Large-Sample Genome-Wide Association Study of Resistance to Retained Placenta in U.S. Holstein Cows.

A genome-wide association study of resistance to retained placenta (RETP) using 632,212 Holstein cows and 74,747 SNPs identified 200 additive effects with p-values < 10-8 on thirteen chromosomes but no dominance effect was statistically significant. The regions of 87.61-88.74 Mb of Chr09 about 1.13 Mb in size had the most significant effect in LOC112448080 and other highly significant effects in CCDC170 and ESR1, and in or near RMND1 and AKAP12. Four non-ESR1 genes in this region were reported to be involved in ESR1 fusions in humans. Chr23 had the largest number of significant effects that peaked in SLC17A1, which was involved in urate metabolism and transport that could contribute to kidney disease. The PKHD1 gene contained seven significant effects and was downstream of another six significant effects. The ACOT13 gene also had a highly significant effect. Both PKHD1 and ACOT13 were associated with kidney disease. Another highly significant effect was upstream of BOLA-DQA2. The KITLG gene of Chr05 that acts in utero in germ cell and neural cell development, and hematopoiesis was upstream of a highly significant effect, contained a significant effect, and was between another two significant effects. The results of this study provided a new understanding of genetic factors underlying RETP in U.S. Holstein cows.

Gene expression profile of placentomes and clinical parameters in the cows with retained placenta.

BACKGROUND: Retained placenta (RP) is a prevalent disorder in cattle with many health-related and economic costs for the farm owners. Its etiology has not been clarified yet and there is no definite therapy for this disorder. In this study we conducted RNA-seq, hematologic and histologic experiments to survey the causes of RP development. METHODS: Blood samples were collected from 4 RP and 3 healthy cows during periparturtion period for hematological assessments followed by placentome sampling within 30 min after parturition. Cows were grouped as RP and control in case the placenta was retained or otherwise expelled, respectively. Total RNA was extracted from placentome samples followed by RNA-sequencing. RESULTS: We showed 240 differentially expressed genes (DEGs) between the RP and control groups. Enrichment analyzes indicated immune system and lipid metabolism as prominent over- and under-represented pathways in RP cows, respectively. Hormonal assessments showed that estradiol-17ß (E2) was lower and cortisol tended to be higher in RP cows compared to controls at the day of parturition. Furthermore, histologic experiment showed that villi-crypt junctions remain tighter in RP cows compared to controls and the crypts layer seemed thicker in the placentome of RP cows. Complete blood cell (CBC) parameters were not significantly different between the two groups. CONCLUSION: Overall, DEGs derived from expression profiling and these genes contributed to enrichment of immune and lipid metabolism pathways. We suggested that E2 could be involved in development of RP and the concentrations of P4 and CBC counts periparturition might not be a determining factor.

Genetic analysis of retained placenta and its association with reproductive disorder, production, and fertility traits of Iranian Holstein dairy cows.

To facilitate breeding for improved resistance to the reproductive disorder of retained placenta (RP), genetic parameters were estimated for RP and its genetic correlation with other reproductive disorders as well as with production and fertility traits of Iranian Holstein dairy cows. Data were 154,048 lactation records collected between 2011 and 2018 from 59,610 Holstein dairy cows in 9 Iranian herds. Other reproductive disorders included dystocia, stillbirth, and twinning. Fertility records were available for days from calving to first service (DFS), days open (DO), number of inseminations per conception (NIC), and success of first insemination (SFI). Genetic parameters for RP were estimated using univariate linear and logistic animal models with ASREML software. The univariate linear animal model was used to implement bivariate analysis to investigate potential genetic correlations of RP with other reproductive disorders and with production and fertility traits. Heritability estimates for RP were low from both linear (0.031) and logistic (0.092) animal models. Estimated genetic correlations with RP were -0.04 for twinning, 0.32 for stillbirth, and 0.34 for dystocia, which indicates that selection against RP could indirectly select against dystocia and stillbirth. Estimated genetic correlations between RP and production traits (milk, fat, and protein yields) at 100, 200, and 305 d in milk ranged from -0.12 to -0.29; the greatest correlation (-0.29) was for the first 100 d in milk. A moderate positive genetic correlation (0.25) was found for RP and DO, DFS, and NIC, whereas a low negative genetic correlation (-0.09) was found between RP and SFI. The pedigree-based genetic analysis of RP showed that this trait has a low heritability, is linked to other reproductive disorders, and generally has an unfavorable relationship with production and fertility traits. Selection against RP can reduce the incidence of reproductive disorders and improve fertility and production traits.

Effect of retained placenta and clinical mastitis on reproduction parameters, immune response, and steroidogenic receptors gene expression in postpartum crossbred dairy cows.

The purpose of this study was to evaluate the effect of retained placenta (RP) and clinical mastitis (CM) on the reproductive efficiency of crossbred dairy cows during the postpartum period and the effect in some innate immune system indicators. For this, two experiments were carried out. In the first, a total of 232 cows were evaluated and divided as: healthy control (n = 184), RP (n = 22), and CM (n = 26) groups. The RP and CM was evaluated until 30 days postpartum (DPP) and reproductive rates were measured. In experiment 2, cows were divided in control (n = 10), RP (n = 10), and CM (n = 30) groups. Between 40 and 50 DPP, clinical, gynecological examination and endometrial cytobrush were performed to evaluate subclinical endometritis (SE) and gene expression of interleukins 1ß (IL-1ß) and 6 (IL-6), chemokine ligand 5 (CCL5), estrogen α (ESR1), and progesterone (PGR) receptors by qRT-PCR analysis. In experiment 1, the conception rate at 1st artificial insemination (AI) was lower in RP and CM groups and pregnancy rate at 150 days decreased in CM group. Calving-to-1st AI interval and days open were shorter in healthy cows. In experiment 2, the occurrence of SE was 26.7% and higher in RP and CM groups. The expression of IL-1ß increased in RP and CM groups, while IL-6 was less expressed in RP group. The CCL5, ESR1, and PGR were similar between groups. In conclusion, cows with RP and CM had their reproductive efficiency negatively affected and had they initial pro-inflammatory response improved by the increase of IL-ß.

Genetics and genomics of reproductive disorders in Canadian Holstein cattle.

In Canada, reproductive disorders known to affect the profitability of dairy cattle herds have been recorded by producers on a voluntary basis since 2007. Previous studies have shown the feasibility of using producer-recorded health data for genetic evaluations. Despite low heritability estimates and limited availability of phenotypic information, sufficient genetic variation has been observed for those traits to indicate that genetic progress, although slow, can be achieved. Pedigree- and genomic-based analyses were performed on producer-recorded health data of reproductive disorders, including retained placenta (RETP), metritis (METR), and cystic ovaries (CYST) using traditional BLUP and single-step genomic BLUP. Genome-wide association studies and functional analyses were carried out to unravel significant genomic regions and biological pathways, and to better understand the genetic mechanisms underlying RETP, METR, and CYST. Heritability estimates (posterior standard deviation in parentheses) were 0.02 (0.003), 0.01 (0.004), and 0.02 (0.003) for CYST, METR, and RETP, respectively. A moderate to strong genetic correlation of 0.69 (0.102) was found between METR and RETP. Averaged over all traits, sire proof reliabilities increased by approximately 11 percentage points with the incorporation of genomic data using a multiple-trait linear model. Biological pathways and associated genes underlying the studied traits were identified and will contribute to a better understanding of the biology of these 3 health disorders in dairy cattle.

miRNA-185 regulates the VEGFA signaling pathway in dairy cows with retained fetal membranes.

Retention of fetal membranes (RFM) of cows is an important reproductive disturbance, and is related to miRNAs. Vascular endothelial growth factor (VEGF)A, regulated by miRNA-185, can activate arachidonic acid (ARA) release via the VEGFA signaling pathway, which influences RFM. The aim of this study was to explore the pathogenic mechanism of RFM by investigating the regulatory relationship between miRNA-185 and the VEGFA signaling pathway. Serum samples of healthy Holstein dairy cows (n = 20) and RFM cows (n = 12), with a similar age, parity, weight, and milk yield, were collected to detect VEGFA and ARA concentrations at 6, 12, and 24 h after calving. Caruncle tissues were collected from healthy (n = 6) and RFM cows (n = 6) at 12 h after calving. Quantitative polymerase chain reaction (qPCR) and western blotting (WB) were performed to detect the mRNA and proteins levels, respectively, of genes involved in the VEGFA signaling pathway. Uterine caruncle epithelial (UCE) cells were cultured by the explant culture method, further purified, and subsequently treated with miRNA-185 mimics, miRNA-185 mimics + MEK inhibitor, or left untreated as a control for detection of the mRNA and protein levels of genes involved in the VEGFA signaling pathway. The cellular supernatant was collected for measurement of ARA levels at 12, 24 and 48 h after treatment. Serum levels of VEGFA and ARA from RFM cows were abnormally increased at 12 h after calving, as compared to those in healthy dairy cows. Expression levels of most of the investigated genes (VEGFA, PLC, PRK, RAF, MEK, MAPK, and PLA) were down-regulated in the caruncle tissue of RFM cows. However, P-p44/42 MAPK was up-regulated in the caruncle tissues of cows with RFM (p < .01). In UCE cells treated with the miRNA-185 mimics, expression of VEGFA, PLC, RAF, MEK, MAPK and PLA was significantly down-regulated, while that of P-p44/42 MAPK was significantly up-regulated. Expression of genes involved in the VEGFA signaling pathway was similar to that in the in vivo assay. In UCE cells treated with the miRNA-185 mimics + MEK inhibitors, expression of VEGFA, PLC, RAF, MEK, MAPK and P-p44/42 MAPK was significantly down-regulated, while that of PLA was significantly up-regulated. Meanwhile, the release of ARA was increased (p < .01). These results demonstrate that miRNA-185 can regulate the VEGFA signaling pathway, especially via abnormal expression of P-p44/42 MAPK, which influences the release of the fetal placenta after calving.

The inherited risk of retained placenta: a population based cohort study.

OBJECTIVE: To investigate whether retained placenta in the first generation is associated with an increased risk of retained placenta in the second generation. DESIGN: Population-based cohort study. SETTING: Sweden. POPULATION: Using linked generational data from the Swedish Medical Birth Register 1973-2012, we identified 494 000 second-generation births with information on the birth of the mother (first-generation index birth). For 292 897 of these births there was information also on the birth of the father. METHODS: Risk of retained placenta in the second generation was calculated as adjusted odds ratios (aOR) by unconditional logistic regression with 95% confidence intervals (95% CI) according to whether retained placenta occurred in a first generation birth or not. MAIN OUTCOME: Retained placenta in the second generation. RESULTS: The risk of retained placenta in a second-generation birth was increased if retained placenta had occurred at the mother's own birth (aOR 1.66, 95% CI 1.52-1.82), at the birth of one of her siblings (aOR 1.58, 95% CI 1.43-1.76) or both (aOR 2.75, 95% CI 2.18-3.46). The risk was slightly increased if retained placenta had occurred at the birth of the father (aOR 1.23, 95% CI 1.07-1.41). For preterm births in both generations, the risk of retained placenta in the second generation was increased six-fold if retained placenta had occurred at the mother's birth (OR 6.55, 95% CI 2.68-16.02). CONCLUSION: There is an intergenerational recurrence of retained placenta on the maternal and most likely also on the paternal side. The recurrence risk seems strongest in preterm pregnancies. TWEETABLE ABSTRACT: A population-based cohort study suggests that there is an intergenerational recurrence of retained placenta.

Oxidative stress and inflammation in retained placenta: a pilot study of protein and gene expression of GPX1 and NFκB.

BACKGROUND: Retained placenta is associated with severe postpartum hemorrhage. Its etiology is unknown and its biochemistry has not been studied. We aimed to assess whether levels of the antioxidative enzyme Glutathione Peroxidase 1 (GPX1) and the transcription factor Nuclear Factor κß (NFκß), as markers of oxidative stress and inflammation, were affected in retained placentas compared to spontaneously released placentas from otherwise normal full term pregnancies. METHODS: In a pilot study we assessed concentrations of GPX1 by ELISA and gene (mRNA) expression of GPX1, NFκß and its inhibitor Iκßα, by quantitative real-time-PCR in periumbilical and peripheral samples from retained (n = 29) and non-retained (n = 31) placental tissue. RESULTS: Median periumbilical GPX1 concentrations were 13.32 ng/ml in retained placentas and 17.96 ng/ml in non-retained placentas (p = 0.22), peripheral concentrations were 13.27 ng/ml and 19.09 ng/ml (p = 0.08). Retained placental tissue was more likely to have a low GPX1 protein concentration (OR 3.82, p = 0.02 for periumbilical and OR 3.95, p = 0.02 for peripheral samples). Median periumbilical GPX1 gene expressions were 1.13 for retained placentas and 0.88 for non-retained placentas (p = 0.08), peripheral expression was 1.32 and 1.18 (p = 0.46). Gene expressions of NFκß and Iκßα were not significantly different between retained and non-retained placental tissue. CONCLUSIONS: Women with retained placenta were more likely to have a low level of GPX1 protein concentration in placental tissue compared to women without retained placenta and retained placental tissue showed a tendency of lower median concentrations of GPX1 protein expression. This may indicate decreased antioxidative capacity as a component in this disorder but requires a larger sample to corroborate results.

Multiple-trait estimates of genetic parameters for metabolic disease traits, fertility disorders, and their predictors in Canadian Holsteins.

Producer-recorded health data for metabolic disease traits and fertility disorders on 35,575 Canadian Holstein cows were jointly analyzed with selected indicator traits. Metabolic diseases included clinical ketosis (KET) and displaced abomasum (DA); fertility disorders were metritis (MET) and retained placenta (RP); and disease indicators were fat-to-protein ratio, milk ß-hydroxybutyrate, and body condition score (BCS) in the first lactation. Traits in first and later (up to fifth) lactations were treated as correlated in the multiple-trait (13 traits in total) animal linear model. Bayesian methods with Gibbs sampling were implemented for the analysis. Estimates of heritability for disease incidence were low, up to 0.06 for DA in first lactation. Among disease traits, the environmental herd-year variance constituted 4% of the total variance for KET and less for other traits. First- and later-lactation disease traits were genetically correlated (from 0.66 to 0.72) across all traits, indicating different genetic backgrounds for first and later lactations. Genetic correlations between KET and DA were relatively strong and positive (up to 0.79) in both first- and later-lactation cows. Genetic correlations between fertility disorders were slightly lower. Metritis was strongly genetically correlated with both metabolic disease traits in the first lactation only. All other genetic correlations between metabolic and fertility diseases were statistically nonsignificant. First-lactation KET and MET were strongly positively correlated with later-lactation performance for these traits due to the environmental herd-year effect. Indicator traits were moderately genetically correlated (from 0.30 to 0.63 in absolute values) with both metabolic disease traits in the first lactation. Smaller and mostly nonsignificant genetic correlations were among indicators and metabolic diseases in later lactations. The only significant genetic correlations between indicators and fertility disorders were those between BCS and MET in both first and later lactations. Results indicated a limited value of a joint genetic evaluation model for metabolic disease traits and fertility disorders in Canadian Holsteins.

Information from later lactations improves accuracy of genomic predictions of fertility-related disorders in Norwegian Red.

Our aim was to investigate whether including information from later lactations improves accuracy of genomic breeding values for 4 fertility-related disorders: cystic ovaries, retained placenta, metritis, and silent heat. Data consisted of health records from 6,015,245 lactations from 2,480,976 Norwegian Red cows, recorded from 1979 to 2012. These were daughters of 3,675 artificial insemination bulls. The mean frequency of these disorders for cows in lactation 1 to 5 ranged from 0.6 to 2.4% for cystic ovaries, 1.0 to 1.5% for metritis, 1.9 to 4.1% for retained placenta, and 2.4 to 3.8% for silent heat. Genomic information was available for all sires, and the 312 youngest bulls were used for validation. After standard editing of a 25K/54K single nucleotide polymorphism data set that was imputed both ways, a total of 48,249 single nucleotide polymorphism loci were available for genomic predictions. Genomic breeding values were predicted using univariate genomic BLUP for the first lactation only and for the first 5 lactations and multivariate genomic BLUP with 5 lactations for each disorder was also used for genomic predictions. Correlations between estimated breeding values for the 4 traits in 5 lactations with predicted genomic breeding values were compared. Accuracy ranged from 0.47 and 0.51 for cystic ovaries, 0.50 to 0.74 for retained placenta, 0.21 to 0.47 for metritis, and 0.22 to 0.60 for silent heat. Including later lactations in a multitrait genomic BLUP improved accuracy of genomic estimated breeding values for cystic ovaries, retained placenta, and silent heat, whereas for metritis no obvious advantage in accuracy was found.

Genetic contribution to postpartum haemorrhage in Swedish population: cohort study of 466,686 births.

OBJECTIVE: To investigate the familial clustering of postpartum haemorrhage in the Swedish population, and to quantify the relative contributions of genetic and environmental effects. DESIGN: Register based cohort study. SETTING: Swedish population (multi-generation and medical birth registers). MAIN OUTCOME MEASURE: Postpartum haemorrhage, defined as >1000 mL estimated blood loss. PARTICIPANTS: The first two live births to individuals in Sweden in 1997-2009 contributed to clusters representing intact couples (n = 366,350 births), mothers with separate partners (n = 53,292), fathers with separate partners (n = 47,054), sister pairs (n = 97,228), brother pairs (n = 91,168), and mixed sibling pairs (n = 177,944). METHODS: Familial clustering was quantified through cluster specific tetrachoric correlation coefficients, and the influence of potential sharing of known risk factors was evaluated with alternating logistic regression. Relative contributions of genetic and environmental effects to the variation in liability for postpartum haemorrhage were quantified with generalised linear mixed models. RESULTS: The overall prevalence of postpartum haemorrhage after vaginal deliveries in our sample was 4.6%. Among vaginal deliveries, 18% (95% confidence interval 9% to 26%) of the variation in postpartum haemorrhage liability was attributed to maternal genetic factors, 10% (1% to 19%) to unique maternal environment, and 11% (0% to 26%) to fetal genetic effects. Adjustment for known risk factors only partially explained estimates of familial clustering, suggesting that the observed shared genetic and environmental effects operate in part through pathways independent of known risk factors. There were similar patterns of familial clustering for both of the main subtypes examined (atony and retained placenta), though strongest for haemorrhage after retained placenta. CONCLUSIONS: There is a maternal genetic predisposition to postpartum haemorrhage, but more than half of the total variation in liability is attributable to factors that are not shared in families.

Heritable and non-heritable genetic effects on retained placenta in Meuse-Rhine-Yssel cattle.

Failure of the timely expulsion of the fetal membranes, called retained placenta, leads to reduced fertility, increased veterinary costs and reduced milk yields. The objectives of this study were to concurrently look at the heritable and non-heritable genetic effects on retained placenta and test the hypothesis that a greater coefficient of relationship between dam and calf increases the risk of retained placenta in the dam. The average incidence of retained placenta in 43,661 calvings of Meuse-Rhine-Yssel cattle was 4.5%, ranging from 0% to 29.6% among half-sib groups. The average pedigree based relationship between the sire and the maternal grandsire was 0.05 and ranged from 0 to 1.04. Using a sire-maternal grandsire model the heritability was estimated at 0.22 (SEM=0.07) which is comparable with estimates for other dual purpose breeds. The coefficient of relationship between the sire and the maternal grandsire had an effect on retained placenta. The coefficient of relationship between the sire and the maternal grandsire was used as a proxy for the coefficient of relationship between dam and calf, which is correlated with the probability of major histocompatibility complex (MHC) class I compatibility between dam and calf. MHC class I compatibility is an important risk factor for retained placenta. Although the MHC class I haplotype is genetically determined, MHC class I compatibility is not heritable. This study shows that selection against retained placenta is possible and indicates that preventing the mating of related parents may play a role in the prevention of retained placenta.

Genetic parameters for producer-recorded health data in Canadian Holstein cattle.

Health traits are of paramount importance for economic dairy production. Improvement in liability to diseases has been made with better management practices, but genetic aspects of health traits have received less attention. Dairy producers in Canada have been recording eight health traits (mastitis (MAST), lameness (LAME), cystic ovarian disease (COD), left displaced abomasum (LDA), ketosis (KET), metritis (MET), milk fever (MF) and retained placenta (RP)) since April 2007. Genetic analyses of these traits were carried out in this study for the Holstein breed. Edits on herd distributions of recorded diseases were applied to the data to ensure a sufficient quality of recording. Traits were analysed either individually (MAST, LAME, COD) or were grouped according to biological similarities (LDA and KET, and MET, MF and RP) and analysed with multiple-trait models. Data included 46 104 cases of any of the above diseases. Incidence ranged from 2.3% for MF to 9.7% for MAST. MET and KET also had an incidence below 4.0%. Variance components were estimated using four different sire threshold models. The differences between models resulted from the inclusion of days at risk (DAR) and a cow effect, in addition to herd, parity and sire effects. Models were compared using mean squared error statistic. Mean squared error favoured, in general, the sire and cow within sire model with regression on DAR included. Heritabilities on the liability scale were between 0.02 (MET) and 0.21 (LDA). There was a moderate, positive genetic correlation between LDA and KET (0.58), and between MET and RP (0.79).

Term placenta shows methylation independent down regulation of Cyp19 gene in animals with retained fetal membranes.

Retention of fetal membranes (RFM) is the major post-partum disorder in dairy cattle. Cyp19 gene encodes the aromatase enzyme responsible for catalyzing the rate limiting step in estrogen biosynthesis, an important hormone for placental maturation and expulsion. The present study was aimed for comparative analysis of Cyp19 gene expression and its epigenetic regulation in placental cotyledons of animals with and without RFM. Significantly lower expression of Cyp19 gene was found in placental samples of RFM affected animals in comparison to normal animals. Methylation analysis of 5 CpG dinucleotides of placenta specific Cyp19 gene promoter I.1 and proximal promoter, PII showed hypo-methylation of both PI.1 and PII in term placenta of normal and diseased animals. In conclusion, a mechanism other than promoter methylation is responsible for decreased aromatase expression in placental cotyledons of animals suffering from RFM.

Two-way calf to dam major histocompatibility class I compatibility increases risk for retained placenta in cattle.

PROBLEM: In cattle, retained placenta (RP) is suggested to arise from failure of immune-mediated rejection of the fetal membranes by the maternal immune system and is associated with major histocompatibility (MHC) class I compatibility between calf and dam. METHOD OF STUDY: To study the association between RP and different MHC class I compatibilities between calf-dam-granddam combinations, massively parallel pyrosequencing was used to determine the MHC class I haplotypes of cows with and without RP. RESULTS: Two-way calf to dam MHC class I compatibility gave a high risk for RP. There was a tendency for a higher risk for RP with calf to dam MHC class I compatibility. CONCLUSIONS: We concluded that in two-way compatible pregnancies, the maternal immune system fails to reject the fetal membranes, and the fetal immune system does not mount an immune response against maternal MHC class I antigens that could influence the immune-mediated rejection of the fetal membranes by the maternal immune system. The lack of immune-mediated rejection of the fetal membranes by the maternal immune system increases the risk of occurrence of RP.

Association between interleukin-8 receptor-α (CXCR1) polymorphism and disease incidence, production, reproduction, and survival in Holstein cows.

The objective was to evaluate the association between the single nucleotide polymorphism at position +735 in the interleukin-8 receptor-α (CXCR1) gene (CXCR1c.735) and disease incidence, milk production, reproductive performance, and survival in Holstein cows. Three-hundred fifty Holstein cows were enrolled. No association was found between CXCR1c.735 genotype and retained fetal membranes, metritis, or endometritis. Incidence rate of clinical mastitis was associated with CXCR1c.735 genotype; cows with genotypes CC and GC had a decreased incidence rate of clinical mastitis compared with GG cows. Milk yield was associated with CXCR1c.735 genotype; cows with genotype GC had greater milk yield than GG cows. Hazard of pregnancy was not associated with CXCR1c.735 genotype. Cows that had clinical mastitis had decreased hazard of pregnancy, and cows that had endometritis tended to have a decreased hazard of pregnancy. Hazard of death or culling was not associated with CXCR1c.735 genotype. Multiparous cows and cows that had mastitis had increased hazard of death or culling. In contrast to what we expected, cows with the genotype GG had an increased incidence rate of clinical mastitis and decreased milk yield.

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes.

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin-eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher (P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower (P<0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher (P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker.