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Communication

Iodoform as an Anti-Methanogenic Feed Additive in Total Mixed Rations of Dairy Cows

by
Mirka Thorsteinsson
*,
Samantha Joan Noel
,
Peter Lund
,
Martin Riis Weisbjerg
,
Anne Louise Frydendahl Hellwing
and
Mette Olaf Nielsen
Department of Animal and Veterinary Sciences, AU Viborg—Research Centre Foulum, Aarhus University, DK-8830 Tjele, Denmark
*
Author to whom correspondence should be addressed.
Dairy 2025, 6(2), 17; https://doi.org/10.3390/dairy6020017
Submission received: 10 February 2025 / Revised: 17 March 2025 / Accepted: 26 March 2025 / Published: 31 March 2025
(This article belongs to the Section Dairy Animal Nutrition and Welfare)
Browse Figure
Versions Notes

Abstract

:
This pilot study investigated whether reductions in enteric CH4 emissions could be obtained without affecting dry matter intake (DMI) when iodoform was mixed into total mixed rations (TMRs). The experiment consisted of four periods of 14 d with four rumen-cannulated Holstein dairy cows. In the pre-period, no iodoform was added to TMR, while either 8, 16, or 20 mg iodoform/kg DM was added to TMR in the remaining periods in a change-over design. However, the experiment was not balanced across treatments and periods due to unexpected animal responses in the second period. Dry matter intake and gas exchange were measured the last 3 d in each period using respiration chambers. Rumen grab samples were collected for microbial analyses on d 14. Dry matter intake was unaffected by the addition of iodoform to TMR at or below 20 mg/kg DMI. Methane and H2 yields (g/kg DMI) quadratically decreased (up to 46%) and increased (up to 1127%), respectively, with the increasing dose. This pilot study indicated that CH4 reductions can be obtained with an addition of up to 20 mg iodoform/kg DM in the diets of dairy cows without affecting DMI. However, high iodine concentration in iodoform limits its use in commercial herds within the EU.
Keywords:
cattle; iodine; halomethane

1. Introduction

The use of anti-methanogenic feed additives is an effective strategy to reduce enteric CH4 emissions from ruminants [1]. Such feed additives could be halomethanes (CH4 analogs), which are known to possess CH4-mitigating properties, with several modes of action being suggested [2]. The most widely recognized theory involves the irreversible binding of the halomethane with reduced B12 to inhibit the cobamide-dependent methyl group transfer in methanogenesis [3]. However, it has also been suggested that halomethanes can inhibit the function of corrinoid enzymes in the various stages of methanogenesis and act as competing terminal electron acceptors [2].
During the last two decades, numerous in vivo studies have demonstrated the anti-methanogenic properties of halomethanes. For instance, up to 91% reductions in CH4 emissions have been reported when bromochloromethane (CH2BrCl) was fed to ruminants [4,5]. Similar inhibitory properties of another halomethane, chloroform (CHCl3), have also been observed [6,7]. Unfortunately, both bromochloromethane and chloroform are classified as possible human carcinogens [8]. Moreover, due to its ozone-depleting capacity, bromochloromethane is banned from commercial use in many countries [2,9]. A more recent dose-response study, in which the halomethane iodoform was pulse-dosed intra-ruminally to dairy cows twice daily, reported up to 66% reductions in CH4 yield (g/kg dry matter intake) with large diurnal variations in emission. However, the study also found up to a 48% reduction in dry matter intake (DMI) with increasing iodoform dose [10]. In contrast to the other aforementioned halomethanes, iodoform is not considered to be ozone-depleting or carcinogenic [9]. Hence, the aim of this pilot study was to investigate whether reductions in enteric CH4 emissions could be obtained without affecting the DMI when iodoform was added to the total mixed rations (TMR) of dairy cows. It was hypothesized that increasing the inclusion level of iodoform would cause increasing reductions in CH4 emissions without affecting the DMI.

2. Materials and Methods

The experiment was conducted at Aarhus University, AU Viborg—Research Centre Foulum, Denmark. The experiment was planned to be a 3 × 3 Latin square design with periods of 14 days using 3 rumen-cannulated Danish Holstein dairy cows. The experiment included a pre-period with no treatment (0 mg iodoform), while the cows received iodoform on all days in the remaining periods. In the first period, the cows were randomly assigned to 1 of 3 levels of iodoform (CAS no. 75-47-8; Merck KGaA, Darmstadt, Germany) mixed into the TMR (16, 24, and 32 mg iodoform/kg dry matter (DM)). However, due to gradual undesired decreases in DMI on the two highest doses (up 48% on 24 mg/kg DM and 54% on 32 mg/kg DM on d 14), the doses were reduced to 8 and 16 mg iodoform/kg DM in the second period. Furthermore, a fourth rumen-cannulated cow was also included in the experiment and fed 0 mg iodoform/kg DM in period 2. The cow receiving the highest dose (32 mg iodoform/kg DM) in the first period was not fed any iodoform in the second period to ensure full recovery, although no health issues were observed besides the reduced DMI. In the third period, the doses were adjusted to 16 and 20 mg iodoform/kg DM due to only minor reductions in enteric CH4 emission on 8 mg iodoform/kg DM. In total, one 2nd parity and three 4th parity cows were used. Hence, one cow (4th parity) received 0, 8, 16, and 20 mg iodoform/kg DM, one cow (4th parity) received 0, 8, and 16 iodoform/kg DM, one cow (2nd parity) received 0 and 16 iodoform/kg DM, and one cow (4th parity) received 0, 16, and 20 iodoform/kg DM. The data from period 1 for the cow fed 24 mg iodoform/kg DM and for periods 1 and 2 for the cow fed 32 mg iodoform/kg DM in period 1 were not included in the dataset. At the beginning of the experiment, the average ± SD milk yield was 27.1 ± 6.41 kg/d, days in milk was 160 ± 148 d, and BW was 716 ± 84.6 kg. The cows were housed in individual pens (4.0 × 4.5 m) with a slatted floor and a cubicle bedded with a mattress and sawdust during the adaptation periods. The cows were milked twice daily at 05.15 and 16.30 h.
The TMR with iodoform was prepared and fed once a day to the cows on an ad libitum basis at 16.30 h. Refusals were weighed daily before the feeding. The iodoform was mixed into 50 g of wheat flour and subsequently mixed into a pre-mixture consisting of sugar beet pulp, rapeseed cake, rapeseed meals, barley, and mineral supplements. Corn silage and both spring growth and third regrowth grass/clover silage (perennial ryegrass, hybrid ryegrass, red clover, and white clover) were added to the pre-mixtures to form the TMR (Table 1). The cows had free access to water. Feed intake was recorded throughout the experiment, while DMI was determined by weighing the amount of allotted feed, and the refusals were followed by the determination of DM on d 12–14.
Gas exchange was measured on d 12–14 in each period using four individual transparent polycarbonate respiration chambers based on open-circuit indirect calorimetry (modified from Hellwing, Lund [11]). The chambers were placed in a square in a separate barn, allowing visual contact between the cows. The cows were assigned to the same specific respiration chamber for gas measurements throughout the experiment. Airflow, concentrations of gases (CH4, CO2, O2, and H2) in outlet air, temperature, humidity, and pressure in the chambers were measured as described by Thorsteinsson and Weisbjerg [12]. Before, during, and after the experiment, recovery tests (n = 21 for CO2 and n = 21 for CH4) were performed by infusing a known amount of pure CO2 or CH4 into the chambers and comparing it with the amount of gas measured by the system. Across chambers, average recovery values ± SD were 99.7 ± 1.28% for CO2 and 99.9 ± 1.38% for CH4. Recovery tests were used to correct the measured gas concentrations. The average of CH4 and CO2 recoveries was used to correct O2 and H2 concentrations.
Table 1. Dietary and chemical composition (in percentage of DM, unless otherwise stated) of TMR with four different inclusion levels of iodoform (0, 8, 16, or 20 mg/kg DM).
Table 1. Dietary and chemical composition (in percentage of DM, unless otherwise stated) of TMR with four different inclusion levels of iodoform (0, 8, 16, or 20 mg/kg DM).
Item0 mg8 mg16 mg20 mg
Dietary composition
Maize silage35.035.035.035.0
Grass/clover silage, spring growth7.377.367.367.36
Grass/clover silage, 3rd regrowth5.535.525.525.52
Sugar beet pulp pellets w/o molasses16.616.616.616.6
Rapeseed cake, 10.5% fat11.411.411.411.4
Rapeseed meal, 4% fat11.411.411.411.4
Barley10.710.710.710.7
Mineral premix 11.921.921.921.92
Wheat flour mixed with iodoform-0.180.180.18
Chemical composition
DM, % of fresh feed40.739.940.539.0
Ash6.907.217.047.10
Crude protein17.017.016.916.8
Crude fat3.703.703.773.90
Neutral detergent fiber32.833.332.334.1
Starch16.216.216.016.1
NEL20, MJ/kg of DM 26.366.366.366.36
1 Vilofoss Komix Type 3, declared macro mineral composition (g/kg DM): Ca = 147, Mg = 141, Na = 116, S = 1. Added vitamins and micro minerals (per kg DM): vitamin A = 600,000.10 IU, vitamin D3 = 190,000.10 IU, vitamin E = 4000 IU, Mn = 4000 mg, Cu = 1500 mg, Zn = 4500 mg, I = 225, Co = 25 mg, Se = 50 mg in combination with Vilofoss Suplex ADE, analyzed/declared macro mineral composition (g/kg DM): Ca = 139, Mg = 91, Na = 95. Added vitamins and microminerals (per kg DM): vitamin A = 900,000 IU, vitamin D3 = 200,000 IU, vitamin E = 2000 IU, Se = 50 mg. 2 Standard feed value for NEL at 20 kg DMI. Calculated according to NorFor [13].
For ruminal microbiota analyses, grab samples from the dorsal, ventral, cranial, and caudal rumen were collected on d 14 at 12.00 h through the rumen cannula, pooled, and mixed as described by Thorsteinsson and Lund [10]. The samples were immediately placed in liquid nitrogen for later storage at −70 °C. DNA was extracted from ~100 mg of the wet rumen sample (actual weight recorded) with the NucleoSpin DNA Stool Kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s directions and eluting in 150 µL of the elution buffer. The concentration of DNA was determined with the Qubit Broad Range Kit (Thermo Fisher Scientific, Wilmington, DE, USA). Total bacteria and archaea were quantified with qPCR as described by Thorsteinsson and Lund [10].
Dry matter concentration of fresh feed and refusals was determined by drying at 60 °C for 48 h. Prior to chemical analysis, TMR samples were freeze-dried and ground on a 1-mm screen, except a 0.5 mm screen was used on samples for starch analysis (Ultra Centrifugal Mill ZM 200, Verder Scientific, Hann, Germany). Ash concentration was determined by combustion at 525 °C for 6 h. Total nitrogen in TMR was analyzed using the Dumas principle in a Vario Max CN (Elementar Analysesysteme GmbH, Langenselbold, Germany), and crude protein (CP) was calculated as total nitrogen × 6.25. Neutral detergent fiber was determined on an organic matter basis by adding heat-stable amylase and sodium sulfite [14] in the ANKOM2000 Fiber Analyzer and following corrected for ash [15]. Crude fat was determined via Soxhlet extraction with petroleum ether (Soxtec 2050, Foss Analytical, Hillerød, Denmark) after hydrolysis with HCl [16]. Starch was digested with heat-stable α-amylase and amyloglucosidase, and the released glucose was measured by using a YSI model 2900 analyzer (YSI Inc., Yellow Springs, OH, USA). Milk iodine was analyzed in milk samples from the pre-period, where no iodoform was administrated (n = 4), and from the third and last period, where the cows received 16 (n = 2) and 20 mg iodoform/kg DM (n = 2), using inductively coupled plasma mass spectrometry (iCAPq ICP-MS, Thermo Fischer, Bremen, Germany).
Gas exchange was measured as flows at standard temperature and pressure (STP (0 °C (273.15 K) and 101.325 kPa)). The respiratory coefficient (RQ) was calculated as the ratio between CO2 produced and oxygen consumed (L/L). Gas amounts in L/d were converted to g/d by using the density of each gas at STP, which were 0.716, 1.963, 0.0899, and 1.428 (L/g) for CH4, CO2, H2, and O2, respectively. Data were deleted when chambers were opened due to milking and feeding. The daily emission was calculated as accumulated gas production over the measuring period divided by accumulated measuring time in minutes to obtain gas production per minute and multiplied by 1440 min. Observations of all variables were averaged within cow and period. Hence, 12 observations were included in the dataset (4 observations for 0 mg iodoform/kg DM, 2 observations for 8 mg iodoform/kg DM, 4 observations for 16 mg/kg DM, and 2 observations for 20 mg iodoform/kg DM). Statistical analyses were conducted in R 4.4.1 [17]. The effect of treatment on the various animal responses was analyzed with the following linear mixed model:
Ytpc = μ + αt + γp + Ac + ɛtpc,
where Ytpc is the dependent response variable, μ is the overall mean, α is the fixed effect of treatment (t = 0, 8, 16 or 20 iodoform/kg DM), γ is the fixed effect of period (p = pre-period, 1, 2, or 3), A is the random effect of cow (c = 1 to 4), and ɛtpc is the random residual error assumed to be independent with constant variance and normally distributed. The linear and quadratic effects of the iodoform level were estimated using orthogonal polynomial contrasts.
Unfortunately, the microbiota sample from the additional cow included in the experiment in period 1 was lost during snap-freezing. Hence, it was not possible to analyze the data with the effect of the period as only data from one cow in period 1 were included in the dataset. Instead, microbiota data were analyzed with the following mixed linear model:
Ytpc = μ + αt + Ac + ɛtc.
Analysis of variance (ANOVA) was used to compute the p-values for the fixed effect of the treatment. Statistical significance was declared when p ≤ 0.05, and statistical tendency was declared when 0.05 < p ≤ 0.10.

3. Results

Dry matter intake was unaffected by the supplementation of iodoform (Table 2). Methane yield (g CH4/kg DMI) decreased quadratically with increasing iodoform dose by up to 46% (0 vs. 20 mg iodoform/kg DM; Figure 1a). Simultaneously, the H2 yield (g H2/kg DMI) increased by up to 1127% on the highest dose compared to the control treatment (Figure 1b). The supplementation of iodoform did not affect the methanogen population (Table 2). Milk iodine concentrations were 183 ± 32.1 (average ± SD), 1155 ± 71.7, and 1350 ± 353.5 μg/kg milk from cows fed diets with 0, 16, and 20 mg iodoform/kg DM, respectively.

4. Discussion

As reported in previous studies [10,18], iodoform supplementation inhibited methanogenesis. In the current study, a concentration of 20 mg/kg DM corresponds to a daily intake of 506 mg/d of iodoform, which resulted in a 46% reduction in CH4 yield. Thorsteinsson and Lund [10] reported a 40% reduction in CH4 yield by supplementing 640 mg/d (app. 42 mg/kg DM) as pulse doses into the rumen. The constant supply of the CH4-mitigating compound to the rumen environment via the feed rather than a twice daily pulse dosing into the rumen appeared to increase the efficacy of iodoform. The reduction efficiency of 20 mg/kg DM has the same CH4-mitigating efficacy as studies with 3-nitrooxypropanol (pulse-dosing 2500 mg/d: 7% reduction in CH4 yield vs. TMR supply of 2500 mg/d: 60% reduction in CH4 yield [19,20]). Although supplementation via the feed might increase the efficacy of iodoform, all treatments in the current study exceeded the maximum iodine level for cattle feed, defined by the European Commission as 55–287% (5 mg/kg mixed feed with 88% DM; [21]), based on the iodine contribution from iodoform alone and ignoring contribution from all other sources (iodine accounts for 96.7% of the molar mass of iodoform). Based on daily milk production and iodine intake originating from iodoform, 9.4 ± 3.8 and 11.4 ± 2.1% of the daily iodine intake was secreted into milk for 16 and 20 mg/kg DM, respectively. A review by Niero and Visentin [22] reported milk iodine levels ranging from 91 to 489 μg/kg of milk for cows fed traditional rations, implying a remarkable increase in milk iodine through iodoform supplementation. The recommended daily intake of iodine differs according to age but, in general, ranges from 90 to 150 µg/d, with an upper limit for iodine consumption ranging from 200 to 600 µg/d [23]. Therefore, an intake of 250 mL of milk from cows fed 16 and 20 mg/kg DM would result in intakes of 289 and 338 µg iodine, respectively, and, thus, exceed the recommended daily intake by 93–125%. Similar increases in milk iodine have been observed in studies with seaweed, which also often has high concentrations of iodine [24,25]. Thus, the high iodine concentration in iodoform will be a constraint for future use of the compound as an anti-methanogenic feed additive due to negative effects on product quality and potentially also animal health.
In contrast to the large reductions in DMI (up to 48%) observed simultaneously with decreasing CH4 and increasing H2 emissions in the previous study by Thorsteinsson and Lund [10], no effect was observed on DMI in the current study at the 16 and 20 mg/kg DM inclusion levels. However, it is important to stress that the experimental design was not well-balanced. Hence, only two cows were fed 8 and 20 mg/kg DM, while four cows received 0 and 16 mg/kg DM. No difference in DMI was observed between 0 and 16 mg/kg DM. Hydrogen accumulation has been suggested to suppress rumen fermentation and microbial synthesis and thereby indirectly affect DMI [26]. The continuous supplementation of iodoform in the TMR may have resulted in smaller fluctuations in H2 emissions originating from suppressing the methanogenesis as compared to the fluctuations observed in response to intra-ruminal pulse dosing [10].

5. Conclusions

Methane yield (g CH4/kg DMI) was quadratically reduced by up to 46% while H2 yield was simultaneously increased by up to 1127%, with increasing doses of iodoform (up to 20 mg/kg DM in the TMR). Dry matter intake, total counts of bacteria, and methanogens were unaffected by the supplementation of 8–20 mg iodoform/kg DM in the TMR. Hence, this pilot study indicated that reductions in enteric CH4 yield can be obtained by using a more continuous supply to the rumen during the day by adding iodoform to the TMR of dairy cows without affecting DMI. However, the high iodine concentration in iodoform will be a constraint for the use of the compound as an anti-methanogenic feed additive in commercial herds.

Author Contributions

Conceptualization, M.O.N.; methodology, M.T., A.L.F.H., P.L., M.R.W., S.J.N. and M.O.N.; validation, M.T., A.L.F.H. and S.J.N.; formal analysis, M.T., A.L.F.H. and S.J.N.; investigation, M.T., A.L.F.H. and S.J.N.; resources, M.O.N.; data curation, M.T., A.L.F.H. and S.J.N.; writing—original draft preparation, M.T.; writing—review and editing, S.J.N., P.L., M.R.W., A.L.F.H. and M.O.N.; visualization, M.T.; supervision, M.O.N.; project administration, M.O.N.; funding acquisition, M.O.N. All authors have read and agreed to the published version of the manuscript.

Funding

The experiment was funded by Innovation Fund Denmark (Grant-ID: 0244-00011B), Vilofoss A/S, Denmark, and DLG a.m.b.a., Denmark.

Institutional Review Board Statement

The study complied with the guidelines set out by the Danish Ministry of Environment and Food with respect to animal experimentation and care of animals under study (Act 474 of 15 May 2014 and Executive Order 2028 of 14 December 2020) and under consideration of the ARRIVE Guidelines.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors express their thanks to the barn staff, especially Tanja Skovbo, Michael Christensen, Nina Engelbreth, and Jørgen Nielsen, for expert caretaking of the animals during the experiment and lab technicians for skillfully performing the laboratory analyses (AU Viborg—Research Centre Foulum, Tjele, Denmark). Also, sincere thanks to Torkild Jakobsen and Ester Bjerregaard (AU Viborg—Research Centre Foulum, Tjele, Denmark) for assisting in experimental planning.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DMDry matter
DMIDry matter intake
TMRTotal mixed ration

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Dairy 06 00017 g001
Figure 1. (a) Estimated marginal means and standard error of mean of methane (CH4) yield (g CH4/kg dry matter intake (DMI)), treatment p = 0.03; linear p = 0.05; quadratic p ≤ 0.01 and (b) hydrogen (H2) yield (g H2/kg DMI), treatment p = 0.01; linear p = 0.63; quadratic p ≤ 0.01 of dairy cows supplemented with four different levels of iodoform (0, 8, 16, and 20 mg/kg DM) in the total mixed rations.
Figure 1. (a) Estimated marginal means and standard error of mean of methane (CH4) yield (g CH4/kg dry matter intake (DMI)), treatment p = 0.03; linear p = 0.05; quadratic p ≤ 0.01 and (b) hydrogen (H2) yield (g H2/kg DMI), treatment p = 0.01; linear p = 0.63; quadratic p ≤ 0.01 of dairy cows supplemented with four different levels of iodoform (0, 8, 16, and 20 mg/kg DM) in the total mixed rations.
Dairy 06 00017 g001
Table 2. Dry matter intake (DMI), gas exchange, and microbiota of dairy cows fed total mixed rations supplemented with either 0, 8, 16, or 20 mg iodoform/kg DM.
Table 2. Dry matter intake (DMI), gas exchange, and microbiota of dairy cows fed total mixed rations supplemented with either 0, 8, 16, or 20 mg iodoform/kg DM.
Treatments, mg/kg DM p-Values
081620SEM 1TreatmentLinear EffectQuadratic Effect
DMI, kg/d23.921.524.325.31.800.280.140.39
RQ 21.131.121.121.120.0140.900.370.77
Microbiota, log10 copies/g
Total bacteria10.110.19.9210.30.1530.270.410.23
Methanogens8.398.378.248.410.0920.120.790.13
1 Highest standard error of mean is reported. 2 Respiration quotient calculated as CO2 (g/d) produced divided by O2 (g/d) consumed.
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MDPI and ACS Style

Thorsteinsson, M.; Noel, S.J.; Lund, P.; Weisbjerg, M.R.; Hellwing, A.L.F.; Nielsen, M.O. Iodoform as an Anti-Methanogenic Feed Additive in Total Mixed Rations of Dairy Cows. Dairy 2025, 6, 17. https://doi.org/10.3390/dairy6020017

AMA Style

Thorsteinsson M, Noel SJ, Lund P, Weisbjerg MR, Hellwing ALF, Nielsen MO. Iodoform as an Anti-Methanogenic Feed Additive in Total Mixed Rations of Dairy Cows. Dairy. 2025; 6(2):17. https://doi.org/10.3390/dairy6020017

Chicago/Turabian Style

Thorsteinsson, Mirka, Samantha Joan Noel, Peter Lund, Martin Riis Weisbjerg, Anne Louise Frydendahl Hellwing, and Mette Olaf Nielsen. 2025. "Iodoform as an Anti-Methanogenic Feed Additive in Total Mixed Rations of Dairy Cows" Dairy 6, no. 2: 17. https://doi.org/10.3390/dairy6020017

APA Style

Thorsteinsson, M., Noel, S. J., Lund, P., Weisbjerg, M. R., Hellwing, A. L. F., & Nielsen, M. O. (2025). Iodoform as an Anti-Methanogenic Feed Additive in Total Mixed Rations of Dairy Cows. Dairy, 6(2), 17. https://doi.org/10.3390/dairy6020017

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