Assessment of systemic inflammation following oral calcium supplementation in healthy postpartum multiparous dairy cows-A randomized controlled trial.

Around parturition, dairy cows inevitably and perhaps necessarily experience some degree of systemic inflammation, but when excessive or dysregulated, it may contribute to health disorders. As immune activation decreases blood Ca, greater extracellular Ca availability may potentiate or sustain inflammation. We hypothesized that in clinically healthy multiparous cows, postpartum administration of supplemental Ca would increase serum concentrations of inflammatory markers. The objective of this randomized controlled trial was to investigate a possible effect of supplementing calcium (Ca) on postpartum systemic inflammation in dairy cows. Healthy cows (n = 101) from 2 commercial dairy farms in Ontario calving into parity 2, 3, or 4 were enrolled. Cows were blocked by parity and randomly assigned to receive an oral bolus of Ca (42 g of Ca) within 12 h after calving and a second bolus 12 h later (TRT; n = 51), or no Ca supplementation (CON; n = 49). Concentrations in serum of total Ca (tCa), haptoglobin (Hp), and albumin (ALB) were assessed at d 0 (within 12 h postpartum), 0.5 (12 h later), 1, 2, 4, 6, and 8 postpartum; ionized calcium was assessed at d 0, 0.5, 2, and 4, and lipopolysaccharide-binding protein (LBP) and serum amyloid A (SAA) were assessed at d 0, 2, and 4. Multivariable linear regression models of each outcome accounting for repeated measures included treatment, parity (2 vs. 3 or 4), farm, sampling day, baseline concentration (d 0), and interactions of treatment with farm, parity, and day. Results are presented as least squares means and 95% confidence intervals. Concentration of tCa tended to be greater at d 0.5 (TRT 2.07 mmol/L [2.03-2.12]; CON 2.01 [1.96-2.06]) but was lesser at d 2 (TRT 2.18 [2.13-2.23]; CON 2.27 [2.23-2.32]) in TRT than CON cows. Concentrations of LBP were greater in TRT (2.28 ng/mL [2.06-2.50]) than CON (1.99 [1.77-2.21]) in parity 2, but not different in older cows (TRT 2.28 ng/mL [2.06-2.50]; CON 1.99 [1.77-2.21]). Concentrations of SAA were greater in TRT than CON cows at d 2 (TRT 135 ug/mL [124-146]; CON 114 [75-106]). Treatment had no effect on ALB or Hp. In clinically healthy cows, oral Ca supplementation had a small transient effect on blood tCa and little indication of increasing inflammation based on the analytes evaluated.

Associations of postpartum health with progesterone after insemination and endocrine signaling during early pregnancy in dairy cows.

Our objective was to investigate associations of postpartum health with serum progesterone (P4) concentrations after first artificial insemination (AI), expression of interferon-stimulated genes (ISG) in blood leukocytes, and serum pregnancy-associated glycoprotein (PAG) during early pregnancy in dairy cows. In this observational study, Holstein cows (n = 475) from 2 commercial herds were enrolled 3 wk before expected parturition. Body condition score (BCS) and lameness were assessed at enrollment, and total Ca, haptoglobin, and nonesterified fatty acids (NEFA) were measured in serum at 2 and 6 ± 2 d in milk (DIM). Blood ß-hydroxybutyrate and metritis were evaluated at 4, 8, 11, and 15 ± 2 DIM, and purulent vaginal discharge (PVD) and endometritis based on endometrial cytology were diagnosed at 35 DIM. Onset of cyclicity was assessed by biweekly P4 measurements. The BCS was assessed at enrollment and at 63 DIM, and lameness at enrollment and at 21 and 49 DIM. First AI was based on estrus detection until ∼75 DIM, or synchronization for timed AI thereafter. Serum P4 was measured at d 8 and 12 after first AI; expression of ISG (ISG15 and RTP4) in blood leukocytes was assessed at d 19; and PAG in serum of pregnant cows was measured at d 29, 33, and 40. Multivariable linear regression models were built including health variables (with metabolites categorized via receiver operating characteristic curve analysis) and covariates (season, milk yield, AI method, DIM), accounting for pregnancy status (for ISG and PAG models), repeated measures (for P4 and PAG models), and herd as random. Results are reported comparing cows affected versus unaffected by each predictor. Serum P4 concentrations were lesser at d 8 after AI in cows that had total Ca ≤2.09 mM (3.6 vs. 4.0 ± 0.2 ng/mL) at 2 DIM or a clinical disease (3.7 vs. 4.0 ± 0.2 mg/mL), and P4 tended to be lesser at d 8 in cows that had PVD (3.6 vs. 4.0 ± 0.2 ng/mL) or any uterine disease (3.7 vs. 4.1 ± 0.2 ng/mL). Among cows pregnant at d 29, relative ISG15 expression (fold change) at d 19 was greater in cows that had lameness (5.10 [4.18-6.18] vs. 3.14 [3.10-3.19]) but tended to be lesser in cows that had endometritis (3.20 [2.89-3.54] vs. 4.29 [4.20-4.35]), and relative expression of RTP4 tended to be lesser in cows that had displaced abomasum (1.02 [0.62-1.67] vs. 1.75 [1.73-1.78]). Serum PAG was lesser at d 29 (3.8 vs. 4.2 ± 0.2 sample-to-positive ratio [S:P]) in cows that had NEFA ≥0.73 mM at 6 DIM, and lesser at d 33 (3.9 vs. 4.5 ± 0.2 S:P) and 40 (3.1 vs. 3.7 ± 0.2 S:P) in cows that had prepartum BCS ≥3.75. These findings suggest long-term effects of metabolic or reproductive tract disorders on luteal function after first AI, pregnancy recognition signaling, and placental function during early pregnancy. These associations may partially explain some of the pathways linking postpartum health and fertility in dairy cows.

Associations of inflammatory and reproductive tract disorders postpartum with pregnancy and early pregnancy loss in dairy cows.

Our objective was to describe associations of postpartum health with pregnancy and pregnancy loss (P-LOSS) from d 19 to 40 after first postpartum artificial insemination (AI) in lactating Holstein cows. In 2 commercial dairy herds in Ontario, Canada, 468 Holstein cows were enrolled 21 ± 3 d before expected parturition when body condition score (BCS) and lameness were assessed. Serum total Ca, haptoglobin (Hp), and nonesterified fatty acids (NEFA) were measured at 2 and 6 ± 2 d in milk (DIM). Blood ß-hydroxybutyrate (BHB) measurement and metritis detection were done at 4, 8, 11, and 15 ± 2 DIM. Cows were examined for endometritis (ENDO; ≥11.5% polymorphonuclear cells in endometrial cytology) and purulent vaginal discharge (PVD) at 35 ± 3 DIM. Lameness was assessed again at 21 and 49 ± 3 DIM and BCS at 63 ± 3 DIM. First postpartum AI occurred primarily (86%) based on detection of estrus by activity monitors, on average (± standard deviation) at 65 ± 9 DIM, and the remaining cows received timed AI at 86 ± 18 DIM. Serum progesterone (P4) was measured on d 8 and 12 after AI, and pregnancy at first AI (P/AI) was estimated by the expression of ISG15 in peripheral blood leukocytes at d 19 after AI and by pregnancy-associated glycoprotein in serum at d 29, 33, and 40 after AI. Each metabolite (Ca, Hp, NEFA, and BHB) was categorized above or below a cut-point identified with receiver operating characteristic curve analysis associated with P/AI confirmed by ultrasound at d 33 from a larger data set. Data were analyzed using multivariable mixed logistic regression models, accounting for parity, health variables, covariates (season at calving and at AI, milk yield at first Dairy Herd Improvement Association test [categorized into terciles], AI method, and DIM), and herd. The proportions of cows classified pregnant at d 19, 29, 33, and 40 after AI were 64%, 54%, 50%, and 45%, respectively. At d 19 after AI, P/AI was less likely in cows diagnosed with ENDO (52% vs. 69%) or PVD (54% vs. 67%). At d 29, P/AI was less likely in cows with Hp ≥1.54 g/L at 2 DIM (38% vs. 55%) or PVD (35% vs. 56%). Both metritis and ENDO were associated with decreased P/AI at d 40 after AI. Cows diagnosed with metritis had greater risk of P-LOSS from d 19 to 29 (43% vs. 22%) or from d 33 to 40 (37% vs. 7%) than cows without metritis. From d 29 to 33, the risk of P-LOSS was greater in cows with NEFA ≥0.73 mM at 2 DIM (13% vs. 5%) or BCS ≤2.75 at 63 DIM (14% vs. 5%). The concentration of P4 on d 8 after AI was positively associated with P/AI at d 29, 33, and 40, and negatively associated with P-LOSS from d 19 to 29. Postpartum health disorders, particularly reproductive tract disease, can have detrimental effects on early pregnancy establishment and on pregnancy maintenance from d 19 to 40 after AI.

Postpartum health is associated with detection of estrus by activity monitors and reproductive performance in dairy cows.

The objective of this prospective observational study was to investigate associations of postpartum health with estrus detection (ED) by activity monitors and pregnancy outcomes in dairy cows. A total of 1,743 Holstein cows from 2 commercial dairy herds in Ontario, Canada were enrolled 3 wk before expected parturition and examined for health variables until 9 wk postpartum. Body condition score (BCS) and lameness were measured at 3 wk prepartum, and serum concentrations of total Ca, haptoglobin (Hp), and nonesterified fatty acids were measured at 2 and 6 ± 2 d in milk (DIM), and blood ß-hydroxybutyrate (BHB) and metritis were assessed at 4, 8, 11, and 15 ± 2 DIM. Cows were examined for purulent vaginal discharge (PVD) and endometritis (ENDO) by endometrial cytology at wk 5, for lameness at wk 3 and 7, for BCS at wk 9 postpartum, and for time to onset of cyclicity by biweekly serum progesterone (P4) measurements. Additional disease data were obtained from farm records. Reproductive management for first AI was primarily based on ED by activity monitors until at least 75 DIM, and cows not detected in estrus were synchronized. Data were analyzed in multivariable logistic or Cox proportional hazards regression models including blood markers, health variables, potential covariates, and herd as a random effect. Estrus was detected in 77% of primiparous and 66% of multiparous cows between 50 or 55 DIM and 75 DIM. In 1,246 cows, the model-predicted probability of ED (percentage point difference) was lower in cows that had retained placenta (-14%), ENDO (-7%), PVD (-8%), delayed cyclicity (no P4 > 1 ng/mL by wk 9; -12%), or ≥0.5-point BCS loss (-14%) compared with cows without each of these risk factors, and it was negatively associated with blood BHB at 15 DIM. Considering only variables measured on farm (not requiring laboratory analysis), the probability of ED was lower (56 vs. 81%) in cows with >1 risk factor compared with cows without risk factors. The predicted probability of pregnancy at first artificial insemination (percentage point difference) was lower in cows that had ENDO (-7%) or PVD (-7%), and negatively associated with serum Hp at 6 ± 2 DIM. In cows detected in estrus by 75 DIM (n = 888), risk factors for reduced pregnancy rate by 250 DIM (adjusted hazard ratio (AHR); 95% confidence intervals) included difficult calving (AHR: 0.67; 0.45 to 1.00), metritis (AHR: 0.79; 0.61 to 1.01), PVD (AHR: 0.79; 0.65 to 0.97), or lameness (AHR: 0.79; 0.62 to 1.01), and it was negatively associated with serum Hp at 6 ± 2 DIM. Monitoring postpartum health may be used to identify cows that are more or less likely to be detected in estrus by activity monitors and to become pregnant in a timely manner. This would support a selective reproductive management program with targeted interventions.

Association of metabolic markers with neutrophil function in healthy postpartum dairy cows.

The objective of this study was to evaluate the associations of serum markers for systemic inflammation, liver, mineral, and energy status, and blood neutrophil counts with the function of circulating neutrophils in postpartum dairy cows. Blood samples were collected from 21 healthy Holstein cows at 5, 10, 14, and 21 d postpartum. Serum samples were used to measure concentrations of total calcium, phosphorus, magnesium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, haptoglobin (Hp), ß-hydroxybutyrate, non-esterified fatty acids (NEFA), and insulin-like growth factor-1. The shift of percentage of activated neutrophils for phagocytosis (PPC) and oxidative burst (POB) and the median fluorescence intensity (MFI) for PC (MFIPC), OB (MFIOB), and endocytic and proteolytic degradation measured via DQ-ovalbumin (MFIDQ) were evaluated using flow cytometry. Mixed linear regression models were used to assess the associations of serum concentrations of metabolites and blood neutrophil counts with each neutrophil function assay outcome accounting for d postpartum, parity, and body condition score. Pearson correlation tests (r) were used to describe the relationships of metabolites and neutrophil counts with neutrophil function assays that were significant in the regression models. Greater serum concentrations of Hp were associated with lesser OB function (POB and MFIOB; r = -0.2 for both), but greater PPC and MFIDQ (r = 0.3 for both). Greater serum NEFA and cholesterol were associated with lesser (r = -0.3) and greater (r = 0.2) POB function, respectively. Blood neutrophil counts were associated with PPC (r = -0.2) and MFIDQ (r = -0.3). At 5 d postpartum, greater serum total protein was associated with greater OB function (POB and MFIOB; r = 0.4 for both). Other markers were not associated with neutrophil function. Some metabolic and inflammatory markers in healthy postpartum dairy cows were associated with circulating neutrophil function. However, these associations only explained a small proportion of the variance in neutrophil function. Serum Hp concentration was most associated with neutrophil function changes but had opposite directions of association with OB- and PC-related functions. Future studies should focus on understanding the mechanisms by which Hp and other metabolic indicators affect neutrophil function in healthy and diseased postpartum dairy cows.

Differing planes of pre- and postweaning phase nutrition in Holstein heifers: I. Effects on feed intake, growth efficiency, and metabolic and development indicators.

The objectives of this study were to determine the effects of pre- and postweaning planes of nutrition on feed and metabolizable energy (ME) intake, growth, concentrations of glucose, insulin, insulin-like growth factor 1 (IGF-1), and ß-hydroxybutyrate (BHB) in blood and rumen volatile fatty acids (VFA) from birth to 25 wk of age in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low (5 L/d of whole milk) or high (10 L/d of whole milk) preweaning plane of nutrition from 1 to 7 wk of age, and either a low (70% concentrate dry total mixed ration) or high (85% concentrate dry total mixed ration) postweaning plane of nutrition from 11 to 25 wk of age. From birth to 25 wk of age, feed intake was recorded daily, and body measures were obtained weekly. Circulating hormone and metabolite concentrations were measured biweekly and total ruminal VFA, fecal starch, and body condition were assessed monthly. Overall, average daily gain and body weight were greater for heifers offered increased planes of nutrition during both the pre- and postweaning phases. Heifers offered the high preweaning plane had greater milk intake (7.7 ± 0.1 vs. 4.5 ± 0.1 L/d) but lower starter intake (0.3 ± 0.04 vs. 0.7 ± 0.04 kg/d) during the preweaning phase than those offered the low plane. High preweaning plane heifers also had greater ME intake from wk 1 to 7, but less ME intake at wk 9 (5.3 ± 0.3 vs. 6.6 ± 0.2 Mcal/kg) than those offered the low plane. Furthermore, overall glucose (118.8 ± 2.9 vs. 110.1 ± 2.9 mg/dL) and IGF-1 (101.6 ± 3.6 vs. 75.9 ± 3.6 ng/mL) concentrations were greater for high versus low preweaning plane heifers, although circulating insulin and BHB did not differ between preweaning plane groups. However, heifers offered the high preweaning plane had reduced total rumen VFA concentrations compared with heifers offered the low plane in the preweaning phase (47.3 ± 2.0 vs. 55.6 ± 2.1 mM). During the postweaning phase, dry matter intake and ME were consistently greater in heifers offered the high postweaning plane. Overall insulin (2.0 ± 0.1 vs. 1.8 ± 0.1 ng/mL), glucose (97.1 ± 0.6 vs. 92.1 ± 0.6 ng/mL), IGF-1 (178.0 ± 4.8 vs. 155.8 ± 4.8 ng/mL), and BHB concentrations (8.8 ± 0.2 vs. 8.1 ± 0.2 ng/mL) were greater in heifers offered the high than the low postweaning plane. In addition, heifers offered the high postweaning plane had increased VFA concentrations in the postweaning phase (73.4 ± 1.3 vs. 63.9 ± 1.3 mM) compared with heifers offered the low postweaning plane. The results indicated that increasing the pre- and postweaning planes of nutrition along with energy levels positively influenced several indicators associated with heifer development before 25 wk of age. Nevertheless, there was limited interaction in growth and development indicators between the 2 phases.

Differing planes of pre- and postweaning phase nutrition in Holstein heifers: II. Effects on circulating leptin, luteinizing hormone, and age at puberty.

The objectives were to investigate the effects of differing planes of pre- and postweaning nutrition on prepubertal serum leptin concentrations, LH profiles, and age at puberty in Holstein heifers. Thirty-six Holstein calves were randomly assigned to either a low (5 L) or high (10 L of whole milk/d) preweaning plane of nutrition from 1 to 7 wk of age, a transition diet (a dry total mixed ration with 85% of concentrate) from wk 8 to 10, and to either a low (70%) or high (85% of concentrate dry total mixed ration) postweaning plane from 11 to 25 wk of age. Serum leptin concentrations were measured every 2 wk from 1 to 25 wk of age, and LH profiles were determined both at wk 15 and 25 based on sequential blood samples taken every 12 min over 10 h. Starting at 26 wk of age, ovaries were examined weekly by transrectal ultrasonography until first ovulation (puberty) was confirmed. Heifers that received the high preweaning plane diet had greater mean (± standard error; SE) leptin concentrations during the preweaning phase than those fed the low plane (2.9 ± 0.1 vs. 2.6 ± 0.1 ng/mL). During the postweaning phase, mean circulating leptin was greater in heifers fed the high compared with the low postweaning diet [3.2 ng/mL (95% confidence interval; CI = 2.7 to 3.4) vs. 2.5 ng/mL (95% CI = 2.3 to 2.8)]. Mean (± SE) amplitude (2.1 ± 0.1 vs. 1.7 ± 0.1 ng/mL), peak (2.2 ± 0.1 vs. 1.8 ± 0.1 ng/mL), and duration (35.6 ± 2.1 vs. 28.7 ± 2.0 min) of LH pulses assessed at wk 15 were greater in heifers offered the high than those offered the low preweaning plane, but no nutritional effects were observed on LH pulses at wk 25. Mean (± SE) age at puberty was 250 ± 9 d and was not influenced by planes of nutrition. However, the likelihood of attaining puberty by 30 wk of age was greater (hazard ratio = 3.8; 95% CI = 1.0 to 14.4) in heifers fed the high postweaning plane compared with the low plane. Heifers achieving puberty by 30 wk also had greater leptin concentrations from wk 1 to 25, whereas for every 1 ng/mL increase in circulating leptin at wk 15 and 25, days to puberty were estimated to decrease by 22 d (95% CI = 1 to 44) and 13 d (95% CI = 1 to 24), respectively. Increasing the preweaning plane from 5 to 10 L/d of whole milk increased serum leptin concentrations at wk 1, 3, and 5 and LH pulse amplitude, peak, and duration at wk 15. Increasing the postweaning plane from 70 to 85% of concentrate resulted in greater circulating leptin concentrations, which may be linked to an earlier onset of puberty.

Carryover effects of pre- and postweaning planes of nutrition on reproductive tract development and estrous cycle characteristics in Holstein heifers.

The objectives of this study were to investigate the carryover effects of differing pre- and postweaning planes of nutrition on prepubertal reproductive tract development and postpubertal estrous cycle characteristics in Holstein heifers. Heifer calves (n = 36) were randomly assigned to receive either a low or a high (5 or 10 L of whole milk/d, respectively) preweaning diet from 1 to 7 wk of age and either a low or a high (70 or 85% of concentrate dry total mixed ration, respectively) postweaning diet from 11 to 25 wk of age. Starting at wk 26 of age, heifers were subjected to weekly transrectal ultrasonography until wk 33 or until first ovulation to assess endometrial thickness and ovarian follicular count and size in the prepubertal phase. In a subset of heifers (n = 28), ovarian ultrasonography continued weekly until at least the second ovulation was confirmed; thereafter, ovarian dynamics (through ultrasonography) and blood progesterone (P4) were assessed every 2 d throughout 1 complete estrous cycle in the postpubertal phase. In the prepubertal phase, endometrial thickness (12.0 ± 0.4 vs. 10.8 ± 0.3 mm) and largest follicle size (11.8 ± 0.3 vs. 10.9 ± 0.2 mm) were greater in heifers fed the high postweaning diet than in those fed the low postweaning diet. Furthermore, the number of class 2 (6-9 mm) follicles was greater in heifers fed the high preweaning diet than in those fed the low preweaning diet (1.6 ± 0.1 vs. 1.1 ± 0.1), whereas the number of class 3 (>9 mm) follicles was greater in heifers fed the high postweaning diet than in those fed the low postweaning diet (1.2 ± 0.1 vs. 1.0 ± 0.1). In the postpubertal phase, overall corpus luteum and P4 dynamics did not differ among pre- or postweaning treatments; however, P4 at 4 d preceding luteolysis was lesser in heifers fed the high postweaning diet than in those fed the low postweaning diet (6.1 ± 0.4 vs. 7.7 ± 0.4 ng/mL). In addition, compared with heifers fed the low postweaning diet, those fed the high postweaning diet had a greater number of antral follicles (31.4 ± 2.2 vs. 21.4 ± 2.3) and tended to have more class 3 follicles (3.6 ± 0.3 vs. 2.7 ± 0.3). Results indicate positive carryover effects of increasing the preweaning plane of nutrition from 5 to 10 L of whole milk/d on prepubertal follicular growth in Holstein heifers. Furthermore, an increased postweaning plane of nutrition (85 vs. 70% of concentrate dry total mixed ration) advanced reproductive development through greater endometrial thickness and follicular growth in the prepubertal phase and increased the population of antral follicles in the postpubertal estrous cycle.

Effects of supplementing a Saccharomyces cerevisiae fermentation product during the periparturient period on performance of dairy cows fed fresh diets differing in starch content.

The objective of this study was to evaluate the effects of supplementing a Saccharomyces cerevisiae fermentation product (SCFP; NutriTek, Diamond V, Cedar Rapids, IA) during the periparturient period (d -28 ± 3 to 44 ± 3 relative to calving) on dry matter intake (DMI), milk production, apparent total-tract nutrient digestibility, and postpartum ovarian activity of dairy cows fed fresh diets varying in starch content. From d 28 ± 3 before the expected calving date until d 44 ± 3 after calving, 117 Holstein cows were fed diets with SCFP (SCFP; n = 59) or without (control, CON; n = 58). A common, basal, controlled-energy close-up diet (net energy for lactation: 1.43 Mcal/kg; 13.8% starch) was fed before calving. Cows within each treatment (CON or SCFP) were fed either a low- (LS; 22.1% starch) or high-starch (HS; 28.3% starch) diet from d 1 to 23 ± 3 after calving (fresh period), resulting in 4 treatment groups: LS-CON (n = 30), LS-SCFP (n = 29), HS-CON (n = 28), and HS-SCFP (n = 30). All cows were fed the HS diets from d 24 ± 3 to 44 ± 3 after calving (post-fresh period). Cows were assigned to treatment balanced for parity, body condition score, body weight, and expected calving date. Milk yield was higher for cows fed the LS diets compared with those fed the HS diets during the fresh period (34.1 vs. 32.1 kg/d), whereas DMI and 3.5% fat-corrected milk yield (FCM) were not affected by dietary starch content, and LS cows tended to lose more body condition than HS cows (-0.42 vs. -0.35 per 21 d) during the fresh period. Overall DMI during the close-up and fresh periods did not differ between SCFP and CON cows. However, SCFP supplementation transiently increased DMI on d 1 (13.0 vs. 11.9 kg/d) and 5 (15.5 vs. 14.1 kg/d) after calving compared with CON. During the post-fresh period, SCFP cows tended to eat less than CON cows (19.8 vs. 20.6 kg/d) but had similar 3.5% FCM (44.9 vs. 43.6 kg/d), resulting in greater feed efficiency for SCFP cows (FCM/DMI; 2.27 vs. 2.13). Neither starch content of fresh diets nor SCFP supplementation affected the interval from calving to first ovulation or the incidence of double ovulation. These findings suggest that feeding low-starch diets during the fresh period can increase milk production of dairy cows during the fresh period, and that supplementation of SCFP may increase feed intake around calving and feed efficiency in the post-fresh period.

Technical note: Validation of an automated in-line milk progesterone analysis system to diagnose pregnancy in dairy cattle.

The in-line milk analysis system (IMAS) is an automated biosensor technology that samples and quantifies milk progesterone concentrations (P4c) at frequent intervals starting early postpartum until pregnancy. The objective was to validate the use of pregnancy notifications (PregN) generated by an IMAS based on P4c profiles after artificial insemination (AI) to determine pregnancy and nonpregnancy status in dairy cows. Records of 1,821 AI events from 715 Holstein cows that had milk P4c (ng/mL) measured every 2.2 ± 1.9 d (mean ± standard deviation) between 24.5 ± 8.2 and 173.4 ± 49.3 d in milk through a real-time IMAS (Herd Navigator, DeLaval International, Tumba, Sweden) were evaluated. Based on variations in adjusted milk P4c (< vs. ≥ the 5.0 ng/mL threshold), the system determined the sampling frequency, onset and cessation of luteal phases, and pregnancy. If a luteal phase initiated (P4c increased to ≥5.0 ng/mL) after AI and remained uninterrupted, a PregN was generated starting at (mean ± standard deviation) 31.0 ± 4.3 d until 53.4 ± 7.9 d after AI, when sampling stopped, unless a decline in P4c (to <5.0 ng/mL) occurred indicating nonpregnancy and imminent estrus. The assessment of IMAS PregN at 4 weekly intervals was tested, and a confirmed calving occurrence between 262 and 296 d after AI, with no other subsequent AI recorded, was the gold standard for pregnancy. In total, 14.1 (256/1,821), 41.0 (746/1,821), and 50.7% (924/1,821) of AI events were followed by a decline in P4c before 19, 23, and 30 d after AI, respectively. Frequency of the last 3 sampling events preceding P4c decline was greater if P4c decline occurred between 18 and 25 d after AI (1.4 ± 0.5 samples per day) compared with before 17 or beyond 26 d after AI (1.0 ± 0.5 samples per day). At 30 ± 3 (27 to 33) d after AI, PregN occurred in 46.8% (853/1,821) of AI events, of which 15.2% (130/853) had a decline in P4c between 30 and 55 d after AI and 17.1% (146/853) was later confirmed nonpregnant based on the gold standard. A total of 40.7% (742/1,821) of AI events was confirmed pregnant by the gold standard, which was no different than the proportion of PregN at 51 ± 3 (48 to 54) d (40.9%; 744/1,821). At any time point between 27 and 54 d after AI, sensitivity and negative predictive values for PregN were greater than 95.0 and 96.0%, respectively, whereas specificity values were less than 90.0% for PregN before 40 d but greater than 94.0% for PregN beyond 41 d after AI. In conclusion, IMAS is able to diagnose pregnancy based on P4c profiles with high precision and determine early nonpregnancy based on the spontaneous cessation of the luteal phase. However, for accuracy greater than 95.0%, pregnancy declaration based on IMAS notifications alone should occur no earlier than 41 d after AI.

Using in-line milk progesterone data to characterize parameters of luteal activity and their association with fertility in Holstein cows.

Our objectives were to characterize parameters of luteal activity based on milk progesterone concentration (P4c) data from before and after artificial insemination (AI) and to evaluate their potential association with fertility in Holstein cows. Records of AI events (n = 4,353) and of milk P4c (n = 158,961) obtained through an in-line milk analysis system (Herd Navigator, DeLaval International, Tumba, Sweden) from 1,891 lactations of 1,423 Holstein cows were evaluated. Milk P4c (ng/mL) were measured every 2.2 ± 1.9 d (mean ± standard deviation) between 23.6 ± 7.3 and 185.3 ± 56.7 d in milk. Variations in milk P4c of consecutive records were used to determine onset of luteal phase (increase in P4c from <5.0 to ≥5.0 ng/mL), luteal phase length (period, in days, of P4c ≥5.0 ng/mL), cessation of luteal phase (decline from ≥5.0 to <5.0 ng/mL, designated as P4c-decline), and pregnancy (AI followed by a luteal phase that remained uninterrupted until 50 d post-AI). The length of the luteal phase preceding AI, the highest P4c (P4c peak) during the luteal phase preceding AI, the lowest P4c preceding AI (P4c pre-AI) that followed a P4c-decline, and the interval between P4c-decline and AI were evaluated, as well as the interval between AI and onset of luteal phase, and P4c at early diestrus (4.5 ± 0.6 d post-AI), mid diestrus (10.0 ± 0.6 d post-AI), and late diestrus (14.1 ± 0.6 d post-AI). Data were analyzed using logistic regressions, and comparisons made based on quartiles and cut-points established by receiver operating characteristic curve analysis. Overall probability of pregnancy was 32.0%. Parameters associated with reduced probability of pregnancy (represented as percentage points decrease in the probability of pregnancy) were (1) luteal phase length >14.4 d (7.6% decrease), (2) P4c peak ≤24.7 ng/mL (4.5% decrease), (3) P4c pre-AI >0.5 ng/mL (5.5% decrease), (4) interval between P4c-decline and AI of >1.6 d (4.0% decrease), (5) interval between AI and onset of luteal phase of <7 or >11 d (9.3 and 12.1% decrease, respectively), and (6) P4c at early diestrus ≤0.7 or >3.5 ng/mL (15.2 and 6.7% decrease, respectively), (7) P4c at mid diestrus ≤12.4 ng/mL (12.5% decrease), and (8) P4c at late diestrus ≤22.7 ng/mL (9.7% decrease). The parameters of luteal activity associated with reduced probability of pregnancy established here could be used as benchmarks while developing recommendations to improve reproductive performance in herds using in-line milk progesterone monitoring.

The relationship between anogenital distance and fertility, and genome-wide associations for anogenital distance in Irish Holstein-Friesian cows.

The evaluation of anogenital distance (AGD), the distance from the center of the anus to base of the clitoris, as a potential fertility trait for genetic selection in dairy cows has generated recent interest. The objectives of this cross-sectional observational study were to (1) characterize the distribution and variability of AGD, (2) determine factors associated with AGD, (3) estimate heritability for AGD, (4) identify single nucleotide polymorphisms (SNP) associated with phenotypic variation of AGD, and (5) validate the relationship between categories of AGD and fertility in Irish Holstein-Friesian cows. Anogenital distance was measured using digital calipers in 1,180 Holstein cows (mean ± standard deviation: 225 ± 79 d in milk) from 10 dairy herds located in Munster, Ireland. In addition, age (yr), weight (kg), height at hip (cm), and body condition score (BCS) at the time of AGD measurement were determined in a subset of 281 cows. Genotype information available from 908 cows was subsequently imputed to the Illumina Bovine High Density BeadChip (Illumina Inc., San Diego, CA) for genome-wide association analysis of phenotypic variation in AGD. Overall, AGD had a normal distribution and high variability (mean ± standard deviation; 119.2 ± 11.6 mm). Anogenital distance was weakly but positively associated with cow age, hip height, and body weight, and negatively associated with BCS; the phenotypic variation in AGD that was explainable by these variables was small (coefficient of determination; R2 = 0.09, 0.06, 0.10, and 0.02, respectively). The estimated heritability for AGD was 0.37 (standard error of mean ± 0.08). Six SNP of suggestive significance were identified on Bos taurus autosomes 6, 15, 20, and 26; however, none of these SNP was related to previously identified candidate genes for fertility. Cows were categorized into quartiles (Q1; 86 to 111 mm; n = 311, Q2; 112 to 120 mm; n = 330; Q3; 121 to 127 mm; n = 265, and Q4; 128 to 160 mm; n = 274) based on AGD and the association with reproductive outcomes examined (21-d submission rate, pregnancy to first AI, pregnancy rate within 21, 42 and 84-d after the farm mating start date, and number of times bred). None of the reproductive variables differed significantly between AGD categories. In summary, despite identification of high variability and moderate heritability for AGD in Irish Holstein-Friesian cows, reproductive outcomes did not differ between categories of AGD. This latter result differs from our previous finding of an inverse relationship between AGD and pregnancy outcomes in first- and second-parity Canadian Holstein cows, emphasizing the need to test and validate this new phenotype in diverse cow populations.

Characterization of anogenital distance and its relationship to fertility in lactating Holstein cows.

Anogenital distance (AGD) serves as a marker for prenatal androgenization, reproductive development, and fertility in humans and rodents. The primary objectives of this observational study in lactating dairy cows were to (1) characterize the distribution and variability of AGD, (2) determine the relationship among AGD and potential postnatal AGD determinants of age and height, and (3) evaluate the associations between AGD and pregnancy to first artificial insemination (P/AI) and cumulative pregnancy by 250 d in milk (DIM) within parity groups (first, second, and third+ parities). The secondary objective was to evaluate the association between AGD and testosterone concentrations. The AGD (mm), age (yr), and height at hip (cm) at the time of AGD determination, and aforesaid reproductive outcomes were determined in 921 Holstein cows (first, second, and third+ parity; n = 360, 256, and 305, respectively). Plasma concentrations of testosterone were determined in a subset of 93 cows. Overall, AGD had a normal distribution and high variability [mean (±standard deviation); 131.0 ± 12.2 mm], was weakly associated with cow age and height (coefficient of determination = 0.09 and 0.04, respectively), and had an inverse relationship with P/AI in first- and second-parity cows, but not in third+ parity cows. For every 1 mm increase in AGD, the odds of P/AI decreased by 3.4 and 2.4% for first- and second-parity cows, respectively. The optimal AGD threshold to predict probability of P/AI was 127.1 mm for both first- (sensitivity: 66.4; specificity: 56.6%) and second-parity cows (sensitivity: 46.0; specificity: 70.4%). Accordingly, first- and second-parity cows were categorized into either short or long AGD (≤ or >127.1 mm), and associations with reproductive outcomes were evaluated. First-parity cows with long AGD had lower P/AI (30.9 vs. 53.6%) and decreased likelihood (hazard ratio: 0.68) of pregnancy by 250 DIM than those with short AGD. Similarly, second-parity cows with long AGD had reduced P/AI (28.3 vs. 44.4%) and a tendency for decreased likelihood (hazard ratio: 0.76) of pregnancy by 250 DIM than in cows with short AGD. The association between AGD and testosterone was weak and nonsignificant. In summary, AGD in Holstein cows was normally distributed, highly variable, and weakly associated with age and height. Besides, AGD had an inverse relationship with P/AI and cumulative pregnancy by 250 DIM in first- and second-parity cows; however, such a relationship was not evident in older (third+ parity) cows.

Characterization of the variability and repeatability of gonadotropin-releasing hormone-induced luteinizing hormone responses in dairy cows within a synchronized ovulation protocol.

The primary objective was to determine the variability and repeatability of GnRH-induced LH responses. The secondary objective was to evaluate the associations among plasma LH, FSH, estradiol (E2), and progesterone (P4) concentrations. One hundred lactating Holstein cows (35 primiparous, 65 multiparous) were initially subjected to a presynchronization protocol (d 0, PGF2α; d 3, GnRH) followed 7 d later by Ovsynch (d 10, GnRH; d 17, PGF2α; 56 h later, GnRH) and timed artificial insemination 16 h after the last GnRH. Blood samples were collected immediately before the GnRH injection of presynchronization and the second GnRH of Ovsynch to determine plasma concentrations of LH, FSH, and P4. A second blood sample was collected 2 h after each of the above GnRH injections to determine GnRH-induced LH and FSH concentrations. Plasma concentrations of E2 were also determined in samples collected immediately before the second GnRH of Ovsynch. Cows that (1) had higher LH concentrations at 0 h than at 2 h after GnRH, (2) showed an ongoing spontaneous LH surge, (3) did not respond to GnRH, and (4) had P4 ≥ 0.5 ng/mL at GnRH of presynchronization and the second GnRH of Ovsynch were excluded from the analysis. The variability (coefficient of variation) and repeatability [between animal variance/(within animal variance + between animal variance)] of GnRH-induced LH response were determined from samples collected 2 h after the GnRH of presynchronization and the second GnRH of Ovsynch. The associations among plasma LH, FSH, E2, and P4 were determined at the second GnRH of Ovsynch. Mean (±SEM) LH concentrations before GnRH were 0.5 ± 0.04 and 0.6 ± 0.03 ng/mL, whereas mean LH concentrations 2 h after GnRH were 9.8 ± 1.0 and 12.1 ± 0.8 ng/mL at GnRH of presynchronization and the second GnRH of Ovsynch, respectively. The variability of GnRH-induced LH was 76.1 and 52.1% at GnRH of presynchronization and the second GnRH of Ovsynch, respectively. The repeatability estimate for GnRH-induced LH concentration between GnRH of presynchronization and Ovsynch assessments was 0.10. Plasma concentrations of LH were positively associated with FSH and E2 (r = 0.61 and 0.30, respectively) and negatively associated with P4 (r = -0.46) at the second GnRH of Ovsynch. In summary, GnRH-induced LH responses were highly variable and unrepeatable, and LH concentrations were positively associated with FSH and E2 and negatively associated with P4.

Repeatability of antral follicle counts and anti-Müllerian hormone and their associations determined at an unknown stage of follicular growth and an expected day of follicular wave emergence in dairy cows.

The objectives were to determine (1) the repeatability of antral follicle counts (AFC) and anti-Müllerian hormone (AMH) concentrations at an unknown stage of follicular growth (FG) and at an expected day of follicular wave emergence (FWE) in dairy cows, and (2) the association between AFC and AMH at the above two stages. Transrectal ultrasound imaging of the ovaries and blood sample collection were performed on 91 lactating Holstein cows (33 primiparous, 58 multiparous) to determine AFC and serum concentrations of AMH at an unknown stage of FG (mean ± SEM; 14 ± 0.5 d postpartum) and an expected day of FWE (mean ± SEM; 73 ± 0.5 d postpartum, approximately 36 h after the 2nd GnRH of Ovsynch protocol). The AFC ranged from 10 to 53 (mean ± SEM; 26.1 ± 1.0) and 6 to 45 (mean ± SEM; 23.4 ± 1.0) at an unknown stage of FG and expected day of FWE, respectively (P = 0.02). Serum concentrations (pg/mL) of AMH ranged from 13.9 to 528.8 (mean ± SEM; 187.3 ± 13.1) and 38.2 to 774.1 (mean ± SEM; 218.7 ± 14.5) at an unknown stage of FG and expected day of FWE, respectively (P < 0.01). The repeatability estimates for AFC and serum AMH concentrations between an unknown stage of FG and the expected day of FWE were 0.37 and 0.73 (P < 0.01), respectively. The correlation between AFC and AMH was moderate at an unknown stage of FG (r = 0.54; P < 0.01) and at an expected day of FWE (r = 0.59; P < 0.01). In summary, both AFC and AMH were repeatable when measured at an unknown stage of FG and an expected day of FWE, but the repeatability was greater for AMH. Thus, if cows are tested at an unknown stage of FG, serum AMH may be a more reliable phenotype than AFC to test association with reproductive outcomes. Furthermore, AFC and AMH were moderately correlated at the two stages examined.