Enhancing bovine abortion surveillance: A learning experience.

Abortions and perinatal mortalities (APM) substantially affect cattle industry efficiency. Various infectious and noninfectious factors have been associated with bovine APM worldwide. Infections are often considered pivotal due to their abortifacient potential, leading laboratories to primarily investigate relevant infectious agents for APM cases. Some infectious causes, such as Brucella abortus, have also a zoonotic impact, necessitating monitoring for both animal and human health. However, underreporting of bovine APM is a global issue, affecting early detection of infectious and zoonotic causes. Previous studies identified factors influencing case submission, but regional characteristics may affect results. In Belgium, farmers are obliged to report cases of APM within the context of a national brucellosis monitoring program. The inclusion criteria for this monitoring program cover abortions (gestation length of 42-260 d) and perinatal mortalities of (pre)mature calves following a gestation length of more than 260 d, which were stillborn or died within 48 h after birth. The objective of the present study was to describe the evolution in submission of APM cases within a mandatory abortion monitoring program in relation to subsidized initiatives in the northern part of Belgium. Based on the proportion of APM submissions versus the proportion of bovine reproductive females, an APM proportion (APMPR) was calculated, and factors at both animal and herd level that may influence this APMPR were explored by using linear models. This evaluation revealed that the APMPR increased with the introduction of an extensive analytical panel of abortifacient agents and a free on-farm sample collection from 0.44% to 0.94%. Additionally, an increase of the APMPR was associated with an outbreak of an emerging abortifacient pathogen (Schmallenberg virus; 1.23%), and the introduction of a mandatory eradication program for bovine viral diarrhea virus (BVDv; 1.20%). The APMPR was higher in beef compared with dairy cattle, and it was higher in winter compared with fall, spring, and summer. Smaller herds categorized in the first quartile had a higher APMPR compared with larger herds. Herds that submitted an APM in the previous year had a higher APMPR in the next year compared with herds without an APM submission. Finally, herds for which there was evidence of the presence of BVDv had a higher APMPR compared with herds without evidence of the presence of BVDv. In conclusion, the number of APM submissions increased after the introduction of a free on-farm sample collection and an extensive pathogen screening panel. Production type (beef), season (winter), smaller herd size, previous APM, and presence of BVDv seemed to have a positive effect on APMPR. However, even under mandatory circumstances, APM still seems to be underreported, since the APMPR was lower than the expected minimal rate of 2%. Therefore, further research is necessary to identify the drivers that convince farmers to submit APM cases to improve submission rates and ensure an efficient monitoring program for APM and eventually associated zoonotic pathogens.

Maternal metabolic health and fertility: we should not only care about but also for the oocyte!

Metabolic disorders due to obesity and unhealthy lifestyle directly alter the oocyte's microenvironment and impact oocyte quality. Oxidative stress and mitochondrial dysfunction play key roles in the pathogenesis. Acute effects on the fully grown oocytes are evident, but early follicular stages are also sensitive to metabolic stress leading to a long-term impact on follicular cells and oocytes. Improving the preconception health is therefore of capital importance but research in animal models has demonstrated that oocyte quality is not fully recovered. In the in vitro fertilisation clinic, maternal metabolic disorders are linked with disappointing assisted reproductive technology results. Embryos derived from metabolically compromised oocytes exhibit persistently high intracellular stress levels due to weak cellular homeostatic mechanisms. The assisted reproductive technology procedures themselves form an extra burden for these defective embryos. Minimising cellular stress during culture using mitochondrial-targeted therapy could rescue compromised embryos in a bovine model. However, translating such applications to human in vitro fertilisation clinics is not simple. It is crucial to consider the sensitive epigenetic programming during early development. Research in humans and relevant animal models should result in preconception care interventions and in vitro strategies not only aiming at improving fertility but also safeguarding offspring health.

Low microbial biomass within the reproductive tract of mid-lactation dairy cows: A study approach.

The microbiome from the reproductive tract is being investigated for its putative effect on fertility, embryo development, and health status of the human or animal host postpartum. Besides the presence of a vaginal microbiome, recent studies have claimed the existence and putative role of the uterine microbiome. Yet, the extremely low bacterial numbers and high eukaryotic/prokaryotic DNA ratio make this a highly challenging environment to study with next-generation sequencing (NGS) techniques. Here, we describe the methodological challenges that are typically encountered when performing an accurate analysis of low microbial biomass samples, illustrated by data of our own observational study. In terms of the research question, we compared the microbial composition throughout different parts of the reproductive tract of clinically healthy, mid-lactation Holstein-Friesian cows. Samples were collected from 5 dairy cows immediately after killing. Swabs were taken from the vagina, and from 4 pre-established locations of the uterine endometrium. In addition to the conventional DNA extraction blank controls, sterile swabs rubbed over disinfected disposable gloves and the disinfected surface of the uterus (tunica serosa) before incision were taken as sampling controls. The DNA extraction, DNA quantification, quantitative PCR of the 16S rRNA genes, and 16S rRNA gene sequencing were performed. In terms of NGS data analysis, we performed prevalence-based filtering of putative contaminant operational taxonomic units (OTU) using the decontam R package. Although the bacterial composition differed between the vagina and uterus, no differences in bacterial community structure (α and ß diversity) were found among the different locations in the uterus. At phylum level, uterine samples had a greater relative abundance of Proteobacteria, and a lesser relative abundance of Firmicutes than vaginal samples. The number of shared OTU between vagina and uterus was limited, suggesting the existence of bacterial transmission routes other than the transcervical one to the uterus. The mid-lactation bovine genital tract is a low microbial biomass environment, which makes it difficult to distinguish between its constitutive versus contaminant microbiome. The integration of key controls is therefore strictly necessary to decrease the effect of accidentally introduced contaminant sequences and improve the reliability of results in samples with low microbial biomass.

Cytological endometritis diagnosed at artificial insemination in repeat breeder dairy cows.

This study aimed to (i) assess the prevalence of cytological endometritis (CYTO) diagnosed at artificial insemination (AI); (ii) evaluate the effect of CYTO on the pregnancy outcome of the same AI sample; and (iii) determine the risk factors associated with CYTO diagnosed at AI in repeat breeder (RB) dairy cows. We analysed the productive and reproductive performances of 146 RB Holstein-Friesian cows. To obtain a CYTO sample at AI, we used the cytotape technique. Generalized mixed effect models were computed to find the risk factors associated with the pregnancy and CYTO outcome. Based on ≥1% PMN cut-off point, the CYTO prevalence at AI in RB cows was 25.3%. The overall pregnancy at AI was 44.2%. The conception rate in CYTO-positive (n = 37) RB cows was 29.7% versus 49.5% for CYTO-negative (n = 109) cows. A RB cow diagnosed CYTO positive at AI had 0.47 [odds ratio (OR)] odds to become pregnant in comparison with a CYTO-negative cow. Cows that produced more milk than their counterparts in this study had increased odds (OR = 1.01) to be CYTO positive at AI. A novel risk factor positively associated with CYTO diagnosed at AI in RB cows was the level of daily milk urea (OR = 1.11). To conclude, CYTO at the moment of AI had a significantly negative effect on the pregnancy outcome in RB dairy cows. However, as only one of fourth of RB cows is affected with CYTO at AI, it may not be considered a key element associated with the RB syndrome.

Cytological endometritis at artificial insemination in dairy cows: Prevalence and effect on pregnancy outcome.

The aims of our field study in dairy cows were (1) to consolidate cytotape (CT) as a valid technique to diagnose cytological endometritis (CYTO) during artificial insemination (AI); (2) to establish a cutoff point concerning the polymorphonuclear cells (PMN) proportion to diagnose CYTO at AI; (3) to assess the prevalence of CYTO at AI; and (4) to evaluate the effect of CYTO on the pregnancy outcome of that AI. The investigation was performed using 1,625 AI-CT samples harvested from 873 Holstein-Friesian cows from 18 dairy farms in the Flemish region of Belgium. The CT device consisted of adapting a 1.5-cm piece of paper tape on the top of a conventional AI catheter covered with a double guard sheet, allowing an endometrial cytology sample to be taken when performing an AI. A receiving operator characteristic curve was built to assess the threshold level above which the PMN proportion significantly affected the AI success. Multilevel generalized mixed-effect models were built to identify factors affecting the pregnancy outcome of the AI under investigation. Only 7 samples (0.4%) harvested in 5 cows were discarded because of low-quality parameters. The cutoff point for CYTO at AI was set at ≥1% PMN (sensitivity=33.8%, specificity=88.6%). Prevalence of CYTO at AI was 27.8%. The conception rate for CYTO-positive samples was 32.7%, whereas it was 47% for CYTO-negative samples. A CYTO-negative AI had 1.8 [odds ratio (OR)] more chances to become pregnant than a CYTO-positive one. Other factors identified as detrimental for the pregnancy outcome were body condition score ≤1.5 (OR=0.6), relative 305-d milk yield (OR=0.9), dystocia (OR=0.3), parity ≥2 (OR=0.7), and warm months of the year. In conclusion, CT is a consolidated technique to diagnose CYTO at AI, PMN 1% is the threshold level to diagnose CYTO at AI, around one-quarter of inseminated uteri suffer from CYTO, and affected uteri having a significantly lower chance to become pregnant from that insemination.

Distribution of inflammation and association between active and chronic alterations within the endometrium of dairy cows.

Objectives of this study were twofold: (i) to assess the association between polymorphonuclear (PMN) counts and chronic alterations within the bovine endometrium and (ii) to determine the distribution of inflammation throughout the endometrium of clinically healthy dairy cows. Holstein-Friesian cows (n = 32) from a single dairy farm were selected for this experiment. Before slaughtering, a complete reproductive examination was performed to discard any type of clinical disease. After slaughtering, reproductive tracts were collected, and the endometrium was sampled at 8 pre-defined locations. At each location, endometrial biopsies (EBs) and cytology (CY) samples were harvested. Histopathology samples were stained with haematoxylin-eosin (EB-HE) and naphthol-AS-D-chloroacetate-esterase (EB-naphthol), while CY samples were stained with Wright-Giemsa. In the EB-HE samples, parameters assessed were epithelium height, mononuclear cells infiltration, lymphocytic aggregates, periglandular fibrosis, angiosclerosis and haemorrhage. In EB-naphthol and CY slides, PMNs counts were evaluated. Binomial logistic regression was used to assess the association between the number of PMNs present in both the EB-naphthol and CY samples and alterations identified in the EB-HE samples and to analyse the distribution of the histopathological alterations (EB-HE). A Poisson mixed-effect model was used to analyse the distribution of PMNs within the endometrium. A significant positive association was found between the PMN counts and the mononuclear cells infiltration. The presence of erythrocytes was associated with higher odds to detect PMNs in the stratum compactum. Significantly, higher infiltration of PMNs and mononuclear cells were detected in the uterine body and the right horn region. Concluding, CY is a technique that allows the evaluation of PMN counts and therefore only evaluates active inflammation. A complete assessment of endometrial health can only be obtained using EB. To optimize the sensitivity to diagnose endometrial inflammation in cows, adjacencies of the corpus uteri should be considered as the preferred region to harvest samples.

Machine learning modeling to predict causes of infectious abortions and perinatal mortalities in cattle.

A plethora of infectious and non-infectious causes of bovine abortions and perinatal mortalities (APM) have been reported in literature. However, due to financial limitations or a potential zoonotic impact, many laboratories only offer a standard analytical panel, limited to a preestablished number of pathogens. To improve the cost-efficiency of laboratory diagnostics, it could be beneficial to design a targeted analytical approach for APM cases, based on maternal and environmental characteristics associated with the prevalence of specific abortifacient pathogens. The objective of this retrospective observational study was to implement a machine learning pipeline (MLP) to predict maternal and environmental factors associated with infectious APM. Our MLP based on a greedy ensemble approach incorporated a standard tuning grid of four models, applied on a dataset of 1590 APM cases with a positive diagnosis that was achieved by analyzing an extensive set of abortifacient pathogens. Production type (dairy/beef), gestation length, and season were successfully predicted by the greedy ensemble, with a modest prediction capacity which ranged between 63 and 73 %. Besides the predictive accuracy of individual variables, our MLP hierarchically identified predictor importance causes of associated environmental/maternal characteristics of APM. For instance, in APM cases that happened in beef cows, season at APM (spring/summer) was the most important predictor with a relative importance of 24 %. Furthermore, at the last trimester of gestation Trueperella pyogenes and Neospora caninum were the most important predictors of APM with a relative importance of 22 and 17 %, respectively. Interestingly, herd size came out as the most relevant predictor for APM in multiparous dams, with a relative importance of 12 %. Based on these and other mix of predicted environmental/maternal and pathogenic potential causes, it could be concluded that implementing our MLP may be beneficial to design a more cost-effective, case-specific diagnostic approach for bovine APM cases at the diagnostic laboratory level.

Single closed-tube quantitative real-time PCR assay with dual-labelled probes for improved sex determination of equine embryos.

In addition to fulfilling many breeders' curiosity, equine embryonic sex determination can have a profound commercial impact. However, the application of currently described assays for equine embryonic sexing has rendered variable diagnosis and validation rates, with sensitivity being the main problem. In addition, while pregnancy results of in vivo-flushed equine embryos following a needle aspiration biopsy equal those of non-biopsied embryos, the effect on in vitro-produced embryos is unknown. Here, we aimed to develop a highly sensitive and specific assay for equine sex determination that can be directly performed on few embryonic cells, and to test the effect of a needle aspiration biopsy on the viability of the in vitro-produced embryo. To this end, a multiplex quantitative real-time PCR (qPCR) assay with dual-labelled probes was designed to allow the simultaneous generation of both male-specific and control fragments in a single closed-tube reaction, avoiding potential sample loss or contamination. To improve sensitivity, multicopy and polymeric genes were chosen to be specifically amplified, i.e., eight copies of Y-chromosomal ETSTY5 as male-specific and four autosomal UBC monomers as control fragment. Specificity was enhanced by the equine-specific character of ETSTY5 and by using dual-labelled probes. The assay was optimised with equine male and female genomic DNA and demonstrated a 100% accuracy and a >95% qPCR efficiency down to 10 pg of DNA. The assay was subsequently applied to determine the sex of 44 in vitro-produced embryos, collecting trophectoderm biopsies by means of a needle aspiration biopsy and herniating cells. Of all trophectoderm biopsies and herniating cell samples (n = 54), 87% could be diagnosed. Assay results were validated on a second sample obtained from the biopsied embryo (n = 18) or, by ultrasound-based sex determination of the foetus (n = 7) following the transfer of the biopsied embryo to a recipient mare, with about half of the embryos being fillies and colts. The needle aspiration biopsy procedure did not impair initial pregnancy rate or early pregnancy losses as compared to non-biopsied embryos. In conclusion, we report a safe, reliable, fast, and cost-effective assay for equine sex determination which was validated for the sex determination of in vitro-produced embryos based on few embryonic cells, and needle aspiration biopsy did not impair the embryo's viability. The assay and safe biopsy strategy hold potential for other applications.

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.

Effects of parity, blood progesterone, and non-steroidal anti-inflammatory treatment on the dynamics of the uterine microbiota of healthy postpartum dairy cows.

This study evaluated the effects of treatment with meloxicam (a non-steroidal anti-inflammatory drug), parity, and blood progesterone concentration on the dynamics of the uterine microbiota of 16 clinically healthy postpartum dairy cows. Seven primiparous and 9 multiparous postpartum Holstein cows either received meloxicam (0.5 mg/kg SC, n = 7 cows) once daily for 4 days (10 to 13 days in milk (DIM)) or were untreated (n = 9 cows). Endometrial cytology samples were collected by cytobrush at 10, 21, and 35 DIM, from which the microbiota analysis was conducted using 16S rRNA gene sequence analysis. A radioimmunoassay was used to measure progesterone concentration in blood serum samples at 35 DIM and cows were classified as ˃ 1 ng/mL (n = 10) or ≤ 1 ng/mL (n = 6). Alpha diversity for bacterial genera (Chao1, Shannon-Weiner, and Camargo's evenness indices) were not affected by DIM, meloxicam treatment, parity, or progesterone category. For beta diversity (genera level), principal coordinate analysis (Bray-Curtis) showed differences in microbiota between parity groups. At the phylum level, the relative abundance of Actinobacteria was greater in primiparous than multiparous cows. At the genus level, there was lesser relative abundance of Bifidobacterium, Lactobacillus, Neisseriaceae, Paracoccus, Staphylococcus, and Streptococcus and greater relative abundance of Bacillus and Fusobacterium in primiparous than multiparous cows. Bray-Curtis dissimilarity did not differ by DIM at sampling, meloxicam treatment, or progesterone category at 35 DIM. In conclusion, uterine bacterial composition was not different at 10, 21, or 35 DIM, and meloxicam treatment or progesterone category did not affect the uterine microbiota in clinically healthy postpartum dairy cows. Primiparous cows presented a different composition of uterine bacteria than multiparous cows. The differences in microbiota associated with parity might be attributable to changes that occur consequent to the first calving, but this hypothesis should be investigated further.

Effect of lycopene supplementation to bovine oocytes exposed to heat shock during in vitro maturation.

We investigated the effect of the antioxidant lycopene supplemented into the in vitro maturation medium (TCM-199 with 20 ng/mL epidermal growth factor and 50 mg/mL gentamycin) in a heat shock (HS) model to mimic in vivo heat stress conditions. Bovine cumulus-oocyte complexes were supplemented with 0.2 µM lycopene (or not supplemented; control) under HS (40.5 °C) and non-HS (NHS; 38.5 °C) during maturation. After 22 h of maturation, we evaluated the nuclear status of the oocytes, the level of reactive oxygen species (ROS) production, and the respective blastocyst development and quality (via differential staining). Data were fitted in logistic and linear regression models, and the replicates were set as a random effect. The nuclear maturation was higher in NHS (84.0 ± 3.2%; least square mean ± standard error) than HS control (60.4 ± 4.3%; P < 0.001). Remarkably, the nuclear maturation in HS lycopene (71.7 ± 4.1%) was similar to NHS control (P = 0.7). Under HS conditions lycopene reduced ROS production (27.4 ± 4.8; relative fluorescence units (RFU)) in comparison to HS control (33.8 ± 1.8 RFU; P = 0.009). However, the ROS production in NHS lycopene (18.9 ± 2.0 RFU) was similar to NHS control (18.7 ± 1.8 RFU; P = 0.9). The cleavage rate in HS lycopene (76.1 ± 3.3%) was not lower than NHS lycopene (83.3 ± 2.5%; P > 0.1). On the day 8 of embryo development, the blastocyst rate was higher for NHS lycopene (55.2 ± 4.7%) versus NHS control (44.5 ± 4.7%; P = 0.04), but under HS the day 8 blastocyst rate was similar between control (29.9 ± 4.2%) and lycopene (32.3 ± 4.2%; P = 0.9). Lycopene supplementation increased the cell number of the embryos (total cell, trophectoderm, and inner cell mass numbers) under NHS conditions (P > 0.03). The apoptotic cell ratio was lower in lycopene (NHS and HS) versus control (NHS and HS) (P > 0.04). Lycopene has the ability to scavenge oocyte ROS and improved the cleavage rate of embryos under HS conditions. However, this could not be translated to a higher blastocyst development, which remained lower under HS. Results of our study indicate that antioxidant supplementation like lycopene during the maturation of bovine cumulus-oocyte complexes may be routinely used to improve blastocyst rate and quality under standard maturation conditions.

Metabolic markers for purulent vaginal discharge and subclinical endometritis in dairy cows.

This study compares serum markers for systemic inflammation, and liver, mineral, and energy status in samples obtained -7, 1, 3, 5, 7, 14 ± 1, 21 ± 1, and 35 d relative to calving from healthy dairy cows and those diagnosed with purulent vaginal discharge (PVD) or subclinical endometritis (SCE). Metabolites and markers measured in serum were total calcium, total protein, albumin, globulin, cholesterol, urea, glucose, gamma-glutamyl transferase, aspartate aminotransferase, glutamate dehydrogenase, ß-hydroxybutyrate (BHB), non-esterified fatty acids (NEFA), haptoglobin (Hp), and insulin-like growth factor-1 (IGF-1). Holstein cows with no recorded clinical disease were classified healthy (neither PVD nor SCE; n = 38), PVD (n = 10) or SCE (n = 10) at 35 d postpartum. The cut-point for PVD was mucopurulent vaginal discharge or worse, measured with Metricheck, and for SCE > 5% endometrial polymorphonuclear cells (PMN). Purulent vaginal discharge and SCE were mutually exclusive categories. The association of each serum marker with reproductive tract health classification was fitted in mixed linear regression models, accounting for repeated measures, sampling day, parity, BCS, and interactions of reproductive tract status and day. Serum Hp concentrations were greater at 3, 5, 7, and 14 ± 1 d postpartum for SCE and at 7 and 35 d postpartum for PVD than in healthy cows. Albumin concentrations were lesser for PVD than healthy at 14 ± 1 d postpartum and lesser for SCE than healthy at 35 d postpartum. The week before calving, SCE had lesser total calcium than healthy cows, and at 7 and 14 ± 1 d postpartum PVD had lesser total calcium than healthy cows. At 14 ± 1 d postpartum, serum NEFA, BHB, and globulin were greater, and IGF-1 lower for SCE than PVD or healthy cows. For all other metabolites, no differences were found. Before diagnosis, PVD or SCE had more indication of postpartum systemic inflammation (high Hp and low albumin) than healthy cows, and markers of energy status were more compromised in SCE than in PVD or healthy cows. This supports the hypothesis that SCE is associated with maladaptation to postpartum metabolic demands and with metabolic inflammation.

Assessment of associations between transition diseases and reproductive performance of dairy cows using survival analysis and decision tree algorithms.

This study aimed to evaluate the associations between transition cow conditions and diseases TD with fertility in Holstein cows, and to compare analytic methods for doing so. Kaplan-Meier, Cox proportional hazard, and decision tree models were used to analyze the associations of TD with the pregnancy risk at 120 and 210 DIM from a 1-year cohort with 1946 calvings from one farm. The association between TD and fertility was evaluated as follows: 1 cows with TD whether complicated with another TD or not TD-all, versus healthy cows, and 2 cows with uncomplicated TD TD-single, versus cows with multiple TD TD+; complicated cases, versus healthy cows. The occurrence of twins, milk fever, retained placenta, metritis, ketosis, displaced abomasum, and clinical mastitis were recorded. Using Kaplan-Meier models, in primiparous cows the 120 DIM pregnancy risk was 62% (95% CI: 57-67 %) for healthy animals. This was not significantly different for TD-single (58%; 95% CI: 51-66 %) but was reduced for TD+ (45%; 95% CI: 33-60 %). Among healthy primiparous cows, 80% (95% CI: 75-84 %) were pregnant by 210 DIM, but pregnancy risk at that time was reduced for primiparous cows with TD-single (72%; 95% CI: 65-79 %) and TD+ (62%; 95% CI: 49-75 %). In healthy multiparous cows, the 120 DIM pregnancy risk was 53% (95% CI: 49-56 %), which was reduced for TD-single (36%; 95% CI: 31-42 %) and TD+ (30%; 95% CI: 24-38 %). The 210 DIM pregnancy risk for healthy multiparous cows was 70% (95% CI: 67-72 %), being higher than the 210 DIM pregnancy risk for multiparous cows with TD-single (47%; 95% CI: 42-53 %) or TD+ (46%; 95% CI: 38-54 %). Cows with TD-all presented similar pregnancy risk estimates as for TD + . Cox proportional hazards regressions provided similar magnitudes of effects as the Kaplan-Meier estimates. Survival analysis and decision tree models identified parity as the most influential variable affecting fertility. Both modeling techniques concurred that TD + had a greater effect than TD-single on the probability of pregnancy at 120 and 210 DIM. Decision trees for individual TD identified that displaced abomasum affected fertility at 120 DIM in primiparous while metritis was the most influential TD at 120 and 210 DIM for multiparous cows. The data were too sparse to assess multiple interactions in multivariable Cox proportional hazard models for individual TD. Machine learning helped to explore interactions between individual TD to study their hierarchical effect on fertility, identifying conditional relationships that merit further investigation.

Comparison of cow-side diagnostic techniques for subclinical endometritis in dairy cows.

A limited number of cow-side diagnostic techniques exist for the diagnosis of subclinical endometritis (SCE) in dairy cattle. The objectives of this study were to compare results of endometrial cytology from samples collected by cytobrush (CB) and low-volume lavage (LVL) and to assess leukocyte esterase (LE) test strips and Brix refractometry as surrogate cow-side tests for SCE. Two samples were consecutively collected from 248 Holstein cows between 29 and 35 days postpartum, using CB and LVL techniques. Each sample was analyzed using cytology with a cut-point of ≥5% polymorphonuclear (PMN) cells, LE strips using cut-points of ≥1 and ≥ 2, and a Brix refractometer. Each diagnostic technique was compared intra-sample using the respective cytology as a gold standard and inter-sample using CB samples as the referent. The concordance correlation coefficient (CCC, ρc) for PMN% between CB and LVL was ρc = 0.59 [95% confidence interval (CI): 0.50 to 0.67] and the Kappa (κ) for agreement was κ = 0.35 [sensitivity (Se) = 0.88, specificity (Sp) = 0.45]. The optimal cut-point of LE ≥ 2 resulted in moderate agreement between CB and LVL samples, κ = 0.56 (Se = 0.89, Sp = 0.65). Agreement between LE and cytology using CB (κ = 0.49; Se = 0.89, Sp = 0.57) and LVL (κ = 0.44; Se = 0.77, Sp = 0.67) were similar. The correlation between Brix values from CB and LVL was ρc = 0.12 (CI -0.01 to 0.26). The correlation between CB cytology and Brix was ρc = 0.33 (CI 0.20 to 0.45) but ρc = -0.07 (CI -0.21 to 0.06) between LVL cytology and Brix. While LE strips with a cut-point of LE ≥ 2 had moderate agreement with cytology, Brix refractometry had poor performance for the diagnosis of SCE. Samples taken by CB and LVL produced comparable cow-side diagnostic results and either is a viable method for the diagnosis of SCE.

An improved vitrification protocol for equine immature oocytes, resulting in a first live foal.

BACKGROUND: The success rate for vitrification of immature equine oocytes is low. Although vitrified-warmed oocytes are able to mature, further embryonic development appears to be compromised. OBJECTIVES: The aim of this study was to compare two vitrification protocols, and to examine the effect of the number of layers of cumulus cells surrounding the oocyte during vitrification of immature equine oocytes. STUDY DESIGN: Experimental in vitro and in vivo trials. METHODS: Immature equine oocytes were vitrified after a short exposure to high concentrations of cryoprotective agents (CPAs), or a long exposure to lower concentrations of CPAs. In Experiment 1, the maturation of oocytes surrounded by multiple layers of cumulus cells (CC oocytes) and oocytes surrounded by only corona radiata (CR oocytes) was investigated. In Experiment 2, spindle configuration was determined for CR oocytes vitrified using the two vitrification protocols. In Experiment 3, further embryonic development was studied after fertilisation and culture. Embryo transfer was performed in a standard manner. RESULTS: Similar nuclear maturation rates were observed for CR oocytes vitrified using the long exposure and nonvitrified controls. Furthermore, a lower maturation rate was obtained for CC oocytes vitrified with the short exposure compared to control CR oocytes (P = 0.001). Both vitrification protocols resulted in significantly higher rates of aberrant spindle configuration than the control groups (P<0.05). Blastocyst development only occurred in CR oocytes vitrified using the short vitrification protocol, and even though blastocyst rates were significantly lower than in the control group (P<0.001), transfer of five embryos resulted in one healthy foal. MAIN LIMITATIONS: The relatively low number of equine oocytes and embryo transfer procedures performed. CONCLUSIONS: For vitrification of immature equine oocytes, the use of 1) CR oocytes, 2) a high concentration of CPAs, and 3) a short exposure time may be key factors for maintaining developmental competence.