Copper and manganese levels are associated with infectious abortions, stillbirths, and early neonatal deaths in upper Midwest beef cattle herds

Abstract

Objective

To determine the incidence of select mineral and vitamin deficiencies in beef cattle abortions, stillbirths, and neonates less than 24 hours old and to evaluate whether nutrient deficiencies are associated with causes of abortion.

Methods

A retrospective study of abortion cases from laboratory-performed necropsies and field-collected tissues submitted to the North Dakota State University Veterinary Diagnostic Laboratory over a 5-year period was conducted. Abortion investigations included gross and microscopic examinations, bacterial and fungal cultures, PCR assays, and quantitative analysis of the liver for copper, manganese, zinc, iron, selenium, cobalt, vitamin A, and vitamin E. Fisher exact tests, χ² tests, and logistic regression analyses were performed to evaluate associations between minerals and vitamins and causes of abortion.

Results

Of 251 animals, 34% of abortion cases were attributable to a known cause. All but 4 animals were late gestational or full term. There was no sex predilection. When evaluated independently, minerals and vitamins were not associated with whether the cause of abortion was known. However, using logistic regression with mineral concentration (wet weight), there was an increased risk of infectious abortion with lower levels of fetal liver copper and higher levels of fetal liver manganese.

Conclusions

Copper and manganese levels are associated with infectious abortions, stillbirths, and neonatal deaths within 24 hours of birth in upper Midwest beef cattle herds. Incorporating liver trace mineral analysis into abortion investigations is crucial for a comprehensive diagnostic strategy.

Clinical Relevance

Practitioners should consider the impact of mineral status when evaluating the cause of abortion in beef cattle.

The causes of bovine abortion are numerous and varied and can involve a combination of infectious and noninfectious (ie, genetic, toxic, and nutritional) causes. While histopathology, bacterial and fungal cultures, and PCR assays are standard diagnostic tests often used to identify infectious abortifacients, investigating underlying causes of noninfectious abortion remains more challenging. In cases that lack gross or histologic lesions, pathologists must rely on clinical histories to determine the strategy for additional diagnostic testing. Analyzing nutritional deficiencies is one common strategy employed at the North Dakota State University Veterinary Diagnostic Laboratory (NDSU VDL).

Evaluation of nutritional deficiencies, particularly trace minerals and vitamins, has garnered more attention in recent decades.^1,2 Minerals such as copper (Cu), iodine (I), iron (Fe), manganese (Mn), selenium (Se), and zinc (Zn) play critical roles in embryonic and fetal development, as well as overall reproductive and growth performance in mammals.³ Because the liver functions as a repository to supply trace minerals and vitamins to other tissues, the determination of trace mineral and vitamin status of the animal is best accomplished by quantitation of these nutrients in this organ.

Given the essential role trace elements and vitamins play in fetal development, we sought to determine the incidence of select mineral and vitamin deficiencies in bovine beef abortion cases, which included aborted fetuses, stillbirths, and dead neonates less than 24 hours old that were submitted to the NDSU VDL over a 5-year period. Associations between nutrients and causes of abortion were examined to investigate if liver mineral or vitamin concentrations increase the risk of abortion in beef cattle in the upper Midwest.

Methods

Animals

Beef cattle abortion cases defined as aborted fetuses, stillbirths, and neonates younger than 24 hours old were submitted to the NDSU VDL from Montana, North Dakota, South Dakota, and Minnesota between December 2019 and May 2024. Cases included in the study were whole animals with or without fresh placenta from necropsies conducted at the laboratory by pathologists and trained diagnosticians, and fresh and formalin-fixed fetal tissues collected via field necropsies performed by referring veterinarians. For whole fetus specimens, fetal ages were estimated by crown-rump length (CRL) as previously described,⁴ and sex was recorded. The measurement of CRL was used to determine the trimester the fetus was aborted (first trimester, < 17 cm CRL; second trimester, 17 to 54 cm; and third trimester, > 54 to 100 cm).⁴ For field necropsy–submitted tissue cases, gestational or neonatal ages and sex were documented when provided. All specimens were examined grossly by either the submitting veterinarian or NDSU VDL veterinarian and histologically by a pathologist. Liver and mineral analysis was performed on specimens when requested or authorized by the submitter, and routine infectious abortion screening panels were performed on all specimens.

Infectious disease testing

Infectious disease workup at the NDSU VDL included bacterial and fungal cultures and PCR multiplex assay for bovine herpesvirus-1 and bovine viral diarrhea virus. For most of the years, Leptospira was also performed, but this assay was discontinued in 2023 due to the paucity of detections over the previous decade of testing for this pathogen. In addition, ancillary PCR testing for Ureaplasma diversum and Neospora caninum was performed when indicated by clinical history and gross and histologic lesions or if the test was specifically requested by the submitter. Abortions were classified as infectious when a pathogenic bacterium, fungus, virus, or protozoal parasite was identified and histological lesions supported infectious diagnosis or when histologic lesions of infection were noted but no causative agent was identified.

Inductively coupled plasma liver mineral and heavy metal analysis

For determining the nutritional status of Cu, Fe, Mn, molybdenum (Mo), Se, cobalt (Co), and Zn using inductively coupled plasma optical emission spectrometry, 1 to 2 g of fetal liver were used.⁵ Liver tissue samples were digested on a heat block with 3 mL of nitric acid and 2 mL of perchloric acid and analyzed with both axial and radial view inductively coupled plasma optical emission spectrometry. The method calibration detection limit for the minerals Co, Cu, Fe, Mo, and Zn was 0.10 ppm and for Se was 0.25 ppm. The results were reported on a ppm wet weight basis.

Additionally, when vitamin analysis was requested by the submitter, 5 g or more of liver were referred to the American Association of Veterinary Laboratory Diagnosticians-accredited California Animal Health and Food Safety (CAHFS) Laboratory for vitamin A and E analyses via reverse-phase HPLC with fluorescence detection. This test method is based on work by Barbas et al⁶ with in-house modifications.

Reference values for bovine mineral and vitamin analytes Cu, Mn, Zn, Fe, Se, Co, and vitamins A and E are listed in Table 1. Mineral and vitamin reference values are based on the published data of Puls,^7–15 but vitamin reference values from CAHFS are converted from dry weight to wet weight values (R. Poppenga, DVM, PhD, DABVT, University of California–Davis, Davis, CA, email, December 13, 2024). No universally accepted liver mineral and vitamin reference values have been established for fetuses; therefore, adult reference values are frequently used.

Table 1—

Liver reference values for trace minerals and vitamins A and E.^7–15

Analyte	Reference values (ppm wet weight)
Copper	30.0–150.0
Zinc	25.0–100.0a
Iron	40.0–400.0
Manganese	2.5–6.0a
Iodine	0.1–0.2a
Selenium	0.3–1.2
Cobalt	0.0–0.1a
Vitamin A	1.5–4.5b
	> 15.0b,c
Vitamin E	NA
	> 3.6b,c

NA = Not applicable.

^aAduIt cattle reference value.

^bCalifornia Animal Health and Food Safety reference values denote dry weight values converted to wet weight by dividing by 3 to 3.3.

^cNeonate cattle reference value.

Data analysis

Cases were omitted from data analysis if they did not include mineral and/or vitamin analysis, the animal was not a beef cattle breed, or there was clinical or gross evidence of colostrum ingestion. A subset of cases with elevated vitamin A and E levels, indicative of colostrum intake, were excluded from analysis because the focus of the study was on in utero nutritional status of the fetus. Consumption of colostrum would confound vitamin data interpretation. Descriptive analyses included sex, estimated gestational trimester, mineral and vitamin numerical values, and abortion etiology. Two main analyses were conducted using Fisher exact and χ² tests and logistic regression models to answer the following questions: (1) are there associations between mineral deficiencies in abortions with known versus unknown causes, and (2) are there associations between mineral deficiencies in abortions that have an infectious cause versus those with no evidence of an infectious cause. All statistical analyses were conducted using R, version 4.4.1 (The R Foundation), using a significance level of 0.05 for all statistical analyses.

Results

Number of animals/cases

A total of 598 individual whole animals or animal tissues were submitted for abortion workup from December 2019 through May 2024 to reflect 5 abortion seasons, which typically occur from early winter to late spring each year. Two hundred fifty-one cases, encompassing 144 field- and 107 laboratory-performed necropsies, requested additional mineral and/or vitamin analyses. Sex was documented in 136 cases and consisted of 67 females and 69 males. Only 153 cases identified the gestational age of the animal, mostly from laboratory-performed necropsies. No first-trimester animals were submitted. Four animals fell into the second-trimester category. Third-trimester, stillborn, and neonatal calves comprised the remainder of the 149 cases with a reported age.

Due to several years of drought during the study period (2021 to 2023), the number of cow-calf pairs, the main livestock commodity in North Dakota, decreased from a high of 1,000,000 in 2018 to 890,000 by 2024.¹⁶ Likewise, the number of abortion workup cases that included nutritional analysis decreased over the study period from just over 60 cases in 2019 to 2020 to an average of 46 cases in each of the remaining years.

Clinically relevant findings

A cause for abortion was identified in 34% (86/251) of whole animals or tissues submitted to the laboratory that requested additional nutritional testing. Of those identified causes, an infectious cause of abortion was diagnosed in 95% (82/86) of cases with a 44% (36/82) rate of pathogen detection. Pathogenic agents identified included bacteria (Campylobacter jejuni, Trueperella pyogenes, Escherichia coli, U diversum, and Mannheimia haemolytica), viruses (bovine herpesvirus-1 and bovine viral diarrhea virus), fungus (Aspergilla spp and Zygomycetes spp), and protozoal parasites (N caninum). The remaining 56% (46/82) of infectious cases had suspected underlying infectious disease based on gross or histologic findings, but no specific pathogen was detected on culture or PCR. This included 15 cases of a novel fetal pneumonia syndrome that has been observed by our laboratory and others (C. Burbick, DVM, PhD, DACVM, Washington State University, Pullman WA, [formerly North Dakota State University, Fargo, ND] and D. Loy, DVM, PhD, DACVM, University of Nebraska, Lincoln, NE, email, February 2013 to current). This syndrome is characterized by chronic bronchopneumonia with peribronchiolar lymphoid hyperplasia and amnionitis indistinguishable from the lesions caused by U diversum. The etiology has yet to be established. Interestingly, 2 cases of infectious abortion also exhibited toxic levels of ergot in feed. The other 4 animals of known cause of abortion had gross evidence of developmental fetal or placental anomalies, including 1 case of chondrodysplasia with Mn deficiency. The remaining cases (165/251) were deemed to have an unknown etiology characterized by no evident gross or histologic lesions and negative cultures and PCR results. This number also includes 10 cases of animals with abnormal mineral or vitamin levels based on reference ranges (Table 1) but no lesions to indicate abortion cause.

Data analysis

In the logistic regression models, vitamin A and E variables were excluded due to the absence of values for fetuses in the study. Descriptive analyses did not reveal any differences in the sex of the animal and the cause of abortion or any specific mineral or vitamin deficiency. Neither the Fisher exact nor χ² test results (Supplementary Tables S1 and S2, respectively) indicated sufficient evidence to support an association between known or unknown abortion causes and the individual levels (deficiency and nondeficiency based on Table 1) of Cu, Mn, Zn, Se, vitamin A, or vitamin E. Likewise, for the logistic regression model using mineral deficiency categories (mineral deficient vs not deficient) as independent variables, there was no statistically significant association between mineral deficiency and known versus unknown causes of abortion (Table 2). However, the logistic regression model using numerical values of minerals as the independent variables and abortion cause as the response variable (Table 3) found higher values of Cu and Fe were significantly associated with a decreased likelihood of abortion, while higher values of Mn were significantly associated with an increased likelihood of abortion. There were also no statistically significant associations between individual mineral deficiencies and infectious versus noninfectious causes of abortion using Fisher exact or χ² tests (Supplementary Tables S3 and S4, respectively). However, again, using the numerical values of minerals as the independent variables and infectious and noninfectious abortions as the response variable (Table 4), the odds of an infectious abortion decrease as Cu levels increase, while Mn increases the odds of an infectious abortion. Both findings are statistically significant.

Table 2—

Logistic regression analysis with abortion cause as the response variable and mineral deficiency categories as independent variables.

Coefficients	Estimate	SE	Z value	OR	P value
Intercept	−0.3635	0.1656	−2.1940		.0282
Manganese: deficiency	−0.4353	0.2920	−1.491	0.6470	.1360
Iron: deficiency	1.0163	0.5780	1.7580	2.7630	.0787

Table 3—

Logistic regression analysis with abortion cause as the response variable and concentrations of minerals in fetal liver as independent variables.

Coefficients	Estimate	SE	Z value	OR	P value
Intercept	0.2846	0.3577	0.7960		.4261
Copper	−0.0135	0.0038	−3.552	0.9866	.0004
Manganese	0.8435	0.2481	3.400	2.3200	.0007
Iron	−0.0030	0.0009	−3.270	0.9970	.0011

Table 4—

Logistic regression analysis with infectious abortion cause as the response variable and mineral concentration in fetal liver as independent variables.

Coefficients	Estimate	SE	Z value	OR	P value
Intercept	−1.1532	0.3579	−3.222		.0013
Copper	−0.0190	0.0061	−3.128	0.9812	.0018
Manganese	0.5346	0.1946	2.7470	1.7068	.0060

Discussion

From this study, Cu is shown to have a significant impact on bovine abortion risk. This finding is in line with previous studies¹⁷ showing Cu deficiency in 15% of aborted beef bovine fetuses in western Canada. While our analysis did not show an increased risk of abortion when the cause of abortion is categorized generally as known or unknown, it did reveal Cu deficiency can be directly linked to an increased risk of infectious abortion, specifically.

The logistic regression model evaluating associations of numerical mineral deficiencies and infectious versus noninfectious abortion identified that each 1-ppm increase in fetal liver Cu is associated with a 1.88% reduction in the odds of abortion (Table 4). This decreased risk of abortion with higher levels of Cu can be explained in part by the role Cu plays in innate immunity. Copper deficiency has been associated with immunosuppression, morbidity, and death in bovine calves due to neutrophil dysfunction related to reduced superoxide dismutase activity and failure to produce oxygen within neutrophil phagosomes, both of which have been documented to be hampered in cattle and sheep with Cu deficiency.¹⁸

While Cu, Mn, Zn, and Se have been identified as critical trace elements for normal fetal development,¹⁹ Cu, especially, is essential for various biological processes, including embryo development, mitochondrial respiration, connective tissue maturation, antioxidant defense, Fe metabolism, and neurotransmitter biosynthesis.²⁰ Marginal Cu deficiency in pregnant bovids has been associated with higher oxidative stress markers during gestation.²¹ Research by Fry et al²² showed that dams fed Cu-deficient diets in turn produce calves with Cu deficiencies. This study also found that the Simmental breed may have a lesser ability to absorb and utilize Cu and, therefore, may be more prone to Cu deficiency compared to Angus breeds. In addition, even when maternal adjustments of Cu intake were corrected, both placental and fetal Cu concentrations remained reduced, suggesting fetuses may be more susceptible to Cu deficiency than their dams. Because the fetus relies entirely on the dam for its supply of these essential nutrients,²³ preventing maternal Cu deficiency is paramount to providing the fetus with sufficient levels of Cu.

One surprising finding in our study is that low Mn levels were not associated with increased abortions. On the contrary, Mn concentrations were higher in cases of infectious abortion with each 1-ppm increase in fetal liver Mn concentration associated with a 70.7% increase in the odds of having an infectious abortion. The literature contains conflicting reports on the role Mn deficiency plays in abortion and neonatal calf health. Abortions and deformities are well documented in dams with Mn deficiency^23,24 and were even observed in one of the cases in our data set. However, in a recent study²¹ where calf Mn levels reflected dam levels and chelated Mn in late gestation improved Cu concentrations, Mn itself did not affect overall calf size and vigor at birth. In a dairy herd, although Mn, among other minerals, was consistently decreased in fetal livers from aborted calves, it was thought to be a nonspecific change and not associated with the cause of abortion itself.²⁵

Unlike Cu and Mn, Fe is not significant in all the regression models performed. In the model where abortion is the response and mineral deficiency categorical variables (deficient versus adequate) are independent, the odds of having an abortion are approximately 2.76 times higher for individuals with Fe deficiency compared to those without (Table 2). However, the OR is decreased to 0.997 when the numerical concentration of iron levels is used as the predictor variable (continuous predictor) and abortion cause is the response variable (Table 3). Iron is not statistically significant in the last model when infection as the specific cause of abortion is the response variable and mineral concentration in the fetal liver is the independent variable (Table 4). Thus, although Fe appears an important factor in abortion, it does not appear to be specific to infectious causes of abortions. Further study is needed in this area to identify if there are any specific associated microscopic lesions in the fetus to high or low levels of Fe.

It is interesting that other minerals did not surface as associations in abortion from our data set. Besides Cu and Mn, I, Fe, Se, and Zn play critical roles in embryonic and fetal survival, as well as overall reproductive performance and growth in mammals.³ Vitamins A and E are also necessary for fetal development but could not be evaluated due to the low numbers in the data set. Vitamin A is required for nervous system functions, while vitamin E is vital for scavenging radical oxygen species.

One limitation of our study is geographical and sample biases in the data set. Mineral and vitamin deficiencies in beef cattle abortions submitted to the NDSU VDL might not be representative of abortions in the greater US cattle population because samples were received primarily from cow-calf operations in the upper Midwest. In addition, most of our samples were from third-trimester fetuses, and therefore, pregnancy loss in the first and second trimesters could not be evaluated in this study.

Another potential limitation is the various cutoff values used to determine vitamin deficiencies in fetal and neonatal liver samples at different diagnostic laboratories. Fetal liver reference values are not well established for all vitamins. However, to mitigate this, our analysis used data from only 1 diagnostic laboratory (CAHFS) and, more importantly, is independent of cutoff reference values in Table 1. Thus, our findings show the relationship between actual numerical mineral levels and the risk of abortion rather than deficiency status, which is subject to change depending on the reference values used.

It is also important to note that the dietary intake of minerals and vitamins by the dam was unknown for fetal and neonatal beef calves, which plays a significant role in the health of both the dam and her calf, in the cases submitted. This highlights a crucial gap in the abortion investigation. Mineral deficiencies, particularly Cu, not only negatively affect the dam’s immune system but also have an adverse effect on calf health. In a study by Hansen et al,²⁶ where the long-term effects of Cu deficiency were evaluated in calves born to cows on Cu-deficient diets throughout their entire gestation, calves had lower average daily gains from birth to weaning, and calves in the severely deficient Cu-treatment group weighed 14% less than controls at harvest. Because dietary intake of minerals affects mineral status, providing adequate mineral and vitamin dietary supplementation during pregnancy is warranted. Minimum dietary nutrient recommendations are listed based on values from the National Academy of Sciences, Engineering, and Medicine’s Nutrient Requirements for Beef Cattle^27–29 shown in Tables 5 and 6.

Table 5—

Nutrient requirements of beef cattle.^24,26

Nutrient	Gestating beef cow (mg/kg dry matter intake)	Stressed calf (weaned) (mg/kg dry matter intake)
Copper	10.00	10.00–15.00
Zinc	30.00
Iron	50.00	100.00–200.00
Manganese	40.00	40.00–70.00
Molybdenum	No values published
Selenium	0.10	0.1–0.20
Cobalt	0.15	0.1–0.20

Table 6—

Vitamin A and E requirements for beef cattle.^25,26

Vitamin	Gestating beef cow (IU/kg body weight)	Stressed calf (weaned) (IU/kg body weight)
Vitamin A	60.0	NA
Vitamin E	NA	1.6–2.0

Finally, in general, the cause of an abortion is more likely to be identified in cases submitted to the laboratory for necropsy compared to tissues sent in from the field. Our data set contained both laboratory and field-conducted necropsies, possibly confounding these studies. Lesions within aborted bovine fetuses can be subtle and missed in field-conducted examinations compared to necropsies performed by trained pathologists and diagnosticians. In addition, field-conducted examinations often do not result in a full set of fresh and formalin-fixed tissues submitted to the laboratory. In general, field-submitted cases often lack tissues beyond the heart, lung, liver, spleen, and kidney. The best chance of determining the cause of abortion if not submitting a whole animal for investigation should include the following samples: (1) fresh brain, heart, lung, liver, kidney, spleen, abomasal fluid, thymus, thyroid, lymph node, fetal eyeball/vitreous humor (for nitrate analysis), placenta, and any tissue with a suspected lesion; (2) formalin-fixed placenta, brain, thymus, heart, lung, liver, kidney, spleen, conjunctiva, adrenal gland, skeletal muscle, and any tissue with a suspected lesion; (3) feed and water; and (4) maternal sera (acute and convalescent).

In conclusion, the results of this study highlight the importance of evaluating nutrition in abortion investigations. Identification of trace elements or vitamin deficiencies in an aborted fetus provides a critical opportunity to correct specific nutritional issues in the herd and mitigate future losses. Appropriate mineral and vitamin supplementation before and during pregnancy may reduce the risk of abortion. Further research is needed to address the understanding of the role of trace minerals in bovine abortion etiology and lead to better diagnostic, preventive, and management practices to reduce the incidence of abortion in cattle.