Variation in protein metabolism biomarkers during the transition period and associations with health, colostrum quality, reproduction, and milk production traits in Holstein cows.

The aims of this study were to assess (1) the variation of protein metabolism biomarkers and factors affecting them during the transition period, (2) the association of each biomarker with skeletal muscle reserves and their changes, and (3) the association of these biomarkers with postpartum health, colostrum quality, reproduction, and milk production. For this purpose, 238 multiparous Holstein cows from 6 herds were used in a prospective cohort study. Plasma concentrations of 3-methylhistidine (3-MH) and 1-methylhistidine (1-MH) and serum concentrations of total protein (TP), albumin (ALB), urea nitrogen (BUN), and creatinine (SCR) were determined for each cow at -21, -7, 7, 21, and 28 d relative to calving. Clinical diseases were recorded during the first 28 d postcalving, and presence of subclinical ketosis (scKET) was investigated at 7 and 21 d. Colostrum quality was estimated by Brix refractometry. Reproduction data by 150 d in milk (DIM) and milk production records were also available. Linear mixed models including the fixed effects of time point, herd, parity, body condition score (-21 d), duration of dry period and postparturient diseases were fitted to assess the variation in each biomarker's concentration. The association between the biomarkers' concentration during the prepartum period with the odds for each postparturient disease and for a combined trait (CD_1-28), defined as the presence of at least one clinical condition during the first 28 d after calving, were assessed with separate binary logistic models for time points -21 d and -7 d. The relationship of each biomarker's concentration with longissimus dorsi thickness (LDT) and the changes in LDT (ΔLDT) was assessed with pairwise correlations. Separate general linear models were used to assess the association of each biomarker with colostrum Brix values and milk production traits. Finally, the associated hazard for first artificial insemination (AI) and for pregnancy by 150 DIM (PREG_150DIM) was assessed with Cox proportional hazard models, whereas odds for pregnancy to the first AI (PREG_1stAI) were assessed with binary logistic models. The level of 3-MH was affected mainly by herd, time points, and their interaction. Higher 3-MH was associated with increased odds for metritis and CD_1-28, increased hazard for PREG_150 DIM and with increased milk production. 1-Methylhistidine was affected mainly by herd, scKET and occurrence of displaced abomasum. Higher 1-MH was associated with better colostrum quality, increased odds for scKET, increased hazard for first AI by 150 DIM and with decreased milk production. Both 3-MH and 1-MH were weakly to moderately negatively correlated with LDT and moderately to strongly negatively correlated with ΔLDT at the corresponding time periods. Additionally, higher TP was associated with increased odds for metritis and CD_1-28 and increased milk production, while higher ALB was associated with increased odds for scKET and increased milk production. Moreover, higher BUN was associated with decreased odds for scKET, increased odds for PREG_1stAI and increased milk production. Higher SCR was associated with decreased odds for retained fetal membranes, metritis, and CD_1-28. Periparturient protein metabolism is significantly associated with postpartum health, colostrum quality, reproduction, and milk production; mechanisms involved require further investigation.

Identification of temporal patterns of environmental heat stress of Holstein dairy heifers raised in Mediterranean climate during their in-utero and post-natal life periods and modelling their effects on age at first calving.

A retrospective study was conducted to evaluate temporal patterns of environmental heat stress during the in-uterus period of development (IUP) and the 3-month post-natal (PN) period of dairy heifers, and to estimate their association with the age at first calving (AFC). Data from 30 dairy herds in Northern Greece including 9098 heifers were extracted from National Cattle Database. Data (2005-2019) regarding 230,100 farm-specific ambient daily temperature and relative humidity records, were obtained from ERA5-Land. Average monthly Temperature-Humidity-Index values (THI; low≤68, and high>68) were calculated and matched for each heifer to their IUP and PN. Subsequently, Cluster Analysis was used with monthly THIs as predictors to allocate heifers to THI clusters. The association of clusters with AFC was assessed with Generalized Linear Mixed Model analysis, an extended form of multiple linear regression. Finally, 8 Heat Stress Clusters (HSC; namely HSC-1 to HSC-8) were identified. Compared to HSC-8 (8th-9th IUP months and 1st PN month) heifers of HSC-5 (4th-7th IUP months) and HSC-6 (6th-8th IUP months) calved 13.8 and 17.8 days later, respectively (P < 0.01-0.001). Moreover, when AFC was treated as a binary variable, heifers of HSC-5 and HSC-6 had 1.15 and 1.34 (P < 0.01-0.001) higher risk of calving for the first time later than 787 days compared to HSC-8, respectively.

Skeletal muscle and adipose tissue reserves and mobilisation in transition Holstein cows: Part 1 Biological variation and affecting factors.

Nutrient deficit during the periparturient period leads to mobilisation of body energy and protein reserves. Research regarding fat reserves and mobilisation is extensive, while, on the contrary, investigation of muscle mobilisation during the periparturient period is limited. The aim of this cohort study was to simultaneously investigate the biological variation of skeletal muscle and subcutaneous fat reserves together with their mobilisation in transition Holstein cows of different herds, using ultrasonography, and to assess potential affecting factors. For this purpose, ultrasound measurements of longissimus dorsi muscle thickness (LDT) and backfat thickness (BFT) from 238 multiparous cows of six dairy farms were obtained at six time points across the transition period (from 21 days pre- to 28 days postpartum). Concentrations of serum creatinine and non-esterified fatty acids were determined in order to confirm the loss of muscle mass and adipose tissue, respectively. Cases of clinical postparturient diseases and subclinical ketosis (scKET) during the first 28 days postcalving were recorded. Cows mobilised on average 32.8% and 37.3% of LDT and BFT reserves, respectively. Large between-cow variation was observed for both the onset and the degree of mobilisation. Time point, initial body condition score and parity were the most important predictors of LDT variation. Cows diagnosed with metritis (MET) had lower LDT postpartum and mobilised more muscle depth compared to cows not diagnosed with MET. Initial BCS, time point, initial BW (estimated by heart girth measurement) and parity were the most important predictors of BFT variation. Cows diagnosed with MET mobilised more backfat between -7d and 7d compared to cows not diagnosed with MET. Cows with scKET mobilised more backfat between 7- and 21 days postpartum compared to healthy ones. Variation of subcutaneous fat and skeletal muscle reserves during the transition period was large and affected by herd and several cow-level factors.

Skeletal muscle and adipose tissue reserves and mobilisation in transition Holstein cows: Part 2 association with postpartum health, reproductive performance and milk production.

The aim of this study was, for the first time, to simultaneously assess the association of skeletal muscle and subcutaneous fat reserves and their mobilisation, measured by ultrasonography, with the incidence of specific postparturient health, reproduction, and milk production traits. For this purpose, ultrasound measurements of longissimus dorsi thickness (LDT) and backfat thickness (BFT) from 238 multiparous cows from 6 dairy farms were obtained at 6 time points during the transition period (from 21 days pre- to 28 days postpartum). In each case, LDT and BFT measurements at each time point and LDT and BFT mobilisation variables at each study period were assessed simultaneously. Cases of specific clinical postparturient diseases and subclinical ketosis were recorded. An additional disease trait was used, defined as the presence or absence of at least one clinical condition after calving (CD_1-28). The associated disease odds with LDT/BFT variables were assessed with binary logistic regression models. The associated hazard for 1st artificial insemination (AI) and for pregnancy by 150 days-in-milk (PREG_150DIM) was assessed with Cox proportional hazard models. Moreover, binary logistic models were used to assess the associated odds for pregnancy to 1stAI (PREG_1stAI). Finally, association with 30d, 100d and 305d milk yield was assessed with linear regression models. Increased muscle depth during transition was negatively associated with odds for metritis and CD_1-28, while associations with odds for subclinical ketosis were inconclusive. Moreover, increased LDT reserves were associated with greater hazard for 1st AI by 150 days-in-milk, but results were inconclusive regarding odds for PREG_1stAI. Increased LDT mobilisation was associated with increased odds for metritis. Increased BFT reserves were positively associated with odds for metritis, CD_1-28 and subclinical ketosis and with decreased hazard for PREG_150DIM. Increased BFT mobilisation was associated with increased odds for subclinical ketosis and with decreased odds for PREG_1stAI and decreased hazard for PREG_150DIM. Cows with moderate BFT reserves performed better. Finally, increased BFT mobilisation during -21d to -7d from parturition was associated with less milk by 30d and 100d. On the contrary, increased BFT mobilisation during -7d to 7d was associated with more milk by 305d. Metabolism of muscle and fat tissue during transition period was differently associated with different postparturient health, reproduction and milk production traits. In general, greater muscle mass and moderate fat reserves with limited muscle and fat mobilisation were associated with better performance.

Association of post-partum serum Ca patterns with reproduction in Holstein cows.

The objective of this study was to evaluate the effects of subclinical hypocalcemia (SCHCa) patterns on reproductive performance in dairy cows. In a prospective observational study 916 cows from 9 herds were blood sampled on DIMs 1, 2, 4 and 8; Ca concentration was measured with atomic absorption and SCHCa was defined as Ca ≤ 2.09 mmol/L. Cluster analysis revealed 2 normocalcemic (NORM and HIGH) and five hypocalcemic (SCH-1 to 5) clusters. Cows in cluster NORM (n = 151) had mean day-to-day serum Ca (DIMs 1-8) between 2.24 and 2.41 mmol/L, and cows in HIGH (n = 167) between 2.42 and 2.53 mmol/L. Cows in SCH-1 (n = 130) and SCH-2 (n = 102) had transient and mostly mild SCHCa on day 1 and day 2, respectively. Cows in SCH-3 (n = 123) had mostly severe SCHCa on days 1 and 2, extending to day 4. Cows in SCH-4 (n = 120) and SCH-5 (n = 145) had SCHCa which culminated on days 4 and 8, respectively. Information on reproductive outcomes including pregnancy status by 120 and 200 DIM and days open were retrieved from farm records. Median (±SE) days to 1st artificial insemination (AI) until 120 DIM estimated with Kaplan-Meier analysis for cows in SCH-3, SCH-4 and SCH-5 were 100.0 ± 7.2, 109.0 ± 6.6 and 120.0 ± 3.2, respectively, and were statistically significantly higher from those in NORM and HIGH (91.0 ± 3.4 and 87 ± 3.7, respectively). Mean days (±SE) to detected pregnancy until 200 DIM estimated with Kaplan-Meier analysis for cows in SCH-3 were 161.2 ± 4.8 and were statistically significantly higher from those in clusters NORM and HIGH (155.5 ± 4.2 and 151.6 ± 4.3, respectively). The association of Ca clusters with the odds of detected pregnancy for the 1st insemination until 120 and 200 DIM was evaluated with Linear Mixed Models. Odds for pregnancy by 120 DIM did not differ among clusters, however, cows in clusters SCH-3 and SCH-5 had lower odds for pregnancy by 200 DIM compared to HIGH (OR = 0.55, P = 0.02, and OR = 0.49, P = 0.004, respectively). Covariate adjusted survival curves generated by multivariable Cox proportional hazards model revealed that: a) clusters SCH-3 and SCH-5 had the highest (36% and 38%, respectively), while NORM and HIGH the lowest (16% and 17%, respectively) proportion of cows not inseminated for the 1st time by 120 DIM, b) compared to HIGH, cows in SCH-5 had a lower hazard of pregnancy by 120 DIM (HR = 0.42, P = 0.005), c) the proportion of open cows by 200 DIM for NORM and HIGH were 57% and 47%, respectively, while for SCH-3 and SCH-5 66% and 68%, respectively, and d) compared to HIGH, SCH-3 (HR = 0.54, P = 0.008) and SCH-5 (HR = 0.50, P = 0.001) presented the lowest hazard for pregnancy by 200 DIM. In conclusion, cows that during the entire first week after calving were continuously normocalcemic had the best reproductive performance, while those of SCH-3 and SCH-5 the worst.

Subclinical hypocalcaemia follows specific time-related and severity patterns in post-partum Holstein cows.

Hypocalcaemia is a common metabolic disorder of post-parturient dairy cows; enhancement of our knowledge regarding Ca dynamics would improve cow health and welfare. This study investigated the presence of time- and severity-related subclinical hypocalcaemia (SCHCa) patterns in Holstein cows after calving and their association with diseases during the first week of lactation. A total of 938 cows from nine herds were blood sampled on days 1, 2, 4 and 8 post-partum, and serum Ca concentration was measured with atomic absorption. Subclinical ketosis (serum ß-hydroxybutyrate≥1.2 mmol/L) and cases of retained foetal membranes, metritis (MET), mastitis, ketosis and displaced abomasum (DA) were recorded. Using receiver operating characteristic (ROC) analysis, the SCHCa cut-off was identified at serum Ca ≤ 2.09 mmol/L. Hierarchical and two-step cluster analysis classified cows to seven clusters based on test day cow Ca records and overall SCHCa status. Two clusters (NORM and HIGH) included 318 normocalcaemic cows and five clusters (SCH-1 to -5) those that were at least once subclinically hypocalcaemic (n = 620). A second ROC analysis was performed in order to distinguish mild from severe cases of SCHCa in these 620 cows; this cut-off was identified at 1.93 mmol/L. The associated risk of disease with Ca clustership was assessed with generalized linear mixed models. Overall incidence of SCHCa was 66.1%. Clusters SCH-1 and SCH-2 included cows with short-term SCHCa of day 1 and day 2, respectively, while SCH-3 included cows with severe and prolonged SCHCa extending to day 4 and beyond. Clusters SCH-4 and SCH-5 included cows with delayed SCHCa, which culminated on days 4 and 8, respectively. Compared to NORM cows in HIGH had lower risk of MET and no cases of DA. Cows in SCH-3 had higher risk of being diagnosed with retained foetal membranes, DA or any disease during the study period. Cows in SCH-5 had higher risk of being diagnosed with ketosis, subclinical ketosis or any disease. In conclusion, there are multiple normocalcaemic and hypocalcaemic patterns that are differently associated with disease risk.

Genetic parameters of subclinical macromineral disorders and major clinical diseases in postparturient Holstein cows.

The main objective of this study was to assess the genetic parameters of subclinical disorders associated with subclinical hypocalcemia, hypophosphatemia, subclinical hypomagnesemia, hypokalemia, and hyperphosphatemia, as well as major clinical diseases after calving in Holstein cows. The secondary objective was to estimate the associated genetic and phenotypic correlations among these subclinical and clinical conditions after calving in Holstein cows. The study was conducted in 9dairy herds located in Northern Greece. None of the herds used any kind of preventive measures for milk fever (MF). A total of 1,021 Holstein cows with pedigree information were examined from November 2010 until November 2012. The distribution across parities was 466 (parity 1), 242 (parity 2), 165 (parity 3), and 148 (parity 4 and above) cows. All cows were subjected to a detailed clinical examination and blood was sampled on d 1, 2, 4, and 8 after calving. Serum concentrations of Ca, P, Mg, and K were measured in all samples, whereas ß-hydroxybutyrate (BHB) was measured only for d 8. The final data set included 4,064 clinical and 16,848 biochemical records (4,020 Ca, 4,019 P, 4,020Mg, 3,792K, and 997 BHB). Data of 1,988 observations of body condition score at d 1 and 8 were also available. All health traits were analyzed with a univariate random regression model. The genetic analysis for macromineral-related disorders included 986 cows with no obvious signs of MF (35 cows with MF were excluded). Analysis for other health traits included all 1,021 cows. A similar single record model was used for the analysis of BHB. Genetic correlations among traits were estimated with a series of bivariate analyses. Statistically significant daily heritabilities of subclinical hypocalcemia (0.13-0.25), hypophosphatemia (0.18-0.33), subclinical hypomagnesemia (0.11-0.38), and hyperphosphatemia (0.14-0.22) were low to moderate, whereas that of hypokalemia was low (0.08-0.10). The heritability of body condition score was 0.20±0.10. Statistically significant daily heritabilities of clinical diseases were those of MF (0.07-0.11), left displaced abomasum (0.19-0.31), and mastitis (0.15-0.41). Results suggest that these health disorders are heritable traits and could be minimized with proper genetic selection. Statistically significant phenotypic correlations were estimated for the first time between macromineral concentrations and almost all transition cow metabolic and infectious health disorders.

Genetic parameters of calcium, phosphorus, magnesium, and potassium serum concentrations during the first 8 days after calving in Holstein cows.

Calcium, Mg, P, and K are of great importance for the health and productivity of dairy cows after calving. So far genetic studies have focused on clinical hypocalcemia, leaving the genetic parameters of these macroelements unstudied. Our objective was to estimate the genetic parameters of Ca, Mg, P, and K serum concentrations and their changes during the first 8d after calving. The study was conducted in 9 herds located in northern Greece, with 1,021 Holstein cows enrolled from November 2010 until November 2012. No herd used any kind of preventive measures for hypocalcemia. Pedigree information for all cows was available. A total of 35 cows were diagnosed and treated for periparturient paresis and, therefore, excluded from the study. The remaining 986 cows were included in genetic analysis. The distribution of cows across parities was 459 (parity 1), 234 (parity 2), 158 (parity 3), and 135 (parity ≥4). A sample of blood was taken from each cow on d1, 2, 4, and 8 after calving and serum concentrations of Ca, P, Mg, and K were measured in each sample. A final data set of 15,390 biochemical records was created consisting of 3,903 Ca, 3,902 P, 3,903Mg, and 3,682K measurements. Moreover, changes of these concentrations between d1 and 4 as well as 1 and 8 after calving were calculated and treated as different traits. Random regression models were used to analyze the data. Results showed that daily heritabilities of Ca, P, and Mg concentrations traits were moderate to high (0.20-0.43), whereas those of K were low to moderate (0.12-0.23). Regarding concentration changes, only Mg change between d1 and 8 after calving had a significant heritability of 0.18. Genetic correlations between Ca, P, Mg, and K concentrations and their concentration changes from d1 to 4 and 1 to 8 after calving were not significantly different from zero. Most phenotypic correlations among Ca, P, Mg, and K concentrations were positive and low (0.09-0.16), whereas the correlation between P and Mg was negative and low (-0.16). Phenotypic correlations among macromineral concentrations on d1 and their changes from d1 to 4 and 1 to 8 after calving varied for each macromineral. This study revealed that genetic selection for normal Ca, P, Mg, and K concentrations in the first week of lactation is possible and could facilitate the management of their deficiencies during the early stages of lactation.