Prediction of key milk biomarkers in dairy cows through milk mid-infrared spectra and international collaborations.

At the individual cow level, suboptimum fertility, mastitis, negative energy balance, and ketosis are major issues in dairy farming. These problems are widespread on dairy farms and have an important economic impact. The objectives of this study were (1) to assess the potential of milk mid-infrared (MIR) spectra to predict key biomarkers of energy deficit (citrate, isocitrate, glucose-6 phosphate [glucose-6P], free glucose), ketosis (ß-hydroxybutyrate [BHB] and acetone), mastitis (N-acetyl-ß-d-glucosaminidase activity [NAGase] and lactate dehydrogenase), and fertility (progesterone); (2) to test alternative methodologies to partial least squares (PLS) regression to better account for the specific asymmetric distribution of the biomarkers; and (3) to create robust models by merging large datasets from 5 international or national projects. Benefiting from this international collaboration, the dataset comprised a total of 9,143 milk samples from 3,758 cows located in 589 herds across 10 countries and represented 7 breeds. The samples were analyzed by reference chemistry for biomarker contents, whereas the MIR analyses were performed on 30 instruments from different models and brands, with spectra harmonized into a common format. Four quantitative methodologies were evaluated to address the strongly skewed distribution of some biomarkers. Partial least squares regression was used as the reference basis, and compared with a random modification of distribution associated with PLS (random-downsampling-PLS), an optimized modification of distribution associated with PLS (KennardStone-downsampling-PLS), and support vector machine (SVM). When the ability of MIR to predict biomarkers was too low for quantification, different qualitative methodologies were tested to discriminate low versus high values of biomarkers. For each biomarker, 20% of the herds were randomly removed within all countries to be used as the validation dataset. The remaining 80% of herds were used as the calibration dataset. In calibration, the 3 alternative methodologies outperform the PLS performances for the majority of biomarkers. However, in the external herd validation, PLS provided the best results for isocitrate, glucose-6P, free glucose, and lactate dehydrogenase (coefficient of determination in external herd validation [R2v] = 0.48, 0.58, 0.28, and 0.24, respectively). For other molecules, PLS-random-downsampling and PLS-KennardStone-downsampling outperformed PLS in the majority of cases, but the best results were provided by SVM for citrate, BHB, acetone, NAGase, and progesterone (R2v = 0.94, 0.58, 0.76, 0.68, and 0.15, respectively). Hence, PLS and SVM based on the entire dataset provided the best results for normal and skewed distributions, respectively. Complementary to the quantitative methods, the qualitative discriminant models enabled the discrimination of high and low values for BHB, acetone, and NAGase with a global accuracy around 90%, and glucose-6P with an accuracy of 83%. In conclusion, MIR spectra of milk can enable quantitative screening of citrate as a biomarker of energy deficit and discrimination of low and high values of BHB, acetone, and NAGase, as biomarkers of ketosis and mastitis. Finally, progesterone could not be predicted with sufficient accuracy from milk MIR spectra to be further considered. Consequently, MIR spectrometry can bring valuable information regarding the occurrence of energy deficit, ketosis, and mastitis in dairy cows, which in turn have major influences on their fertility and survival.

Influence of the concentrate inclusion level in a grass silage-based diet on hepatic transcriptomic profiles in Holstein-Friesian dairy cows in early lactation.

Excessive negative energy balance in early lactation is linked to an increased disease risk but may be mitigated by appropriate nutrition. The liver plays central roles in both metabolism and immunity. Hepatic transcriptomic profiles were compared between 3 dietary groups in each of 40 multiparous and 18 primiparous Holstein-Friesian cows offered isonitrogenous grass silage-based diets with different proportions of concentrates: (1) low concentrate (LC, 30% concentrate + 70% grass silage); (2) medium concentrate (MC, 50% concentrate + 50% grass silage), or (3) high concentrate (HC, 70% concentrate + 30% grass silage). Liver biopsies were taken from all cows at around 14 d in milk for RNA sequencing, and blood metabolites were measured. The sequencing data were analyzed separately for primiparous and multiparous cows using CLC Genomics Workbench V21 (Qiagen Digital Insights), focusing on comparisons between HC and LC groups. More differentially expressed genes (DEG) were seen between the primiparous cows receiving HC versus LC diets than for multiparous cows (597 vs. 497), with only 73 in common, indicating differential dietary responses. Multiparous cows receiving the HC diet had significantly higher circulating glucose and insulin-like growth factor-1 and lower urea than those receiving the LC diet. In response to HC, only the multiparous cows produced more milk. In these animals, bioinformatic analysis indicated expression changes in genes regulating fatty acid metabolism and biosynthesis (e.g., ACACA, ELOVL6, FADS2), increased cholesterol biosynthesis (e.g., CYP7A1, FDPS, HMGCR), downregulation in hepatic AA synthesis (e.g., GPT, GCLC, PSPH, SHMT2), and decreased expression of acute phase proteins (e.g., HP, LBP, SAA2). The primiparous cows on the HC diet also downregulated genes controlling AA metabolism and synthesis (e.g., CTH, GCLC, GOT1, ODC1, SHMT2) but showed higher expression of genes indicative of inflammation (e.g., CCDC80, IL1B, S100A8) and fibrosis (e.g., LOX, LUM, PLOD2). This potentially adverse response to a HC diet in physically immature animals warrants further investigation.

Relationships between metabolic profiles and gene expression in liver and leukocytes of dairy cows in early lactation.

Homeorhetic mechanisms assist dairy cows in the transition from pregnancy to lactation. Less successful cows develop severe negative energy balance (NEB), placing them at risk of metabolic and infectious diseases and reduced fertility. We have previously placed multiparous Holstein Friesian cows from 4 herds into metabolic clusters, using as biomarkers measurements of plasma nonesterified fatty acids, ß-hydroxybutyrate, glucose and IGF-1 collected at 14 and 35 d in milk (DIM). This study characterized the global transcriptomic profiles of liver and circulating leukocytes from the same animals to determine underlying mechanisms associated with their metabolic and immune function. Liver biopsy and whole-blood samples were collected around 14 DIM for RNA sequencing. All cows with available RNA sequencing data were placed into balanced (BAL, n = 44), intermediate (n = 44), or imbalanced (IMBAL, n = 19) metabolic cluster groups. Differential gene expression was compared between the 3 groups using ANOVA, but only the comparison between BAL and IMBAL cows is reported. Pathway analysis was undertaken using DAVID Bioinformatic Resources (https://david.ncifcrf.gov/). Milk yields did not differ between BAL and IMBAL cows but dry matter intake was less in IMBAL cows and they were in greater energy deficit at 14 DIM (-4.48 v -11.70 MJ/d for BAL and IMBAL cows). Significantly differentially expressed pathways in hepatic tissue included AMPK signaling, glucagon signaling, adipocytokine signaling, and insulin resistance. Genes involved in lipid metabolism and cholesterol transport were more highly expressed in IMBAL cows but IGF1 and IGFALS were downregulated. Leukocytes from BAL cows had greater expression of histones and genes involved in nucleosomes and cell division. Leukocyte expression of heat shock proteins increased in IMBAL cows, suggesting an unfolded protein response, and several key genes involved in immune responses to pathogens were upregulated (e.g., DEFB13, HP, OAS1Z, PTX3, and TLR4). Differentially expressed genes upregulated in IMBAL cows in both tissues included CD36, CPT1, KFL11, and PDK4, all central regulators of energy metabolism. The IMBAL cows therefore had greater difficulty maintaining glucose homeostasis and had dysregulated hepatic lipid metabolism. Their energy deficit was associated with a reduced capacity for cell division and greater evidence of stress responses in the leukocyte population, likely contributing to an increased risk of infectious disease.

Genome-wide association for metabolic clusters in early-lactation Holstein dairy cows.

The aim of this study was to detect the genomic region or regions associated with metabolic clusters in early-lactation Holstein cows. This study was carried out in 2 experiments. In experiment I, which was carried out on 105 multiparous Holstein cows, animals were classified through k-means clustering on log-transformed and standardized concentrations of blood glucose, insulin-like growth factor I, free fatty acids, and ß-hydroxybutyrate at 14 and 35 d in milk (DIM), into metabolic clusters, either balanced (BAL) or other (OTR). Forty percent of the animals were categorized in the BAL group, and the remainder were categorized as OTR. The cows were genotyped for a total of 777,962 SNP. A genome-wide association study was performed, using a case-control approach through the GEMMA software, accounting for population structure. We found 8 SNP (BTA11, BTA23, and BTAX) associated with the predicted metabolic clusters. In experiment II, carried out on 4,267 second-parity Holstein cows, milk samples collected starting from the first week until 50 DIM were used to determine Fourier-transform mid-infrared (FT-MIR) spectra and subsequently to classify the animals into the same metabolic clusters (BAL vs. OTR). Twenty-eight percent of the animals were categorized in the BAL group, and the remainder were classified in the OTR category. Although daily milk yield was lower in BAL cows, we found no difference in daily fat- and protein-corrected milk yield in cows from the BAL metabolic cluster compared with those in the OTR metabolic cluster. In the next step, a single-step genomic BLUP was used to identify the genomic region(s) associated with the predicted metabolic clusters. The results revealed that prediction of metabolic clusters is a highly polygenic trait regulated by many small-sized effects. The region of 36,258 to 36,295 kb on BTA27 was the highly associated region for the predicted metabolic clusters, with the closest genes to this region (ANK1 and miR-486) being related to hematopoiesis, erythropoiesis, and mammary gland development. The heritability for metabolic clustering was 0.17 (SD 0.03), indicating that the use of FT-MIR spectra in milk to predict metabolic clusters in early-lactation across a large number of cows has satisfactory potential to be included in genetic selection programs for modern dairy cows.

Reproductive efficiency and survival of Holstein-Friesian cows of divergent Economic Breeding Index, evaluated under seasonal calving pasture-based management.

The objective of the current study was to examine phenotypic fertility performance and survival, and to gain insight into underlying factors that may contribute to greater fertility performance in 2 divergent genetic groups (GG) of Holstein-Friesian, selected using the Irish Economic Breeding Index (EBI). The GG were evaluated across 3 spring calving pasture-based feeding treatments (FT) over 4 yr. The 2 divergent GG were (1) high EBI; representative of the top 5% nationally (elite), and (2) EBI representative of the national average (NA). In each year, 90 elite and 45 NA cows were randomly allocated to 1 of 3 FT: control, lower grass allowance, and high concentrate. No interaction between GG and FT was observed for any of the measures of fertility investigated. The elite cows achieved significantly greater pregnancy rate to first service (+14.9 percentage points), and significantly greater pregnancy rates after 21, 42, and 84 d of breeding (+17.3, +15.2, and +9.6 percentage points, respectively) compared with NA. The number of services per cow was fewer for elite (1.57) compared with NA (1.80). The interval from mating start date to pregnancy was significantly shorter for elite cows compared with NA. The elite cows maintained greater mean body condition score than NA throughout the study (2.91 vs. 2.72), and had greater body condition score at calving, artificial insemination, and drying off compared with NA. The elite cows had greater mean circulating concentrations of insulin-like growth factor-1 compared with NA. No significant effect was observed of GG on commencement of luteal activity, or progesterone profile variables. Greater survival to the start of fifth lactation was observed for elite cows. The elite cows were 43% less likely to be culled than NA by the beginning of the fifth lactation. The results highlight the success of the Economic Breeding Index to deliver reproductive performance and longevity consistent with industry targets across a range of seasonal pasture-based FT. The results also clearly demonstrate the potential of appropriate genetic selection to reverse negative fertility trends incurred during previous decades of selection for milk production alone.

Potential of milk mid-infrared spectra to predict nitrogen use efficiency of individual dairy cows in early lactation.

Improving nitrogen use efficiency (NUE) at both the individual cow and the herd level has become a key target in dairy production systems, for both environmental and economic reasons. Cost-effective and large-scale phenotyping methods are required to improve NUE through genetic selection and by feeding and management strategies. The aim of this study was to evaluate the possibility of using mid-infrared (MIR) spectra of milk to predict individual dairy cow NUE during early lactation. Data were collected from 129 Holstein cows, from calving until 50 d in milk, in 3 research herds (Denmark, Ireland, and the UK). In 2 of the herds, diets were designed to challenge cows metabolically, whereas a diet reflecting local management practices was offered in the third herd. Nitrogen intake (kg/d) and nitrogen excreted in milk (kg/d) were calculated daily. Nitrogen use efficiency was calculated as the ratio between nitrogen in milk and nitrogen intake, and expressed as a percentage. Individual daily values for NUE ranged from 9.7 to 81.7%, with an average of 36.9% and standard deviation of 10.4%. Milk MIR spectra were recorded twice weekly and were standardized into a common format to avoid bias between apparatus or sampling periods. Regression models predicting NUE using milk MIR spectra were developed on 1,034 observations using partial least squares or support vector machines regression methods. The models were then evaluated through (1) a cross-validation using 10 subsets, (2) a cow validation excluding 25% of the cows to be used as a validation set, and (3) a diet validation excluding each of the diets one by one to be used as validation sets. The best statistical performances were obtained when using the support vector machines method. Inclusion of milk yield and lactation number as predictors, in combination with the spectra, also improved the calibration. In cross-validation, the best model predicted NUE with a coefficient of determination of cross-validation of 0.74 and a relative error of 14%, which is suitable to discriminate between low- and high-NUE cows. When performing the cow validation, the relative error remained at 14%, and during the diet validation the relative error ranged from 12 to 34%. In the diet validation, the models showed a lack of robustness, demonstrating difficulties in predicting NUE for diets and for samples that were not represented in the calibration data set. Hence, a need exists to integrate more data in the models to cover a maximum of variability regarding breeds, diets, lactation stages, management practices, seasons, MIR instruments, and geographic regions. Although the model needs to be validated and improved for use in routine conditions, these preliminary results showed that it was possible to obtain information on NUE through milk MIR spectra. This could potentially allow large-scale predictions to aid both further genetic and genomic studies, and the development of farm management tools.

Prediction of metabolic clusters in early-lactation dairy cows using models based on milk biomarkers.

The aim of this study was to describe metabolism of early-lactation dairy cows by clustering cows based on glucose, insulin-like growth factor I (IGF-I), free fatty acid, and ß-hydroxybutyrate (BHB) using the k-means method. Predictive models for metabolic clusters were created and validated using 3 sets of milk biomarkers (milk metabolites and enzymes, glycans on the immunogamma globulin fraction of milk, and Fourier-transform mid-infrared spectra of milk). Metabolic clusters are used to identify dairy cows with a balanced or imbalanced metabolic profile. Around 14 and 35 d in milk, serum or plasma concentrations of BHB, free fatty acids, glucose, and IGF-I were determined. Cows with a favorable metabolic profile were grouped together in what was referred to as the "balanced" group (n = 43) and were compared with cows in what was referred to as the "other balanced" group (n = 64). Cows with an unfavorable metabolic profile were grouped in what was referred to as the "imbalanced" group (n = 19) and compared with cows in what was referred to as the "other imbalanced" group (n = 88). Glucose and IGF-I were higher in balanced compared with other balanced cows. Free fatty acids and BHB were lower in balanced compared with other balanced cows. Glucose and IGF-I were lower in imbalanced compared with other imbalanced cows. Free fatty acids and BHB were higher in imbalanced cows. Metabolic clusters were related to production parameters. There was a trend for a higher daily increase in fat- and protein-corrected milk yield in balanced cows, whereas that of imbalanced cows was higher. Dry matter intake and the daily increase in dry matter intake were higher in balanced cows and lower in imbalanced cows. Energy balance was continuously higher in balanced cows and lower in imbalanced cows. Weekly or twice-weekly milk samples were taken and milk metabolites and enzymes (milk glucose, glucose-6-phosphate, BHB, lactate dehydrogenase, N-acetyl-ß-d-glucosaminidase, isocitrate), immunogamma globulin glycans (19 peaks), and Fourier-transform mid-infrared spectra (1,060 wavelengths reduced to 15 principal components) were determined. Milk biomarkers with or without additional cow information (days in milk, parity, milk yield features) were used to create predictive models for the metabolic clusters. Accuracy for prediction of balanced (80%) and imbalanced (88%) cows was highest using milk metabolites and enzymes combined with days in milk and parity. The results and models of the present study are part of the GplusE project and identify novel milk-based phenotypes that may be used as predictors for metabolic and performance traits in early-lactation dairy cows.

Role of early life nutrition on regulating the hypothalamic­anterior pituitary­testicular axis of the bull

The objective of this study was to examine the effect of nutrition during the first 18 weeks of life on the physiological and transcriptional functionality of the hypothalamic (arcuate nucleus region), anterior pituitary and testes in Holstein­Friesian bull calves. Holstein­Friesian bull calves with a mean (±S.D.) age and bodyweight of 19 (±8.2) days and 47.5 (±5.3) kg, respectively, were assigned to either a HIGH (n = 10) or LOW (n = 10) plane of nutrition, to achieve an overall target growth rate of 1.2 or 0.5 kg/day, respectively. At 126 ± 1.1 days of age, all calves were euthanised. Animal performance (weekly) and systemic concentrations of metabolic (monthly) and reproductive hormones (fortnightly) were assessed. Testicular histology, targeted gene and protein expression of the arcuate nucleus region, anterior pituitary and testes were also assessed using qPCR and immunohistochemistry, respectively. The expression of candidate genes in testicular tissue from post pubertal 19-month-old Holstein­Friesian bulls (n = 10) was compared to that of the 18-week-old calves. Metabolite and metabolic hormone profiles generally reflected the improved metabolic status of the calves on the HIGH (P< 0.001). Calves offered a HIGH plane of nutrition were heavier at slaughter (P < 0.001), had larger testes (P < 0.001), larger seminiferous tubule diameter (P < 0.001), more mature spermatogenic cells (P < 0.001) and more Sertoli cells (P < 0.05) in accordance with both morphological and transcriptional data. Overall, testicular gene expression profiles suggested a more mature stage of development in HIGH compared with LOW and were more closely aligned to that of mature bulls. Ghrelin receptor was the only differentially expressed gene between LOW and HIGH calves in either the anterior pituitary (P < 0.05) or arcuate nucleus region of the hypothalamus (P < 0.10) and was upregulated in LOW for both tissues. This study indicates that an enhanced plane of nutrition during early calfhood favourably alters the biochemical regulation of the hypothalamus­anterior pituitary­testicular axis, advancing testicular development and hastening spermatogenesis.

Plane of nutrition before and after 6 months of age in Holstein-Friesian bulls: II. Effects on metabolic and reproductive endocrinology and identification of physiological markers of puberty and sexual maturation.

The aim of this study was (1) to examine the effect of plane of nutrition during the first and second 6 mo of life on systemic concentrations of reproductive hormones and metabolites in Holstein-Friesian dairy bulls, and (2) to establish relationships with age at puberty and postpubertal semen production potential. Holstein-Friesian bull calves (n = 83) with a mean (standard deviation) age and body weight of 17 (4.4) d and 52 (6.2) kg, respectively, were assigned to a high or low plane of nutrition for the first 6 mo of life. At 24 wk of age, bulls were reassigned, within treatment, either to remain on the same diet or to switch to the opposite diet until puberty, resulting in 4 treatment groups: high-high, high-low, low-low, and low-high. Monthly blood samples were analyzed for metabolites (albumin, urea, total protein, ß-hydroxybutyrate, glucose, nonesterified fatty acid, triglycerides and creatinine), insulin, insulin-like growth factor-1, leptin, adiponectin, FSH, and testosterone. A GnRH challenge was carried out at 16 and 32 wk of age (n = 9 bulls per treatment). Blood was collected at 15-min intervals for 165 min, with GnRH administered (0.05 mg/kg of body weight, i.v.) immediately after the third blood sample. Blood samples were subsequently analyzed for LH, FSH, and testosterone. Stepwise regression was used to detect growth and blood measurements to identify putative predictors of age at puberty and subsequent semen quality traits. Metabolic hormones and metabolites, in general, reflected metabolic status of bulls. Although FSH was unaffected by diet, it decreased with age both in monthly samples and following GnRH administration. Testosterone was greater in bulls on the high diet before and after 6 mo of age. Testosterone concentrations increased dramatically after 6 mo of age. Luteinizing hormone was unaffected by diet following GnRH administration but basal serum LH was greater in bulls on a high diet before 6 mo of age. In conclusion, the plane of nutrition offered before 6 mo of age influenced metabolic profiles, which are important for promoting GnRH pulsatility, in young bulls.

A comparison of serum metabolic and production profiles of dairy cows that maintained or lost body condition 15 days before calving.

Body condition score (BCS) change is an indirect measure of energy balance. Energy balance before calving may affect production and health in the following lactation. It is likely that cows may experience BCS loss before calving due to negative energy balance. The objective of this study was to determine if loss of BCS 15d before calving affected milk production, BCS profile, and metabolic status during the transition period and early lactation. On d -15 to d 0 relative to calving, BCS was assessed (1=emaciated, 5=obese) for 98 Holstein-Friesian cows. The cows were divided into 2groups: those that did not lose BCS between d -15 and d 0 (maintained, BCS-M, n=55) and those that lost BCS from d -15 to d 0 (lost, BCS-L, n=43, average loss of 0.29±0.11 BCS). The fixed effects of BCS group, parity, week (day when analyzing milk production records), their interactions, and a random effect of cow were analyzed using PROC MIXED of SAS (SAS Institute Inc., Cary, NC). Before calving, BCS-L cows tended to have higher concentrations of nonesterified fatty acids than BCS-M cows (0.88 vs. 0.78mmol/L). After calving, BCS-L cows had higher nonesterified fatty acid concentrations in wk 1 (0.93 vs. 0.71mmol/L), wk 2 (0.84 vs. 0.69mmol/L), and wk 4 (0.81 vs. 0.63mmol/L) than BCS-M cows. The BCS-L cows had higher concentrations of ß-hydroxybutyrate (BHB) in wk 1 (0.72 vs. 0.57mmol/L), wk 2 (0.97 vs. 0.70mmol/L), and wk 4 (0.94 vs. 0.67mmol/L) compared with BCS-M cows. We detected significant reductions in insulin concentrations in BCS-L cows from wk -1 (2.23 vs. 1.37 µIU/mL) to wk 2 (1.68 vs. 0.89 µIU/mL) and wk 4 (2.21 vs 1.59 µIU/mL) compared with BCS-M cows. Prevalence of subclinical ketosis increased in BCS-L cows in wk 3 and 4 when BHB was ≥1.4mmol/L and in wk 1, 3, and 4 when BHB was ≥1.2mmol/L. In wk 1, BCS-L cows tended to have lower levels of calcium than BCS-M cows (2.33 vs. 2.27mmol/L). We found no differences between the groups of cows for milk yield and energy-corrected milk. The BCS-L cows had lower BCS up to 75d in lactation. Overall, BCS-L cows had higher somatic cell scores with an elevated somatic cell score on d 45, d 60, and d 75. There was an overall tendency for BCS-L cows to have higher fat yield and an overall significant increase in fat percentage. Overall, BCS-L cows had lower lactose percentage, with a reduction on d 60. This work shows that BCS loss before calving may have significant consequences for metabolic status, milk composition, somatic cell score, and BCS profile in dairy cows.

Fertility and genomics: comparison of gene expression in contrasting reproductive tissues of female cattle.

To compare gene expression among bovine tissues, large bovine RNA-seq datasets were used, comprising 280 samples from 10 different bovine tissues (uterine endometrium, granulosa cells, theca cells, cervix, embryos, leucocytes, liver, hypothalamus, pituitary, muscle) and generating 260 Gbases of data. Twin approaches were used: an information-theoretic analysis of the existing annotated transcriptome to identify the most tissue-specific genes and a de-novo transcriptome annotation to evaluate general features of the transcription landscape. Expression was detected for 97% of the Ensembl transcriptome with at least one read in one sample and between 28% and 66% at a level of 10 tags per million (TPM) or greater in individual tissues. Over 95% of genes exhibited some level of tissue-specific gene expression. This was mostly due to different levels of expression in different tissues rather than exclusive expression in a single tissue. Less than 1% of annotated genes exhibited a highly restricted tissue-specific expression profile and approximately 2% exhibited classic housekeeping profiles. In conclusion, it is the combined effects of the variable expression of large numbers of genes (73%-93% of the genome) and the specific expression of a small number of genes (<1% of the transcriptome) that contribute to determining the outcome of the function of individual tissues.

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

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

Key Factors Affecting Reproductive Success of Thoroughbred Mares and Stallions on a Commercial Stud Farm.

To evaluate factors contributing to fertility of thoroughbred mares, data from 3743 oestrous periods of 2385 mares were collected on a large thoroughbred farm in Ireland. Fourteen stallions (mean age 8.3 years; range 4-15 years) had bred 2385 mares (mean age 9.4 years; range 3-24 years). Maiden mares accounted for 12%, mares with a foal at foot for 64%, and barren, slipped or rested mares for 24% of the total. The mean pregnancy rate per cycle was 67.8% (68.6% in year 1 and 66.9% in year 2). Backward stepwise multivariable logistic regression analysis was utilized to develop two models to evaluate mare factors, including mare age, reproductive status, month of foaling, dystocia, month of cover, foal heat, cycle number, treatments, walk-in status and stallion factors including stallion identity, stallion age, shuttle status, time elapsed between covers and high stallion usage on the per cycle pregnancy rate and pregnancy loss. Old age (p < 0.001) and cover within 20 days post-partum (p < 0.003) were associated with lowered pregnancy rates. High mare age (p < 0.05) and barren, slipped or rested reproductive status (p = 0.05) increased the likelihood of pregnancy loss. Uterine inflammation or infection, if appropriately treated, did not affect fertility. Only high usage of stallions (used more than 21 times in previous week) was associated with lowered (p = 0.009) pregnancy rates. However, shuttle stallions were more likely to have increased (p = 0.035) pregnancy survival, perhaps reflecting a bias in stallion selection. In conclusion, mare age exerted the greatest influence on fertility; nonetheless, thoroughbreds can be effectively managed to achieve high reproductive performance in a commercial setting.

The relationship between activity clusters detected by an automatic activity monitor and endocrine changes during the periestrous period in lactating dairy cows.

The aim of this study was to determine the relationship between observed estrous-related behavior, activity clusters (AC; detected by automatic activity monitor), endocrine profiles, and ovulation time. Twenty-one cows in estrus (after 2 cloprostenol treatments, 11 d apart) and 12 nonsynchronized cows, to establish Heatime (SCR Engineers Ltd., Netanya, Israel) herd baseline activity, were enrolled. Cows had Heatime monitors applied 3 wk before the trial to establish their own baseline activity level. Cows in standing estrus had ultrasonography and phlebotomy carried out every 4 h to determine dominant follicle size, endocrine profiles, and ovulation time. After ovulation, these procedures were repeated once on d 3 to 6. Heatime alerted estrus in 90% of cows, and incorrectly alerted 17% of AC. The mean±SEM duration for standing estrus was 9±1 and 13±1 h for estrous-related behavior. Estrous-related behavior began after the start of the proestrous estradiol-17ß (E2) increase (59±6.5 h). Cows with longer durations of raised proestrous E2 had longer intervals from its onset to the start of standing estrus and AC. The AC duration increased with longer durations of estrous-related behavior. Higher peak E2 occurred with longer standing estrus and estrous-related behavior. As E2 concentration decreased after the peak, 90% of cows still had estrous-related behavior. Duration of estrous-related behavior increased with higher average E2 concentration during the last 8 h before the start of the LH surge. During this surge 90% of cows had all of their standing estrus. As yields increased, so did the magnitude of the preovulatory FSH surges. Higher surges occurred with shorter standing estrus and estrous-related behavior. Cows with shorter LH surges had longer standing estrus. Peak LH preceded the AC peak (6.6±0.8 h). Duration of overlap between the AC start and the LH surge end ranged between 0 and 14 h; 1 cow had none. No association was found between the AC characteristics with the E2, LH, or FSH profiles. In conclusion, the relationship between the timing of the E2 increase and estrous activity may be mediated by other factors (GnRH surge). Estrous-related behavior, but not endocrine profiles, was related to AC duration. Timing of standing estrus during the LH surge ensures that mating allows sperm maturation before ovulation. Based on the interval from the start of an AC to ovulation (27±1 h), the optimum time to artificial insemination is, on average, between 9 and 15 h after the AC start.

Pyrinomonas methylaliphatogenes gen. nov., sp. nov., a novel group 4 thermophilic member of the phylum Acidobacteria from geothermal soils.

An aerobic, thermophilic, moderately acidophilic non-spore-forming bacterium, strain K22(T), was isolated from geothermally heated soil at Mount Ngauruhoe, New Zealand. On the basis of 16S rRNA gene sequence similarity, K22(T) was shown to belong to subdivision 4 of the phylum Acidobacteria and to be most closely related to 'Candidatus Chloracidobacterium thermophilum' (86 %) and Blastocatella fastidiosa (86 %). Cells stained Gram-negative and were catalase and oxidase-positive. The major fatty acids detected were iso-C15 : 0, iso-C17 : 0, iso-C19 : 0 and iso-C21 : 0 when standard lipid extraction protocols were employed. Analysis of the total cell lipid acid hydrolysate also detected membrane-spanning and ether lipids, which made up approximately 40 % of the total membrane composition. These lipids included dicarboxylic (iso-diabolic) acid and the glyceryl ether of alkyl analogues of iso-C15 : 0 and iso-diabolic acid. The G+C content of the genomic DNA was 59.6 mol% and the primary respiratory quinone was MK-8. Strain K22(T) grew at 50-69 °C with an optimum temperature of 65 °C and at pH 4.1-7.8 with an optimum growth pH of 6.5. NaCl tolerance was up to 1 % (w/v). Cells displayed a chemoheterotrophic and obligately aerobic metabolism. Cells grew on nutrient broth, alginate, arabinose, Casamino acids, glucose, lactate, formate, mannose, sodium alginate, peptone, sucrose, tryptone, xanthan, xylan, xylose and yeast extract. Nitrogen sources included nitrate, ammonium, urea, yeast extract and Casamino acids, but not dinitrogen gas. The distinct phylogenetic position and the phenotypic characteristics separate strain K22(T) from all other members of the class Acidobacteria and indicate that it represents a novel species and genus, for which the name Pyrinomonas methylaliphatogenes gen. nov., sp. nov. is proposed. The type strain of the type species is K22(T) ( = DSM 25857(T) = ICMP 18710(T)).

Early pregnancy diagnosis on days 18 to 21 postinsemination using high-resolution imaging in lactating dairy cows.

The aim was to assess the ability of corpus luteum (CL) and uterine ultrasound characteristics on d 18 to 21 to predict pregnancy status in lactating dairy cows. Ultrasound examinations were carried out on cows (n = 164) on d 18 to 21 following artificial insemination (AI). Images of the uterus and CL were captured using a Voluson i ultrasound device (General Electric Healthcare Systems, Vienna, Austria) equipped with a 12-MHz, multi frequency, linear array probe. Serum concentrations of progesterone were determined from blood samples collected at each ultrasound examination. Images of the CL were captured and stored for calculation of CL tissue area and echotexture. Images of the CL and associated blood flow area were captured and stored for analysis of luteal blood flow ratio. Longitudinal B-mode images of the uterine horns were stored for analysis of echotexture. Diagnosis of pregnancy was made at each ultrasound examination based on CL blood flow, CL size, and uterine echotexture. Pregnancy was confirmed by ultrasonography on d 30 after AI. The relationship between ultrasound measures and pregnancy outcome, as well as the accuracy of the pregnancy diagnosis made at each ultrasound examination was assessed. Progesterone concentrations and CL tissue area were greater in pregnant compared with nonpregnant cows on all days. The CL blood flow ratio was higher in pregnant compared with nonpregnant cows on d 20 and 21 after AI. Echotexture measures of the CL and uterus were not different between pregnant and nonpregnant cows on any day of examination. The best logistic regression model to predict pregnancy included scores for CL blood flow, CL size, and uterine echotexture on d 21 following AI. Accuracy of pregnancy diagnosis was highest on d 21, with sensitivity and specificity being 97.6 and 97.5%, respectively. Uterine echotexture scores were similar for pregnant and nonpregnant cows from d 18 to 20. On d 21, pregnant cows had higher uterine echotexture scores compared with nonpregnant cows. The logistic regression equation most likely to provide a correct pregnancy diagnosis in lactating dairy cows included the visual score for CL blood flow, CL size, and uterine echotexture on d 21 after AI. In support of this finding, the diagnostic accuracy for visual scores of CL blood flow, CL size, and uterine echotexture were also highest on d 21.

Risk factors that affect reproductive target achievement in fertile dairy cows.

The aims of the present study were to investigate (1) the risk factors that influence the achievement of reproductive targets postpartum (pp) and (2) the key factors that influence pregnancy rate following first artificial insemination (AI) in dairy cows. Ninety-eight Holstein-Friesian pp cows were blood sampled from wk 1 to 4 pp for hematology and biochemistry. Reproductive tract health was assessed weekly by ultrasonography and vaginal mucus scoring. Body condition score (BCS), lameness score, and milk yield were assessed every 2 wk. Milk samples for progesterone assay were collected twice weekly and on d 4, 5, and 7 after AI. Risk factors associated with achieving reproductive targets depended on (1) increased metabolic activity of the liver (increased glutamate dehydrogenase at calving and increased γ-glutamyl transpeptidase in wk 4), (2) a competent immune system (increased neutrophils in wk 1; decreased α1-acid glycoprotein in wk 1, 2, and 3), (3) an endocrine system that was capable of responding by producing sufficient triiodothyronine in wk 2 and increased insulin-like growth factor I in wk 3 and 4, (4) a lower negative energy balance status (decreased nonesterified fatty acid concentration in wk 1; decreased ß-hydroxybutyrate concentration in wk 2; BCS loss between calving and d 28 pp <0.5), (5) good reproductive tract health [normal uterine scan at d 45 pp; clear vaginal mucus discharge at first ovulation and at d 45 pp; resumed ovarian cyclicity by the end of the voluntary waiting period (≥ d 35 pp)], and (6) adequate diet (to ensure increased glutathione peroxidase in wk 2 and 3 and increased magnesium in wk 4). Risk factors that increased the odds of a successful first AI were previous ovulation(s) (odds ratio=3.17 per ovulation), BCS >2.5 at AI (odds ratio=3.01), and clear vaginal mucus (score=0) compared with purulent mucus (score >0) 4 d after first AI (odds ratio=2.99). In conclusion, this study identified key risk factors in the early pp period that give a higher probability of cows achieving their reproductive targets and of having a first-AI pregnancy.

Altered endometrial immune gene expression in beef heifers with retarded embryos.

The aim of the present study was to compare endometrial gene expression profiles in a group of beef heifers yielding viable or retarded embryos on Day 7 after oestrus as a means of potentially explaining differences in embryo survival rates. Heifers were classified as either: (1) viable, when the embryo collected on Day 7 after oestrus was at the correct developmental stage (i.e. morula/early blastocyst); or (2) retarded, when the embryo was arrested at the 2-16-cell stage. The focus of the present study was on genes that were associated with either the pro- or anti-inflammatory immune response. Endometrial gene expression was determined using quantitative real-time polymerase chain reaction analysis. Expression of the ß-defensin (DEFB1), interferon (IFN)-α (IFNA), IFN-γ (IFNG), interleukin (IL)-6 (IL6), IL-10 (IL10), forkhead box P3 (FOXP3) and natural cytotoxicity triggering receptor 1 (NCR1) genes was lower in endometria from viable than retarded heifers. Expression of the nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (NKFB1), transforming growth factor (TGF)-ß (TGFB), IFN-γ-inducible protein 16 (IFI16) and IL-21 (IL21) genes was higher in viable than retarded heifers. We propose that small disturbances in the expression of immune genes in the endometrium on Day 7 after oestrus can have detrimental effects on embryo survival.