively). Presynchronizing the estrous cycle of lactating dairy cows with a GnRH 7 d before the start of the resynchronization protocol or use of a CIDR insert within the resynchronization protocol resulted in greater P/AI after resynchronized AI compared with control cows.