Novel high throughput screen reports that benzo(a)pyrene overrides mouse trophoblast stem cell multipotency, inducing SAPK activity, HAND1 and differentiated trophoblast giant cells.

INTRODUCTION: Cultured mouse trophoblast stem cells (mTSC) maintain proliferation/normal stemness (NS) under FGF4, which when removed, causes normal differentiation (ND). Hypoxic, or hyperosmotic stress forces trophoblast giant cells (TGC) differentiate. Hypoxic, hyperosmotic, and genotoxic benzo(a)pyrene (BaP), which is found in tobacco smoke, force down-regulation of inhibitor of differentiation (Id)2, enabling TGC differentiation. Hypoxic and hyperosmotic stress induce TGC by SAPK-dependent HAND1 increase. Here we test whether BaP forces mTSC-to-TGC while inducing SAPK and HAND1. METHODS: Hand1 and SAPK activity were assayed by immunoblot, mTSC-to-TGC growth and differentiation were assayed at Tfinal after 72hr exposure of BaP, NS, ND, Retinoic acid (RA), or sorbitol. Nuclear-stained cells were micrographed automatically by a live imager, and assayed by ImageJ/FIJI, Biotek Gen 5, AIVIA proprietary artificial intelligence (AI) software or open source, CellPose artificial intelligence/AI software. RESULTS: BaP (0.05-1µM) activated SAPK and HAND1 without diminishing growth. TSC-to-TGC differentiation was assayed with increasingly accuracy for 2-4 N cycling nuclei and >4 N differentiating TGC nuclei, using ImageJ/FIJI, Gen 5, AIVIA, or CellPose AI software. The AIVIA and Cellpose AI software matches human accuracy. The lowest BaP effects on SAPK activation/HAND1 increase are >10-fold more sensitive than similar effects for mESC. RA induces 44-47% 1st lineage TGC differentiation, but the same RA dose induces only 1% 1st lineage mESC differentiation. DISCUSSION: First, these pilot data suggest that mTSC can be used in high throughput screens (HTS) to predict toxicant exposures that force TGC differentiation. Second, mTSC differentiated more cells than mESC for similar stress exposures, Third, open source AI can replace human micrograph quantitation and enable a miscarriage-predicting HTS.

Development of sustainable performance index based on production, reproduction, health and longevity for genetic improvement of Sahiwal cattle.

An attempt was made for the first time in India to develop the performance indices for quantifying the relative emphasis to be given to production, reproduction, health and longevity traits for selection and genetic improvement of Sahiwal cattle in a sustainable manner. The study was conducted using information related to various production, reproduction, health and longevity traits spread over a period of 29 years in Sahiwal cattle. Using income and expenditure method, the relative economic values for 305 days milk yield (305DMY), average daily milk yield (ADMY), calving to first insemination interval (CFI), days open (DO) and longevity (LNG) were estimated as 1, 1, -6.62, -6.62 and 5.96 in Sahiwal cattle. A total of three performance indices were constructed using three-trait combination, that is production, reproduction and longevity before and after excluding the days suffered by the animals. Correlation between the aggregate genotype and index (RIH ) was computed to determine the accuracy of each performance index, and based on highest RIH value, best performance index was identified for selection of Sahiwal cattle. Developed indices were further assessed for robustness by increasing the relative economic values of the traits by 25% and 50%. The performance index (305DMY, DO and LNG) was found to be the best index before as well as after excluding days suffered by the animals, but in later case emphasis to be given to different traits was found to be more balanced. The best index indicated that about 47, 42 and 11% relative emphasis to be assigned to 305 days milk yield, days open and longevity for selection of Sahiwal animals for sustainable genetic improvement.

Mutations in growth hormone gene affect stability of protein structure leading to reduced growth, reproduction, and milk production in crossbred cattle-an insight.

The GH has a multifaceted role in growth, reproduction, and milk production. Nonsynonymous mutations identified as L153V were observed from GH1 (L) to GH2 (V) with higher genotypic frequency for GH1 being 0.87. GH2 (V) allele was identified as a rare allele and the population followed Hardy-Weinberg equilibrium. LL homozygote variant had significantly better growth, reproduction, and expected milk production at different ages in crossbred (CB) males. Reports are scanty explaining the molecular mechanism of how individuals with LV genotype were phenotypically inferior to that of wild-type LL. In the present study, it was explored that GH peptide with LV heterozygotes of GH gene, were observed to have reduced structural stability thermodynamically and thus functionally leads to reduced economic traits in CB animals. The fact was first time reported and confirmed through genomic analysis, bioinformatics, and later confirmed through immunohistochemistry. Differential expression analysis of the GH gene with respect to other genes in the hypothalamus-pituitary growth axis of CB cattle was also studied to have a complete insight of the GH gene.

Point prevalence & risk factor assessment for hospital-acquired infections in a tertiary care hospital in Pune, India.

BACKGROUND & OBJECTIVES: Hospital-acquired infections (HAIs) are a major challenge to patient safety and have serious public health implications by changing the quality of life of patients and sometimes causing disability or even death. The true burden of HAI remains unknown, particularly in developing countries. The objective of this study was to estimate point prevalence of HAI and study the associated risk factors in a tertiary care hospital in Pune, India. METHODS: A series of four cross-sectional point prevalence surveys were carried out between March and August 2014. Data of each patient admitted were collected using a structured data entry form. Centers for Disease Control and Prevention guidelines were used to identify and diagnose patients with HAI. RESULTS: Overall prevalence of HAI was 3.76 per cent. Surgical Intensive Care Unit (ICU) (25%), medical ICU (20%), burns ward (20%) and paediatric ward (12.17%) were identified to have significant association with HAI. Prolonged hospital stay [odds ratio (OR=2.81), mechanical ventilation (OR=18.57), use of urinary catheter (OR=7.89) and exposure to central air-conditioning (OR=8.59) had higher odds of acquiring HAI (P<0.05). INTERPRETATION & CONCLUSIONS: HAI prevalence showed a progressive reduction over successive rounds of survey. Conscious effort needs to be taken by all concerned to reduce the duration of hospital stay. Use of medical devices should be minimized and used judiciously. Healthcare infection control should be a priority of every healthcare provider. Such surveys should be done in different healthcare settings to plan a response to reducing HAI.

Distribution of Class II integrons and their contribution to antibiotic resistance within Enterobacteriaceae family in India.

BACKGROUND: Integrons are the main contributors to the development of multidrug resistance (MDR) among Gram-negative bacilli. There is a lack of knowledge about the molecular relation between gene cassettes and antibiotic resistance in India. OBJECTIVE: In this study, we have investigated the occurrence of Class II integron and their cassette array among Enterobacteriaceae. MATERIALS AND METHODS: A total of 268 MDR non-duplicate strains of Enterobacteriaceae were collected from Silchar Medical College and Hospital, Silchar, Assam, India, during June 2012 to May 2013. Polymerase chain reaction was performed for detection of the integrase genes and gene cassettes within the Class II integron which were further analysed by sequencing. RESULTS: Class II integron was observed in 47 isolates. Four different gene cassette arrangements were detected: dfrA1-sat2-aadA1; dfrA1-sat2-aadA1-orfX-ybeA-ybfA-ybfB-ybgA; dfrA12-sat2-aadA1; and dfrA1-linF-aadA1. The most prevalent cassette combination was dfrA1-sat2-aadA1. This study has also identified a set of gene cassette associated with linF gene instead of sat2 gene. CONCLUSION: Further investigation is required to determine the current situation and important reservoir of Class II integron for the transmission of drug resistance among Enterobacteriaceae and their contribution to antimicrobial resistance in hospital environment .

Evaluation of fertility in relation to milk production and productivity of Murrah buffaloes.

Intense selection of buffaloes for milk production at organized herds of the country without giving due attention to fertility traits has lead to deterioration in their performances. The study was initiated to assess the relationship between milk production and productivity with fertility in Murrah buffaloes. In the present study, fertility was defined in terms of pregnancy rate of Murrah buffaloes. Pregnancy rate measures the percentage of non-pregnant animals that become pregnant during each oestrous cycle. Data pertaining to 1224 lactation records of Murrah buffaloes spread over a period 22 years from 1993 to 2014 were analyzed in the study. It was observed that pregnancy rate is negatively associated with 305days or less milk yield (-0.08±0.04) and wet average (-0.12±0.02) and positively associated with life time (0.15±0.03) in Murrah buffaloes. Wet average is defined as average daily milk yield per lactation of Murrah buffaloes. To achieve around 2000kg 305days or less milk yield and 7.5kg wet average, the level of pregnancy rate in Murrah buffaloes should vary between 30 and 50%. The per unit change in pregnancy rate with respect to milk yield in Murrah buffaloes, were studied using regression analysis. The results suggest that increasing hundred kilogram in 305days or one kilogram milk yield per day in 305days, pregnancy rate reduced by 0.9% in overall lactations of Murrah buffaloes. By increasing hundred kilogram life time 305days or less milk yield and one kilogram life time wet average, pregnancy rate of Murrah buffaloes reduced by about 0.2% and 0.34%, respectively. The study quantifies the decline of pregnancy rate with increase of lactation milk yield and wet average in Murrah buffaloes and emphasis the importance of fertility i.e. pregnancy rate in the evaluation and breeding programmes of Murrah buffaloes.

Effect of heat stress on reproductive performances of dairy cattle and buffaloes: A review.

Heat stress has adverse effects on the reproductive performances of dairy cattle and buffaloes. The dairy sector is a more vulnerable to global warming and climate change. The temperature humidity index (THI) is the widely used index to measure the magnitude of heat stress in animals. The objective of this paper was to assess the decline in performances of reproductive traits such as service period, conception rate and pregnancy rate of dairy cattle and buffaloes with respect to increase in THI. The review stated that service period in cattle is affected by season of calving for which cows calved in summer had the longest service period. The conception rate and pregnancy rate in dairy cattle were found decreased above THI 72 while a significant decline in reproductive performances of buffaloes was observed above threshold THI 75. The non-heat stress zone (HSZ) (October to March) is favorable for optimum reproductive performance, while fertility is depressed in HSZ (April to September) and critical HSZ (CHSZ) (May and June). Heat stress in animals has been associated with reduced fertility through its deleterious impact on oocyte maturation and early embryo development. The management strategies viz., nutrition modification, environment modification and timed artificial insemination protocol are to be strictly operated to ameliorate the adverse effects of heat stress in cattle and buffaloes during CHSZ to improve their fertility. The identification of genes associated with heat tolerance, its incorporation into breeding program and the inclusion of THI covariate effects in selection index should be targeted for genetic evaluation of dairy animals in the hot climate.

Estimates of genetic parameters for fat yield in Murrah buffaloes.

AIM: The present study was performed to investigate the effect of genetic and non-genetic factors affecting milk fat yield and to estimate genetic parameters of monthly test day fat yields (MTDFY) and lactation 305-day fat yield (L305FY) in Murrah buffaloes. MATERIALS AND METHODS: The data on total of 10381 MTDFY records comprising the first four lactations of 470 Murrah buffaloes calved from 1993 to 2014 were assessed. These buffaloes were sired by 75 bulls maintained in an organized farm at ICAR-National Dairy Research Institute, Karnal. Least squares maximum likelihood program was used to estimate genetic and non-genetic parameters. Heritability estimates were obtained using paternal half-sib correlation method. Genetic and phenotypic correlations among MTDFY, and 305-day fat yield were calculated from the analysis of variance and covariance matrix among sire groups. RESULTS: The overall least squares mean of L305FY was found to be 175.74±4.12 kg. The least squares mean of overall MTDFY ranged from 3.33±0.14 kg (TD-11) to 7.06±0.17 kg (TD-3). The h(2) estimate of L305FY was found to be 0.33±0.16 in this study. The estimates of phenotypic and genetic correlations between 305-day fat yield and different MTDFY ranged from 0.32 to 0.48 and 0.51 to 0.99, respectively. CONCLUSIONS: In this study, all the genetic and non-genetic factors except age at the first calving group, significantly affected the traits under study. The estimates of phenotypic and genetic correlations of MTDFY with 305-day fat yield was generally higher in the MTDFY-5 of lactation suggesting that this TD yields could be used as the selection criteria for early evaluation and selection of Murrah buffaloes.

Lactoferrin gene promoter variants and their association with clinical and subclinical mastitis in indigenous and crossbred cattle.

Lactoferrin (Lf) gene promoter was screened for the presence of single nucleotide polymphism in indigenous and crossbred cattle from North India and to evaluate its association with Mastitis. Study revealed the presence of genetic variation in regulatory region of bovine Lactoferrin gene using PCR-RFLP technique. Three genotypes namely GG, GH and HH were identified. A single nucleotide change, from guanine to adenine at 25th position was found to be significantly associated (p<0.05) with clinical mastitis in indigenous Sahiwal and crossbred Karan Fries cattle maintained at organised herd of National Dairy Research Institute, Karnal. A non-significant association was observed between subclinical mastitis, somatic cell score (SCS), and GG genotype in Karan Fries cattle, however, a lower SCS was observed in animals having GG genotype. Overall a lower incidence of clinical mastitis was recorded in those animals having GG genotype of Lf in Sahiwal and Karan Fries (KF) cattle. The SNP identified in the promoter region may effect expression lactoferrin protein, which may lead to different levels of antibacterial and anti-inflammatory activity of Lf gene. Results from this study indicated the probable role played by Lactoferrin promoter to serve as candidate gene for mastitis susceptibility among indigenous and crossbred milch cattle.

Influence of Temperature and Humidity on Pregnancy Rate of Murrah Buffaloes under Subtropical Climate.

Heat stress has adverse effects on fertility of dairy animals. Decline in fertility is linearly associated with an increase in combination of both temperature and humidity. The purpose of this study was to investigate the relationship between temperature humidity index (THI) and the pregnancy rate of Murrah buffaloes in a subtropical climate. The effects of genetic and non-genetic factors viz., sire, parity, period of calving and age group at first calving were found non-significant on pregnancy rate. The effect of THI was found significant (p<0.001) on pregnancy rate of Murrah buffaloes calved for first time and overall pregnancy rate. The threshold THI affecting the pregnancy rate was identified as THI 75. The months from October to March showed THI<75 and considered as non heat stress zone (NHSZ), while months from April to September were determined as heat stress zone (HSZ) with THI≥75. The lowest overall pregnancy rate (0.25) was obtained in July with THI 80.9, while the highest overall pregnancy rate (0.59) was found in November with THI 66.1. May and June were identified as critical heat stress zone (CHSZ) within the HSZ with maximum decline (-7%) in pregnancy rate with per unit increase in THI. The highest overall pregnancy rate was estimated as 0.45 in NHSZ with THI value 56.7 to 73.2. The pregnancy rate was found to have declined to 0.28 in HSZ with THI 73.5 to 83.7. However, the lowest pregnancy rate was estimated as 0.27 in CHSZ with THI value 80.3 to 81.6.

Contribution of efflux pumps in fluroquinolone resistance in multi-drug resistant nosocomial isolates of Pseudomanas aeruginosa from a tertiary referral hospital in north east India.

BACKGROUND: Pseudomonas aeruginosa is one of the leading opportunistic pathogen and its ability to acquire resistance against series of antimicrobial agents confine treatment option for nosocomial infections. Increasing resistance to fluroquinolone (FQ) agents has further worsened the scenario. The major mechanism of resistance to FQs includes mutation in FQs target genes in bacteria (DNA gyrase and/or topoisomerases) and overexpression of antibiotic efflux pumps. OBJECTIVE: We have investigated the role of efflux pump mediated FQ resistance in nosocomial isolates of P. aeruginosa from a tertiary referral hospital in north eastern part of India. MATERIALS AND METHODS: A total of 234 non-duplicate, consecutive clinical isolates of P. aeruginosa were obtained from a tertiary referral hospital of north-east India. An efflux pump inhibitor (EPI), carbonyl cyanide m-chlorophenylhydrazone (CCCP) based method was used for determination of efflux pump activity and multiplex polymerase chain reaction (PCR) was performed for molecular characterisation of efflux pump. Minimum inhibitory concentration (MIC) reduction assay was also performed for all the isolates. RESULTS AND CONCLUSION: A total number of 56 (23%) have shown efflux mediated FQ resistance. MexAB-OprM efflux system was predominant type. This is the first report of efflux pump mediated FQ resistance from this part of the world and the continued emergence of these mutants with such high MIC range from this part of the world demands serious awareness, diagnostic intervention, and proper therapeutic option.

ABC-VED analysis of expendable medical stores at a tertiary care hospital.

BACKGROUND: The modern system of medicine has evolved into a complex, sophisticated and expensive treatment modality in terms of cost of medicines and consumables. In any hospital, approximately 33% of total annual budget is spent on buying materials and supplies including medicines. ABC (Always, Better Control)-VED (Vital, Essential, Desirable) analysis of medical stores of a large teaching, tertiary care hospital of the Armed Forces was carried out to identify the categories of drugs needing focused managerial control. METHODS: Annual consumption and expenditure data of expendable medical stores for one year was extracted from the drug expense book, followed by classification on its annual usage value. Subsequently, the factor of criticality was applied to arrive at a decision matrix for understanding the need for selective managerial control. RESULTS: The study revealed that out of 1536 items considered for the study, 6.77% (104), 19.27% (296) and 73.95% (1136) items were found to be A, B and C category items respectively. VED analysis revealed that vital items (V) accounted for 13.14% (201), essential items (E) for 56.37% (866) and desirable accounted for 30.49% items (469). ABC-VED matrix analysis of the inventory reveals that only 322 (21%) items out of an inventory of 1536 drugs belonging to category I will require maximum attention. CONCLUSION: Scientific inventory management tools need to be applied routinely for efficient management of medical stores, as it contributes to judicious use of limited resources and resultant improvement in patient care.

Sexual behavior and its relationship with semen quality parameters in Sahiwal breeding bulls.

AIM: The study was conducted at Artificial Breeding Research Centre, NDRI, Karnal, to determine the sexual behavior and its relationship with semen quality parameters in Sahiwal breeding bulls. MATERIALS AND METHODS: A total of 63 ejaculates were collected from six adult Sahiwal bulls (age ~47 mo and bwt ~466 kg), to study the relationship of sexual behavior and semen quality. The degree of association between different variables was estimated by Pearson's correlation coefficient method. RESULTS: The results depicted that, sexual aggressiveness showed significantly high positive correlation with libido score (LS) and sexual behavior score (SBS). Reaction time (RT) and total time taken in mounts (TTTM) had a significant negative correlation with LS and SBS. Penile erection score and penile protrusion score (PPS) both had a significant positive correlation with ejaculatory thrust score, mating ability score, and SBS. Results of correlation among seminal attributes and with sexual behavior depicted that ejaculate volume had positive significant correlation with initial progressive motility (IPM), sperm concentration (SCON), head abnormality, total abnormality, hypo-osmotic swelling test (HOST), acrosomal integrity (AI) whereas, mass activity had positive significant correlation with IPM, SCON, non-eosinophilic spermatozoa count (NESC), HOST, AI, RT and TTTM and IPM had positive significant correlation with SCON, NESC, HOST, AI, and TTTM, whereas and HOST had positive significant correlation with AI. Among seminal attributes, SCON had a positive significant correlation with PPS where as head abnormalities had a positive significant correlation with RT and TTTM. CONCLUSION: It can be concluded that the relationship of sexual behavior and semen quality parameters are reflecting that the sexual behavior of individual bulls is important to harvest good quality and quantity of semen as desired type of sexual preparation can be provided.

Assessment of expected breeding values for fertility traits of Murrah buffaloes under subtropical climate.

AIM: The aim of the present study was to assess the influence of temperature and humidity prevalent under subtropical climate on the breeding values for fertility traits viz. service period (SP), pregnancy rate (PR) and conception rate (CR) of Murrah buffaloes in National Dairy Research Institute (NDRI) herd. MATERIALS AND METHODS: Fertility data on 1379 records of 581 Murrah buffaloes spread over four lactations and climatic parameters viz. dry bulb temperature and relative humidity (RH) spanned over 20 years (1993-2012) were collected from NDRI and Central Soil and Salinity Research Institute, Karnal, India. Monthly average temperature humidity index (THI) values were estimated. Threshold THI value affecting fertility traits was identified by fixed least-squares model analysis. Three zones of non-heat stress, heat stress and critical heat stress zones were developed in a year. The genetic parameters heritability (h(2)) and repeatability (r) of each fertility trait were estimated. Genetic evaluation of Murrah buffaloes was performed in each zone with respect to their expected breeding values (EBV) for fertility traits. RESULTS: Effect of THI was found significant (p<0.001) on all fertility traits with threshold THI value identified as 75. Based on THI values, a year was classified into three zones: Non heat stress zone(THI 56.71-73.21), HSZ (THI 75.39-81.60) and critical HSZ (THI 80.27-81.60). The EBVfor SP, PR, CR were estimated as 138.57 days, 0.362 and 69.02% in non-HSZ while in HSZ EBV were found as 139.62 days, 0.358 and 68.81%, respectively. EBV for SP was increased to 140.92 days and for PR and CR, it was declined to 0.357 and 68.71% in critical HSZ. CONCLUSION: The negative effect of THI was observed on EBV of fertility traits under the non-HSZ and critical HSZ Thus, the influence of THI should be adjusted before estimating the breeding values for fertility traits in Murrah buffaloes.

Optimizing age of bull at first use in relation to fertility of Murrah breeding bulls.

AIM: The aim of the present investigation was to optimize the age at first use (AAFU) of semen of Murrah breeding bulls, which will help in early selection of bulls under progeny testing program for improving the reproductive performance in the herd. MATERIALS AND METHODS: The data on AAFU, conception rate based on first A.I. (CRFAI), overall conception rate (OCR), and birth weight (B.WT) of 57 Murrah bulls during 1993-2014 at NDRI center pertaining to 14 sets of Network Project on Buffalo Improvement at ICAR-National Dairy Research Institute, Karnal, Haryana, India were adjusted for significant environmental influences and subsequently analyzed. Simple and multiple regression models were used for prediction of CRFAI and OCR of Murrah breeding bulls. Comparative evaluation of three developed models (I-III) showed that Model III, having AAFU and B.WT, fulfill the accuracy of model as revealed by high coefficient of determination, low mean sum of squares due to error, low conceptual predictive value, and low Bayesian information criterion. RESULTS: The results revealed that the average predicted CRFAI was highest (39.95%) at <3.5 years and lowest (34.87%) at >4.5 years of age at first A.I/use. Similarly, average predicted OCR was highest (41.05%) at <3.5 years and lowest (39.42%) at >4.5 years of age at first A.I/use of Murrah bulls. CONCLUSION: In organized herd under progeny testing program, Murrah bulls should be used at young age, i.e. prior to 3.5 years, which is expected to result in 5.08% better CRFAI and 1.63% better OCR in comparison to Murrah bulls used after 4.5 years of age.

Identifying factors affecting age at first semen freezing and age at first semen use in Sahiwal bulls.

AIM: The objective of the study was to evaluate the effects of non-genetic factors on reproduction traits viz. age at first semen freezing and age at first semen use of breeding bulls in Sahiwal bulls by fitting least-squares analysis. MATERIALS AND METHODS: The information on reproduction traits of 43 Sahiwal breeding bulls belonging to 8 sets of Sahiwal breeding program at Indian Council of Agricultural Research-National Dairy Research Institute (ICAR-NDRI), Karnal (Haryana), India during 27 years (1987-2013) were analyzed using fixed linear model. The information was collected from AI records, reproduction sheets, and bull AI register maintained at different sections of Institute viz. record room of Dairy Cattle Breeding Division (DCB), Cattle Yard, Artificial Breeding Research Centre, ICAR-NDRI, Karnal. RESULTS: The average age at first semen freezing and age at first semen use of Sahiwal breeding bulls was estimated as 3.17±0.01 years and 5.35±0.01 years, with the coefficient of variation 18.93% and 20%, respectively. The overall least-squares mean for age at first semen freezing and age at first semen use was estimated as 3.14±0.09 years and 5.25±0.02 years, respectively, in Sahiwal breeding bulls. Period of freezing/use had significant effects on reproductive traits (p<0.01). Season had no significant effect on any of the traits considered in this study. CONCLUSION: It can be concluded that management inputs such as nutrition, breeding, and optimum environment should be taken care of to optimize age at first semen freezing and age at first semen use for better utilization of superior germplasm.

Identification of putative fertility markers in seminal plasma of crossbred bulls through differential proteomics.

Sub-fertility is a major problem in crossbred bulls leading to disintegration of breeding systems and huge economic loss. Identification of some potential biomarkers to determine the latent fertility of bulls accurately has long been the interest of researchers. In this study, we analyzed the proteome of seminal plasma (SP) from bulls with varying fertility to identify the fertility-associated proteins. The proteomic profile of high- and low-fertile bulls was compared by two-dimensional difference gel electrophoresis and differentially expressed proteins were identified through matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. Out of the 18 differentially expressed proteins (P < 0.05), 9 were overexpressed in SP of high-fertile bulls and 9 were overexpressed in SP of low-fertile bulls. The differential expressions ranged from 1.5- to 5.5-fold between the two groups, where protection of telomeres-1 protein (POT1) was highly overexpressed (2.9-fold) in high-fertile group and prostaglandin E2 receptor EP3 (PTGER3) was highly abundant (5.5-fold) in low-fertile group. The protein interaction network was elucidated using STRING software tool, and the functional bioinformatics analysis was done using Blast2Go software. Most of the differentially expressed proteins were found to be involved in cellular processes and biological regulation with binding and catalytic function. It is inferred that the expression of certain proteins in the SP varied with bull fertility, and concurrent appraisal of their expression along with other fertility assays may help in determining bull fertility.

Applications of pathology-assisted image analysis of immunohistochemistry-based biomarkers in oncology.

Immunohistochemistry-based biomarkers are commonly used to understand target inhibition in key cancer pathways in preclinical models and clinical studies. Automated slide-scanning and advanced high-throughput image analysis software technologies have evolved into a routine methodology for quantitative analysis of immunohistochemistry-based biomarkers. Alongside the traditional pathology H-score based on physical slides, the pathology world is welcoming digital pathology and advanced quantitative image analysis, which have enabled tissue- and cellular-level analysis. An automated workflow was implemented that includes automated staining, slide-scanning, and image analysis methodologies to explore biomarkers involved in 2 cancer targets: Aurora A and NEDD8-activating enzyme (NAE). The 2 workflows highlight the evolution of our immunohistochemistry laboratory and the different needs and requirements of each biological assay. Skin biopsies obtained from MLN8237 (Aurora A inhibitor) phase 1 clinical trials were evaluated for mitotic and apoptotic index, while mitotic index and defects in chromosome alignment and spindles were assessed in tumor biopsies to demonstrate Aurora A inhibition. Additionally, in both preclinical xenograft models and an acute myeloid leukemia phase 1 trial of the NAE inhibitor MLN4924, development of a novel image algorithm enabled measurement of downstream pathway modulation upon NAE inhibition. In the highlighted studies, developing a biomarker strategy based on automated image analysis solutions enabled project teams to confirm target and pathway inhibition and understand downstream outcomes of target inhibition with increased throughput and quantitative accuracy. These case studies demonstrate a strategy that combines a pathologist's expertise with automated image analysis to support oncology drug discovery and development programs.