In vivo mechanical and in vitro electromagnetic side-effects of a ruminal transponder in cattle

In vivo mechanical and in vitro electromagnetic side-effects of a ruminal transponder in cattle1,2 C. Antonini,* M. Trabalza-Marinucci,* R. Franceschini,† L. Mughetti,* G. Acuti,* A. Faba,‡ G. Asdrubali,† and C. Boiti†3 *Dipartimento di Patologia, Diagnostica e Clinica Veterinaria, †Dipartimento di Scienze Biopatologiche ed Igiene delle Produzioni Animali e Alimentari, Via S. Costanzo 4, 06126 Perugia, Universita` degli Studi di Perugia, Italy; ‡Dipartimento di Ingegneria Industriale, Via Pentima Bassa 21, 05100 Terni, Universita` degli Studi di Perugia, Italy

ABSTRACT: This work was undertaken to assess the long-term impacts of a ruminal transponder, used for electronic identification, on ruminal motility and on health and performance of cattle, as well as to study the electromagnetic effects on ruminal bacteria in vitro. A passive transponder (51.4 g, 67 × 17 mm) was delivered into the forestomachs of 8 calves, 32 bulls, 10 heifers, and 40 dairy cows. Final readability was 87.5% in calves, 96.9% in bulls, 90% in heifers, and 100% in cows at 481, 360, 650, and 601 d, respectively, after transponder administration. The transponder did not affect production or reproduction of cows over a 2-yr period, or performance of bulls, or mortality compared with control animals. Chewing movements per bolus were lower (P < 0.01) in treated animals than in controls (49.6 vs. 52.2, 51.2 vs. 63.6, and 57.0 vs. 59.7 for bulls, heifers, and cows, respectively). Regurgitation frequency (number of boluses/10 min) tended to be greater in treated cattle: 12.4 vs. 11.3 (P = 0.07), 11.3 vs. 10.6, and 11.3 vs. 10.7 (P = 0.08) for bulls, heifers, and cows, respectively. Rumination patterns of calves fitted with transponders within the first weeks of life were similar

to controls. During the experiment, 43 treated animals (8 calves, 29 bulls, and 6 cows) were slaughtered. Thirty transponders were localized in the reticulum (3 calves, 24 bulls, and 3 cows), 11 in the rumen (4 calves, 4 bulls, and 3 cows), and 2 were not recovered (1 calf and 1 bull). Within the calves, 57% of the boluses were found in the rumen. In 8 reticula (2 calves and 6 bulls) and 1 rumen (1 cow), an impression left by physical contact of the transponder was observed, although histological examination did not reveal specific lesions in the mucosa of the dystrophic areas. In strained, whole ruminal contents incubated in vitro, pH values were lower after 24 and 48 h (P < 0.001) of continuous exposure to an electromagnetic field induced by the transponder-reading system. After 48 h of incubation, total bacterial numbers and NH3-N concentration were greater (P < 0.001) in exposed flasks than in controls. These data indicate that the transponder may alter, via mechanical action, the reticuloruminal mucosa and rumination patterns. Furthermore, the transponder may increase, via its electromagnetic action, the growth rate and metabolic activity of ruminal bacteria.

Key words: cattle, electromagnetic field, ruminal transponder, ruminal bacteria, rumination ©2006 American Society of Animal Science. All rights reserved.

INTRODUCTION In recent years, several record systems have been devised to identify livestock for sanitary and genetic programs (Artmann, 1999; Rossing, 1999). Recently,

1 This research was supported by the Italian “Ministero della Istruzione, dell’Universita` e della Ricerca” (Progetto di Ricerca, Sviluppo e Alta Formazione # 12914_2001 and PRIN # 2005078793_004). 2 The authors would like to thank C. Cavalletti for assistance with data collection and care of the animals, E. Del Rossi and E. Cassetta for technical help in laboratory analyses, and Patrick Raymer for revision of the English text.

J. Anim. Sci. 2006. 84:3133–3142 doi:10.2527/jas.2006-136

the traceability concept, linking animal identification to information, has become a very sensitive topic for the consumer and public. In cattle, application of transponders into the rumen for the electronic identification has been successfully tested in several studies since 1976 (Hanton, 1976; Ribo´ et al., 2001; Caja et al., 2003; Saa et al., 2005). However, the biological impact of the device because of its long-term persistence within the forestomachs

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Corresponding author: [email protected] Received March 8, 2006. Accepted June 8, 2006.

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has rarely been studied. In fact, transponder-dependent side effects may arise from the mechanical action of the bolus on ruminal mucosa as well as from exposure to electromagnetic fields generated by the reading procedure. Although these electromagnetic fields are weak and short in duration, their potential for direct or indirect biological effects on both cells and ruminal bacteria cannot be ruled out. Electromagnetic fields of different intensity were recently found to stimulate cell proliferation and to modify gene expression of E. coli cultured in vitro (Potenza et al., 2004b). Therefore, the present work investigated the longterm effects of passive ruminal transponders on 1) health and performance of cattle over a 2-yr period, 2) patterns of reticulorumenal motility, and 3) in vitro growth and metabolism of bacterial populations in the rumen exposed to an electromagnetic field induced by prolonged transponder activation.

MATERIALS AND METHODS Animals and Treatments The experimental procedures and animal care conditions were approved by the Bioethics Committee of the University of Perugia and authorized by the Italian Ministry of Health (D.M. 113/203/a). A total of 16 Italian Holstein-Friesian male calves, 64 Chianina and Italian Holstein-Friesian bulls, and 20 heifers and 82 Italian Holstein-Friesian cows from the experimental farms at the University of Perugia were used. At the beginning of the experiment, the age of the calves and bulls spanned from 3 d to 3 wk and from 6 to 14 mo, respectively; heifers and cows were from 18 to 23 mo and 2 to 4 yr of age, respectively. The calves were kept in individual crates up to 60 d of age and then transferred to pens with outdoor paddocks. Calves received a pelleted feed that averaged (as-fed basis) 14% CP, 17% NDF, and 13% ADF. This concentrate, along with mixed hay (9% CP, 61% NDF, and 40% ADF) and wheat straw (3% CP, 75% NDF, and 48% ADF), was provided in quantities sufficient for ad libitum DMI. Dairy heifers and cows were raised in a conventional loose housing system and had free access to a total mixed ration, which was formulated with corn silage (6% CP, 37% NDF, and 18% ADF), alfalfa hay (15% CP, 45% NDF, and 33% ADF), and concentrate (18% CP, 16% NDF, and 7% ADF), in different proportions according to their physiological state. Bulls were kept in group pens (8 animals/pen; pen dimensions: 10 × 4.5 m) and their ad libitum diet (10.24% CP, 23.38% NDF, 7.95% ADF) contained (as-fed) 80% steam-rolled barley grain and 20% barley silage. A dietary supplement containing vitamins and minerals was available to all of the cattle. Feed was provided at 0830 and 1330 daily. Within each category, the cattle were randomly allocated to 2 groups (control and treated), balanced for age, BW, BCS, lactation number, and genetic merit.

Treated animals (8 calves, 32 bulls, 10 heifers, and 40 cows) were cared for exactly as the controls except that they were provided with a ruminal transponder by means of a metallic bolus gun at the beginning of the trial. The bolus transponder was delivered into the reticulorumen by inducing the swallowing reflex following its delivery over the caudal portion of the tongue. The operators were previously trained. After transponder administration, animals were observed for a 4-h period to detect any behavioral alteration or regurgitation of the transponder. The day of transponder application was considered d 0.

Bolus Transponders and Reading Procedure with Transceivers A passive half-duplex, glass-encapsulated transponder was enclosed in each ceramic bolus (51.4 g, 67 × 17 mm, Innoceramics, Teramo, Italy). The ceramic composition was 91.4% Al2O3, 2.5% CaO, 2.2% MgO, and 3.9% SiO. The specific density was 3.65 g/cm3. Technical specifications of the transponders conformed to the International Organization for Standardization, standards 11784 and 11785 (ISO, 1996), and worked at a frequency of 134.2 kHz. Readability of the transponder was tested throughout the experimental period by the same trained personnel, using a portable reader (Mod P3000, Innoceramics) with a stick antenna (field strength 108 db ␮V/m at 3 m; reading distance ≤25 cm). Reading checks of dairy cows were done in the milking parlor during the afternoon milking session. Reading checks of calves, heifers, and bulls were done in corresponding pens. For each treated animal, readings of the transponder were performed immediately after administration of the bolus and were repeated at 1 h, 24 h, and 1 wk. Continued readings were taken every 2 wk during the first 3 mo and then at approximately monthly intervals for the following 21 mo. For each animal category, the average duration of treatment is reported in Table 1. During the experiment, a total of 2,085 readings were carried out.

Performance Measurements Standard performance and reproductive data as well as any incidental health problems were recorded for each group of animals over a 2-yr period. For dairy cows, milk yield (limits of error of the milk recording device was 2.5%) as well as milk fat and milk protein yield were obtained at monthly intervals following the A4 scheme milking recording (ICAR, 2005). Production records were adjusted to twice daily milking for 305 d, and age-corrected to a mature equivalent basis (Bagnato et al., 1994). Milk fat and protein analyses were carried out by infrared spectroscopy (DairyLab2 A7S Foss Electric, Hillero¨d, Denmark). Reproductive indexes (days open, number of inseminations per pregnancy, conception rate) of heifers and cows were recorded. Chianina bulls were weighed at monthly inter-

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Table 1. Performance, readability, and localization of the transponders in calves, bulls, heifers, and cows Item Transponders applied, No. Age of treatment Duration of treatment, d Readings performed, No. Reading failures, % Readability1 at d 0, % Final readability, % Animals slaughtered, No. Lost boluses, No. Transponder localization Reticulum, No. Rumen, No. Cases of mucosal dysplasia Reticulum, No. Rumen, No.

Calves

Bulls

Heifers

Cows

Total

8 3 d to 3 wk 481 ± 42 191 0.5 100 87.5 8 1

32 6 to 14 mo 360 ± 37 528 0.9 100 96.9 29 1

10 18 to 23 mo 650 ± 67 276 7.2 100 90 0 0

40 2 to 4 yr 601 ± 30 1,090 2.3 100 100 6 0

90 — — 2,085 2.4 100 96.7 43 2

3 4

24 4

0 0

3 3

30 11

2 0

6 0

0 0

0 1

8 1

Readability = (readable transponders/administered transponders) × 100.

1

vals using a digital scale (precision, 500 g) until they reached the slaughter age (approximately 22 mo).

Rumination Behavior One objective of this experiment was to evaluate possible modifications of rumination behavior induced by the presence of the transponder in the forestomachs. All 4 animal categories (calves, bulls, heifers, and cows) were used in this experiment. Observations were carried out at the experimental farms according to the focal animal sampling technique (Lehner, 1996). The number of regurgitations in 10 min and chewing movements for each bolus were recorded during each session in randomly selected animals. The observation sessions (1 in July and 1 in December) began no less than 30 d after application of the ruminal transponder and were conducted for 3 consecutive weeks by 2 trained operators. Observations (250 for calves, 491 for bulls, 112 for heifers, and 520 for cows) were stratified by taking into consideration the time of day (0800 to 0900, 1130 to 1230, and 1530 to 1630), season (July and December), operator (1 or 2), animal category (calves, bulls, heifers, and cows), and group (treated or control). Within each session, each animal was observed at least once.

Postmortem Examination Postmortem localization of ruminal transponders was evaluated in 43 treated animals (8 calves, 29 bulls, and 6 cows) at the abattoir. The forestomachs, opened and cleared of ruminal contents, were carefully examined to find any gross lesion of the mucosa that could be related to the presence of the transponder. Samples from 27 treated animals (8 calves, 14 bulls, 5 cows) were taken from the reticulorumen wall in close proximity to the site of transponder retrieval or where a tissue lesion was evident. Samples of reticulorumen wall were also obtained from the same regions of 23 control animals

(8 calves, 14 bulls, 1 cow). Tissue samples were fixed by immersion in 10% neutral buffered formalin for 48 h and then processed for embedding in paraffin following routine tissue preparation procedures. Serial 5-␮mthick sections were prepared and stained with hematoxylin and eosin (Thompson and Hunt, 1966).

In Vitro Studies of the Electromagnetic Field This in vitro experiment was designed to assess the effects of the electromagnetic field generated by the transponder on rumen microbial numbers and their metabolism. Samples of ruminal contents were collected approximately 30 min before the morning feeding from the same ruminally fistulated dairy cow, which was fed 9 kg daily of mixed hay (12% CP, 62% NDF, 38% ADF) plus 1 kg of concentrate (22% CP, 20% NDF, 5% ADF) containing a vitamin-mineral supplement. The liquid fraction, obtained by filtering the ruminal contents through 4 layers of cheesecloth, was immediately taken to the laboratory, where all subsequent techniques were performed under anaerobic conditions. Ruminal fluid was added to prewarmed (39°C) McDougall’s artificial saliva (McDougall, 1948) at a 1:4 ratio and buffered to between pH 6.8 and 7.0 with 20% NaOH or 20% H3PO4 if needed. Anaerobic conditions were maintained by bubbling with CO2. Five hundred milliliters of buffered ruminal fluid were dispensed into six 500-mL Erlenmeyer flasks (base diameter, 10 cm), each containing 2 g of alfalfa hay (15% CP, 44% NDF, 32% ADF) and 0.2 g of corn (10% CP, 12% NDF, 2% ADF), both ground to pass through a 1-mm screen. Immediately after addition of the inoculum mixture, the flasks were gassed with CO2 and closed with rubber stoppers that were equipped with Bunsen valves. The flasks were randomly allocated to control and treated groups and equally distributed in 2 corresponding 205-L incubators (Termodigit, PBI International, Milan, Italy) at 39°C for 48 h. The 2 incubators were

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close to each other in the same laboratory; nevertheless, the flasks were isolated from radiation interferences by the stainless steel case of the incubator. A transponder was dropped into each treated flask. Inside one of the incubators, the stick antenna (27-cm long) of the portable reader was placed centrally, at the same distance (5 cm) from the 3 surrounding treated flasks, located in the frontal, right-lateral, and left-lateral position. Every 2 h, except between 2400 and 0800, all the flasks were gently mixed by swirling, and their position was changed in a clockwise order. The intensities of the electric and magnetic fields generated by the transceiver were measured using a 9cm-long monopole and a 6.5-cm loop antenna connected to a spectrum analyzer (model E4407B - S/N MY41441068, Agilent Technologies Italia S.p.A., Cernusco sul Naviglio, Italy) through a low-noise preamplifier (model BA 011000-35 - S/N 04-1978, RFPA, Artigues pre`s Bordeaux, France). To improve accuracy, all measurements were performed inside an anechoic chamber by reproducing the same experimental conditions used in the incubators for the in vitro experiments. The effect of reflection from the metallic case of the incubator was considered negligible compared with the direct radiations generated by the radiator caused by the frequency of the electromagnetic field. The power density components were calculated using the electric and magnetic field data. The electric fields were 0.785, 0.558, and 0.300 V/m. The magnetic fields were 0.300, 0.380, and 0.088 A/m. The power density measured was 0.187, 0.173, and 0.017 W/m2 in the right lateral, left lateral, and frontal position, respectively. Differences between the 3 positions were in accordance with the theoretical radiation pattern of a dipole antenna (Paul, 1992). After incubation for 24 or 48 h, fluid samples were collected from treated and control flasks to measure pH and to evaluate NH3, lactate, and VFA production. The pH was evaluated by immersing a probe (Microprocessor pH Meter “pH 213”, Hanna Instruments s.r.l., Ronchi di Villafranca, Italy) into each flask. The NH3-N content was evaluated using a colorimetric assay (Beecher and Whitten, 1970), and the lactate concentration using a commercial kit (Randox Laboratories Ltd., Ardmore, UK) with a UV-Visible spectrophotometer (DMS 90, Varian Techtron Pty., Ltd., Mulgrave, Australia). Volatile fatty acid production was evaluated by gas chromatography (4810 Gas Chromatograph, Perkin-Elmer, Norwalk, CT) according to Huntington et al. (1998). At the end of the incubation period, anaerobic culture media for enumeration of total, amylolytic, and cellulolytic bacteria were inoculated according to the most-probable-number procedure (Dehority et al., 1989). The experiment was replicated 6 times. Exposures to the electromagnetic field were always carried out with alternating incubators so that slight differences between incubators were averaged.

Statistical Analysis The data were statistically evaluated by ANOVA using the GLM procedure of SAS (release 8.02, SAS Inst. Inc., Cary, NC). Performance and reproductive traits were analyzed within each animal category. Adjustment was made for the year of birth, calving month, and age at first parturition, in the case of dairy cattle, or for the BW at the beginning of the experiment in the case of bulls. As for the model used for rumination behavior, independent factors other than experimental treatment and operator (i.e., time of the day, season, and animal category) were not included because they were found to be not significant (P > 0.05). Factors considered in the model for the in vitro fermentation experiment were treatment (with or without electromagnetic stimulation), time of incubation (24 and 48 h), and in vitro run. Interactions among factors were included if significant (P < 0.05). Comparisons among the individual treatments were made by the Tukey test where significance (P ≤ 0.05) had been indicated by the ANOVA.

RESULTS Overall performance, initial and final readability, and localization of the transponders in calves, bulls, heifers, and cows are summarized in Table 1. Readability (readable transponders/transponders administered × 100) at d 0 (100% in all animal categories) differed from that observed at the end of the experiment (Table 1). The portable reader failed to read the transponder in 51 occasions (1 in calves, 5 in bulls, 20 in heifers, and 25 in cows) corresponding to 2.4% of the total readings. Twenty-six of the 51 missing readings were associated with 1 calf and 1 bull whose transponders (unreadable at d 360, 352 kg of BW, and d 270, 645 kg of BW, respectively) were not recovered at the abattoir, and with 1 heifer not slaughtered (unreadable at d 120, 517 kg of BW). The remaining reading failures occurred sporadically within the cows.

Animal Performance Over the 2-yr study period, the presence of the transponder did not affect (P > 0.05) annual milk yield [10,268 ± 81.0 vs. 10,318 ± 141.4 kg of mature equivalent milk (n = 1,567)], milk fat yield [311 ± 1.9 vs. 313 ± 2.1 kg (n = 1,566)] and milk protein yield [285 ± 1.8 vs. 280.2 ± 1.9 kg (n = 1,573)] for treated and control cows, respectively. Similarly, treatment did not influence the reproductive traits (149 ± 1.2 vs. 170 ± 11.9 d open and 2.2 ± 0.15 vs. 2.6 ± 0.22 inseminations per pregnancy, for treated and control cows, respectively) except conception rate, which was greater in treated cows (73 ± 2.6 vs. 66 ± 2.7 %; P < 0.05). Body weight gain of bulls up to 22 mo of age was not influenced by treatments (final BW: 785 ± 73.4 vs. 771 ± 49.8 kg; and ADG: 1.4 ± 0.15 vs. 1.3 ± 0.13 kg; for

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Table 2. Rumination pattern as affected by the presence of the ruminal transponder in calves, bulls, heifers, and cows Chews per digesta bolus Item

No.1

Calves Bulls Heifers Cows

250 491 112 520

Treated 55.9 49.6 51.2 57.4

± ± ± ±

0.6 0.6 0.8 0.6

Regurgitations per 10 min

Control

P-value

± ± ± ±

0.423 0.006 3.3 g/cm3) were characterized by 99.7% readability values. Hasker and Bassingthwaighte (1996) reported 100% retention rate in feedlot steers when similar transponders (65 g, 3 g/cm3) were used. Ghirardi et al. (2003) reported a minimum weight of 65 g and a specific gravity higher than 3.0 g/cm3 to reach the 98% retention rate established by ICAR (2003). In an 8-yr study conducted on 161 beef cows, Ghirardi et al. (2004) found 98.8% readability when using a ceramic transponder with a weight of 75 g (3.36 g/cm3 specific gravity).

Transponder localization in the rumen did not appear to affect its readability. Nevertheless, when the antenna placed beneath the reticulum projection area failed to read the transponder, further attempts were made by moving the stick antenna along the abdomen of the animal. In 1 case (1 heifer), the transponder was unreadable since d 120 after administration, but it was impossible to determine whether it was lost or simply not functioning because the animal was not slaughtered. Administration of transponders to young (3 to 21 d of age) calves was easily carried out by using the same instrument and technique employed for older animals. Nevertheless, our data suggest that some caution is needed when evaluating the feasibility of the system in these young animals; most transponders administered to calves were found in the rumen, or in 1 case, never recovered at slaughter (Table 1). However, because of the relatively low number of animals used in the current study, readability values and slaughterhouse recovery rates are not directly comparable with those obtained in large-scale experiments. Overall performance and reproductive traits were not affected by the presence of ruminal transponders, suggesting that the modification of ruminal motility patterns were not able to exert negative effects on digestive and reproductive physiology. In our in vivo study, effects of the electromagnetic field were probably negligible because each animal was subjected to a maximum of 30 readings over the experimental period. At present, no explanation is available for the increased conception rate found in treated cows and heifers. Similarly, an increase of conception rate, although not significant, was reported in bolused sheep by Caja et al. (1999). However, before excluding any side effects caused by transponder-induced changes in ruminal motility of cattle, performance and reproductive traits should be

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Table 3. Effects of continuous exposure to the electromagnetic field (EMF) generated by a radiofrequency handheld reader on in vitro ruminal conditions Treatment1 Item Bacteria, No./mL (log) Total Amylolytic Cellulolytic pH Total VFA, mM Individual VFA, % Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Acetate:propionate NH3-N, mg/100 mL Lactate, mM

P-value

T-24

C-24

T-48

C-48

SE

EMF

IT2

INT3

— — — 6.82 6.96

— — — 6.89 7.56

7.21 7.22 4.84 6.97 8.30

6.64 5.62 3.98 7.06 6.91

0.09 0.44 0.49 0.02 1.08