Characterization of trace element concentrations in blood samples of dairy cows with uterine torsion and their neonatal calves

doi:10.21203/rs.3.rs-3222186/v1

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Characterization of trace element concentrations in blood samples of dairy cows with uterine torsion and their neonatal calves

https://doi.org/10.21203/rs.3.rs-3222186/v1

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Mineral deficiencies can lead to dystocia and abnormalities in neonates. In dairy cows, the stillbirth of neonatal calves due to dystocia has become an economic problem. Uterine torsion is a common form of dystocia observed in dairy cows. However, to the best of our knowledge, there are no reports on the characteristics of serum trace element concentrations in dairy cows with uterine torsion. Therefore, this study aimed to comprehensively measure and compare the serum trace element concentrations in dairy cows with uterine torsion and their neonates. The study found that calves delivered by cows with uterine torsion had significantly higher fetal mortality rates compared to those delivered from cows without uterine torsion. The odds ratio for fetal mortality in dairy cows with uterine torsion was 7.85. Maternal serum Zinc (Zn) concentrations in cows with uterine torsion were significantly lower than those in cows without uterine torsion (p < 0.01). In addition, the Cu: Zn ratios of cows with uterine torsion were significantly higher than that of cows without torsion (p < 0.05). In contrast, serum cobalt (Co) concentrations in neonates from cows with uterine torsion were significantly higher than those in neonates from cows without torsion (p < 0.01). In conclusion, uterine torsion is associated with a higher fetal mortality rate and greater economic loss than other types of dystocia. Further investigation is required to better understand the relationship between uterine torsion and trace mineral levels.

cattle

copper

dystocia

micro minerals

neonate

zinc

There have been reports indicating a high incidence of dystocia in cows with hypocalcemia and hypophosphatemia during parturition [1]. Furthermore, cows with hypocalcemia during parturition had a higher incidence of stillbirth [1]. One of the abnormalities observed in dairy cows during calving is dystocia, which includes uterine torsion. Uterine torsion is a common cause of dystocia in dairy cows, [2, 3] and can result in local ischemia, fetal death, or even the death of the cow [2]. However, the rate of calcium administration is higher in cows with uterine torsion compared to cows with other forms of dystocia [4]. Additionally, it has been reported that cows with dystocia have lower plasma zinc (Zn) levels compared to cows with eutocia [5]. In humans, the serum levels of copper (Cu) and Zn are lower in women with spontaneous abortion [6]. Moreover, lower serum Zn levels have been observed in subjects with preterm delivery or miscarriage than in controls [7]. It has been reported that not only Zn but also a decrease in blood iron (Fe) is associated with abortions [8]. Although blood levels of Cu and Zn are associated with diseases, it has been reported that the balance of these minerals is also important for maintaining health. Cu/Zn ratios have been reported to be related to spontaneous abortion [6]. On the other hand, cobalt (Co) is an important element needed for fetal growth, and a low level of Co is related to a high probability of macrosomia [9]. Serum selenium (Se) concentrations are lower in premature infants compared to mature infants, and maternal serum Se concentrations are positively correlated with neonatal birth weight [10]. Thus, it has been reported that deficiency of various minerals might lead to dystocia and neonatal abnormalities. However, to the best of our knowledge, there are no reports on the characteristics of serum trace element concentrations in dairy cows with uterine torsion. In this study, the serum trace element concentrations of dairy cows with uterine torsion and dystocia were comprehensively measured and in order to elucidate its characteristics.

2.1 Samples

This study was approved by the Animal Care and Use Committee of Azabu University School of Veterinary Medicine (approval number:171113-1).

Forty-two Holstein cows bred in the Kushiro area of Hokkaido, Japan, were included in the study. The cows were divided into four groups based on their condition at the time of delivery: maternal cows with uterine torsion (MUT, n = 15), maternal cows without uterine torsion (non-MUT, n = 27), neonatal calves from MUT (NUT, n = 9), and neonatal calves from non-MUT (non-NUT, n = 26). Blood samples were collected from the neonatal calves immediately after birth and prior to colostrum feeding. Calving number, gestational age, and fetal mortality in each group are shown in Table 1. Blood samples were collected from maternal and neonatal jugular veins. Specimens could not be collected from some calves as they were stillborn or deceased. The collected blood samples were placed in plain tubes (Terumo Corporation, Tokyo, Japan), and incubate at 37℃ for 15 min to allow for coagulation. Subsequently, the tubes were centrifuged at 1,700 × g for 15 min at 4℃, and the serum was separated and stored at − 20°C until further use.

Table 1

The calving number, gestational age, and fetal mortality of cows with uterine torsion (MUT) and without uterine torsion (non-MUT). *: p < 0.05
	Calving number	Gestational age (days)	Fetal mortality
non-MUT	3.2 ± 1.7	281.14 ± 4.94	2 / 27
MUT	3.5 ± 2.3	280.53 ± 10.24	6 / 15
Odds ratio			7.85^*

2.2 Serum mineral analysis

Serum samples (60 µL) were added to nitric acid (1 mL: 63% wt / wt) in TFM insert and then digested at 50℃ for 2 min, 30℃ for 1 min, and 180℃ for 20 min using START D (Milestone General K.K., Kanagawa, Japan). Digested samples were diluted 250-fold in a decomposition matrix before undergoing inductively coupled plasma mass spectrophotometry (ICP-MS) analysis. Nitric acid (1%, v/v) in distilled deionized water was used as the decomposition matrix.

2.3 Instruments

An ICP-MS instrument (Agilent 7700x, Agilent Technologies Co., Tokyo, Japan), equipped with a quadrupole mass spectrometer and an XXXctupole reaction cell was used to determine the trace elements in each sample. The operating conditions of the ICP-MS instrument are summarized in Table 2. In the ICP-MS measurements, internal standard correction was performed using tellurium (Merk, Tokyo, Japan) and iridium (Merk, Tokyo, Japan) as internal standard elements to account for matrix effects caused by major elements. The purified water (18.2 MQ cm) used throughout the present study was prepared using a Milli Q SP-TOC system (Nihon Millipore Kogyo, Tokyo, Japan).

Table 2

The operating conditions for the ICP-MS instrument
ICP-MS: Agilent 7700x
Plasma conditions:
RF power	1.55 kW
Plasma gas flow rate	15.0 L min^− 1 Ar
Auxiliary gas flow rate	0.90 L min^− 1 Ar
Makeup gas flow rate	0 L min^− 1 Ar
Carrier gas flow rate	1.0 L min^− 1 Ar
Sampling depth (mm from load coil)	He mode: 8.00 mm
	H₂ mode: 10.00 mm
Cell gas	He mode: 4.3 mL min^− 1 Ar
	H₂ mode: 6.0 mL min^− 1 Ar
Nebulizer	Micro Mist
Sample uptake rate	0.45 mL min^− 1
Data acquisition:
Accumulation time	0.3-1.0 s / point
Data point	3 points / peak
Repetition	3 times

2.4 Chemicals

The standard solutions for creating the calibration curves in the ICP-MS measurements were prepared by diluting commercial multi-element standard stock solutions (TraceCERT®, Sigma-Aldrich, Tokyo, Japan) and a Molybdenum ICP-MS Standard (Wako Pure Chemical Industries Inc., Osaka, Japan). The nitric acid solution used was of industry-grade (Kanto Chemical Co., Tokyo, Japan).

2.3 Statistics

The data were expressed as the mean ± SD. The obtained data did not have a normal distribution, as verified using the Shapiro–Wilk test. Serum trace element levels between the UT and non-UT groups were compared using the Mann–Whitney U test. All statistical analyses were performed using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria). EZR is a modified version of the R commander, designed to add statistical functions frequently used in biostatistics. A p value < 0.05 was considered statistically significant and p value < 0.10 was considered as a significant tendency.

Calves delivered from cows with uterine torsion exhibited significantly higher fetal mortality than those delivered from cows without uterine torsion, although there were no differences in calving number or gestational age (Table 1). The odds ratio for fetal mortality in dairy cows with uterine torsion was 7.85.

Table 3

shows the serum trace element concentrations in the maternal cows. The serum Zn concentrations in MUT were significantly lower in MUTs than in non-MUTs (p < 0.01). Serum Se concentrations in MUT tended to be higher than in non-MUT (p = 0.0673). Moreover, the Cu/Zn ratios in MUT were significantly higher than those in non-MUTs (p < 0.05).
able 3 Serum trace element concentrations of maternal cows. ns; not significant.
	MUT (µg / dL)	n	non-MUT (µg / dL)	n	p value
Cr	1.92 ± 1.36	9	2.09 ± 1.74	16	ns
Mn	0.60 ± 0.60	5	1.18 ± 0.98	16	ns
⁵⁶Fe	123.21 ± 49.70	9	157.70 ± 101.10	19	ns
⁵⁷Fe	120.68 ± 44.95	9	154.65 ± 100.98	19	ns
Co	0.12 ± 0.05	7	0.19 ± 0.11	14	ns
Ga	1.42 ± 0.64	9	1.34 ± 0.69	18	ns
Se	4.53 ± 1.64	9	3.62 ± 1.71	18	0.067
Rb	18.08 ± 3.58	9	16.74 ± 6.19	19	ns
Sr	5.66 ± 1.28	9	7.14 ± 4.72	18	ns
Mo	2.86 ± 1.84	9	3.68 ± 4.29	19	ns
Cs	0.17 ± 0.05	9	0.19 ± 0.10	18	ns
Ba	9.21 ± 4.95	9	8.74 ± 3.87	18	ns
Cu	69.83 ± 21.67	9	82.33 ± 44.37	19	ns
Zn	49.84 ± 7.95	5	112.94 ± 61.60	15	0.008
Cu / Zn ratios	1.60 ± 0.45	5	1.10 ± 1.25	15	0.025

The neonatal serum trace element concentrations are shown in Table 4. The serum Co concentrations of NUT were significantly higher than in non-NUT (p < 0.01). Furthermore, NUT tended to have lower Zn concentrations (p = 0.0583) and higher Se concentrations (p = 0.0726) than non-NUT.

Table 4 Serum trace element concentrations of neonatal calves. ns; not significant.
	NUT (µg / dL)	n	non-NUT (µg / dL)	n	p value
Cr	1.59 ± 1.21	5	1.35 ± 1.38	14	ns
Mn	0.49 ± 0.20	2	1.25 ± 1.26	16	ns
⁵⁶Fe	147.43 ± 47.47	6	185.33 ± 96.29	20	ns
⁵⁷Fe	138.40 ± 44.52	6	187.83 ± 96.76	20	ns
Co	0.54 ± 0.23	4	0.17 ± 0.12	14	0.005
Ga	1.14 ± 0.24	5	1.36 ± 0.85	18	ns
Se	3.33 ± 0.68	6	2.36 ± 1.26	20	0.073
Rb	23.44 ± 3.01	6	22.28 ± 8.27	20	ns
Sr	4.72 ± 0.89	6	6.57 ± 4.87	19	ns
Mo	1.87 ± 1.22	6	2.74 ± 3.14	20	ns
Cs	0.23 ± 0.08	6	0.23 ± 0.10	19	ns
Ba	6.70 ± 2.70	6	8.76 ± 5.27	19	ns
Cu	19.14 ± 4.22	5	23.37 ± 7.04	19	ns
Zn	87.75 ± 62.34	5	182.61 ± 105.18	17	0.058
Cu / Zn ratios	0.40 ± 0.38	5	0.18 ± 0.14	17	ns

Uterine torsion can result in local ischemia, leading to fetal death, and potentially causing the death of the cow [2]. The fetal mortality rate in calves born to cows with uterine torsion was 7.85 times higher than that of cows without torsion. Therefore, uterine torsion is considered to cause greater economic losses in calves compared to other types of dystocia.

Zn is an essential component of various enzymes, including those involved in the synthesis of DNA and RNA [11]. Studies in humans and animals have demonstrated that Zn deficiency decreases immunity and resistance to infectious disease [12]. Serum Zn concentrations in dairy cows have been shown to decrease in cases of subclinical ketosis [13]. Serum Zn concentrations are also known to decrease in dairy cows with subclinical hypocalcemia [14]. Therefore, Zn deficiency is suspected to contribute to immunosuppression, decreased productivity, and poor reproductive performance associated with these conditions. In this study, maternal serum Zn concentrations were significantly lower in cows with uterine torsion compared to those without torsion. Blood Zn concentrations also decrease during parturition [15] and from before parturition to one week following parturition [14]. Therefore, the results of this study revealed that cows with uterine torsion showed a marked decrease in serum Zn concentration, whereas healthy cows showed a decreased periparturient Zn concentration. As serum Zn concentration decreased, the Cu/Zn ratios were significantly higher in cows with uterine torsion than in those without torsion. The Cu/Zn ratios have been reported to be associated with malnutrition, increased oxidative stress, inflammation, and a disrupted immune status in humans [16]. The Cu/Zn ratios are considered a clinically useful index compared to the concentration of either Cu or Zn alone [17]. In human studies, the Cu/Zn ratio has been reported to be a useful diagnostic marker for ectopic pregnancy related to oxidative stress [18]. Uterine torsion induces significant oxidative stress at the tissue level owing to the increased production of free radicals, as indicated by the high levels of malondialdehyde, superoxide dismutase, and glutathione [19]. Therefore, the Cu/Zn ratio may provide valuable insights into the pathology of uterine torsion in dairy cows.

Selenium is a trace element that plays an important role in animal health and performance [20]. In cattle, Se deficiency can contribute to the retained placenta [21], incidence of mastitis [22], reduced fertility, and metritis [23], which can have significant economic impacts reduction in milk production and poor reproductive performance. However, a study on pregnant cows fed with Se demonstrated that subclinical Se toxicosis could lead to stillbirth, which may adversely affect both pregnancy outcomes and the bovine immune system [24]. In the present study, serum Se concentrations tended to be higher in both cows with uterine torsion and in calves born to cows with uterine torsion, although the difference was not statistically significant. However, the association between Se and uterine torsion in dairy cows remains unclear and requires further investigation.

Co., is essential for the formation of vitamin B12 [25]. Vitamin B12 deficiency is known to cause macrocytosis [26]. It has been reported that buffaloes with uterine torsion show macrocytosis [27]. However, the results of this study indicated that neonatal calves born to cows with uterine torsion had significantly higher serum Co concentrations (p < 0.01). Cobalt chloride is traditionally used to treat anemia in pregnant women, infants, and patients with chronic anemia undergoing long-term hemodialysis. Cobalt chloride is known to induce hypoxia-like reactions, such as erythropoiesis and angiogenesis, in vivo [28]. Uterine torsion leads to local ischemia [2], which results in insufficient oxygen supply to the fetus and presumed hypoxemia. The hypoxia inducible factor (HIF) activates the expression of genes containing a hypoxia response element and helps cells adapt to hypoxia [29]. HIF is a heterodimeric transcription factor composed of oxygen-regulated alpha subunit (HIF-1α, -2α, -3α) [30]. It is reported that there is a correlation between Co and HIF-1α mRNA in a dose- and time-dependent manner [31]. Therefore, high serum Co concentrations in neonatal calves born to cows with uterine torsion may protect against ischemic hypoxemia. The mechanism underlying high Co levels in neonatal calves requires further investigation.

In conclusion, uterine torsion is associated with a higher fetal mortality rate and greater economic loss compared to other types of dystocia. Cows with uterine torsion have low serum Zn levels, which may predict reduced disease resistance. Calves born to cows with uterine torsion have high serum Co concentrations and can adapt to ischemic hypoxemia. To the best of our knowledge, this is the first study to examine serum trace mineral levels in dairy cows with uterine torsion and their neonatal calves. Further investigation is required to elucidate the relationship between uterine torsion and trace mineral levels.

Statements and Declarations Funding

This study was supported by the JSPS KAKENHI [grant numbers 25450457, 16K08064, and JP19K23709].

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author Contributions

All authors contributed to the study conception and design. Material preparation, data collection were performed by Kei Kazama and Ken Onda, analysis by Kei Kazama and Kazutoshi Sugita. The first draft of the manuscript was written by Kei Kazama and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data Availability

The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study was approved by the Animal Care and Use Committee of Azabu University School of Veterinary Medicine (approval number:171113-1).

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Characterization of trace element concentrations in blood samples of dairy cows with uterine torsion and their neonatal calves

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Version 1

Abstract

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1 Samples

2.2 Serum mineral analysis

2.3 Instruments

2.4 Chemicals

2.3 Statistics

3. RESULTS

4. DISCUSSION

Declarations

References

Additional Declarations

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