Sceleratine nitrogen oxide, when administered together with a dichloromethane extract of Senecio latifolius, successfully conditioned cattle and sheep to avoid milled freeze dried S. latifolius mixed with maize meal. This treatment was effectively applied in conditioning steers to refuse eating S. latifolius grown in pots

Snyman, Leendert D

English

DigitalCommons@USU

Poisonous Plant Research (PPR) Volume 6 Article 4 12-20-2023 Sceleratin Nitrogen Oxide as Aversive Agent in Conditioning Livestock to Avoid Senecio latifolius Leendert D. Snyman Toxicology, Agricultural Research Council, Onderstepoort Veterinary Institute, Private Bag X05, Onderstepoort 0110, South Africa, snymanleendert@gmail.com Follow this and additional works at: https://digitalcommons.usu.edu/poisonousplantresearch Part of the Agriculture Commons, Animal Sciences Commons, Plant Sciences Commons, Social and Behavioral Sciences Commons, and the Toxicology Commons Recommended Citation Snyman, Leendert D. (2023) "Sceleratin Nitrogen Oxide as Aversive Agent in Conditioning Livestock to Avoid Senecio latifolius," Poisonous Plant Research (PPR): Vol. 6, p. 40-48. Available at: https://digitalcommons.usu.edu/poisonousplantresearch/vol6/iss1/4 This Article is brought to you for free and open access by the Journals at DigitalCommons@USU. It has been accepted for inclusion in Poisonous Plant Research (PPR) by an authorized administrator of DigitalCommons@USU. For more information, please contact digitalcommons@usu.edu. Sceleratin Nitrogen Oxide as Aversive Agent in Conditioning Livestock to Avoid Senecio latifolius Abstract Sceleratine nitrogen oxide, when administered together with a dichloromethane extract of Senecio latifolius, successfully conditioned cattle and sheep to avoid milled freeze dried S. latifolius mixed with maize meal. This treatment was effectively applied in conditioning steers to refuse eating S. latifolius grown in pots. Keywords Senecio latifolius, sceleratine-NO, aversive agent, conditioning, livestock Cover Page Footnote Footnote LD Snyman: Retired veterinary research scientist (snymanleendert@gmail.com) Professor CJ Botha of the Faculty Veterinary Science, University Pretoria, is acknowledged for reviewing the veterinary aspects of the manuscript This article is available in Poisonous Plant Research (PPR): https://digitalcommons.usu.edu/poisonousplantresearch/vol6/iss1/4 INTRODUCTION Ingestion of Senecio latifolius and some other Senecio spp. causes seneciosis, which is estimated to be the second largest cause of cattle mortalities due to plant poisoning or mycotoxicosis in South Africa (Kellerman et al. 1996). Cattle, especially young animals newly introduced to S. latifolius infested pastures, may die suddenly within the first few days after ingesting relatively large quantities of the plant. Acute and chronic poisoning of sheep also occurs regularly in areas of abundant plant growth. Young plants in the two-leaf stage of growth appears to be more palatable and most toxic to livestock (Kellerman et al. 1988). The application of conditioned feed aversion potentially might be a means to prevent large scale poisoning of livestock newly introduced to S. latifolius infested pastures. Sceleratine-NO, the toxic principle of S. latifolius, has been shown to be aversive when administered to a sheep (Snyman 2023) and thus could potentially be used as an aversive agent in conditioning livestock to avoid S. latifolius. The sheep, however, did not refuse eating S. latifolius presented as part of a maize meal mixture (Snyman 2023), suggesting that sceleratine-NO as such may not be usable for establishing aversion to S. latifolius. However, a sheep averted to S. latifolius refused eating a dichloromethane extract of S. latifolius. This was ascribed to the sensory characteristics of S. latifolius present in the extract. Thus, the hypothesis was made that livestock treated with a combination of sceleratine-NO and a dichloromethane extract of S. latifolius will associate the aversive effect of sceleratine-NO with the sensory characteristics of S. latifolius extracted by dichloromethane and refuse intake of S. latifolius. This approach would be in accordance with the mechanism of learning in diet selection by herbivores (Provenza et al. 1992). In this study, the aversive effect of sceleratine-NO administered together with a dichloromethane extract of S. latifolius was investigated to determine their capability in conditioning sheep and cattle to refuse S. latifolius containing maize meal mixtures and S. latifolius grown in pots. MATERIALS Plant Material. Senecio latifolius was collected in the eMkhondo (= Piet Retief) (27.0245⁰S, 30.7925⁰E) district and re-established on a plot on the premises of the Onderstepoort Veterinary Research Institute. Senecio latifolius used for pen trials with sheep and cattle was collected at the pre-bloom stage where after it was freeze-dried, milled, and stored at – 20 ⁰C in a conventional deep freezer. Plants from this cultivation were also transplanted to pots to be used for pen trials with steers. Experimental Animals. All procedures with animals were carried out according to the South African National Standard (The Care and Use of Animals for Scientific Purposes [SANS 10386:200X]. Animal trials were approved by the 40Snyman: Sceleratine-NO for inducing aversion to S. latifoliusPublished by DigitalCommons@USU, 2023animal ethics committee of the Agricultural Research Council–Onderstepoort Veterinary Institute. Adult Dorper wethers of approximately 50 kg and Bonsmara and Nguni steers (15 – 18 months old), naïve to S. latifolius, were used as experimental animals. The animals were individually housed in pens and fed Eragrostis curvula hay ad lib. Apparatus. Freshly collected plant material was lyophilized with a Christ laboratory freeze drier (Martin Christ, Germany), and ground with a Wiley cutting mill (Arthur H. Thomas Co., Philadelphia) to pass a 1 mm sieve. Evaporation of solvents was carried out at 40 ⁰C using a Buchi R–100 rotary evaporator (Büchi Labortechnik AG, Flawil, Switzerland). Chemicals. Redistilled analytical grade (Merck Darmstadt, Germany) methanol and dichloromethane were used. METHODS AND RESULTS Preparation of sceleratine-NO and dichloromethane extract containing maize meal mixtures. The sceleratine-NO and dichloromethane extract containing maize meal mixtures presented or dosed to the animals were prepared as follow. The needed amount of sceleratine-NO to be mixed with maize meal for the various trials described below was dissolved in 50 ml methanol, mixed, and the methanol evaporated. For mixtures containing both sceleratine-NO and a dichloromethane extract, sceleratine-NO was mixed with maize meal as described above whereafter the dichloromethane extract (prepared as described by Snyman 2023) was added, mixed and the dichloromethane evaporated. Dichloromethane of mixtures containing only the dichloromethane extract was also evaporated before presentation to animals. Establishing aversion to milled freeze-dried S. latifolius. Establishing and testing for aversion was performed as previously described (Snyman 2023). Treatment with sceleratine–NO. A sheep was offered 6 mg sceleratine-NO in 100 g maize meal on Day 1, which was totally consumed. After consumption, 35 mg sceleratine-NO dissolved in 100 ml water was orally drenched, using a drench gun. Sceleratine-NO ingested plus drenched (41 mg) was equivalent to consumption of 33 g dried S. latifolius which is considered as an aversive dose (Snyman 2023). When challenged with the sceleratine-NO containing meal mixture (6 mg/100g) on days 2, 4 and 6 the mixture was totally refused. Offering the sheep 100 g of a 2% S. latifolius maize meal mixture on days 5 and 6 resulted in partial consumption on Day 5 followed by total consumption on Day 6. The results are shown in Fig. 1. In a second trial a sheep was offered a sceleratine-NO containing maize 41Poisonous Plant Research (PPR), Vol. 6 [2023], Art. 4https://digitalcommons.usu.edu/poisonousplantresearch/vol6/iss1/4meal mixture (50 g) equivalent to 2% S. latifolius. After consumption of the mixture the sheep was orally drenched with sceleratine-NO dissolved in water at the equivalent to 18 g S. latifolius. The sceleratine-NO containing meal mixture (equivalent to 2% S. latifolius) was totally refused when presented to the sheep on days 3 and 29. However, a 2% S. latifolius containing maize meal mixture (100 g) presented on days 4, 7, 11, and 12 was totally consumed on each of these days. Reduced consumption of the mixture recorded on days 13, 14, 15 and 16 might be 0204060801001 2 3 4 5 6Intake (%)DayFigure 1. Effect of sceleratine nitrogen oxide ingested (6 mg) plus drenched (35 mg) on intake of sceleratine nitrogen oxide (6 mg/100g) and S. latifolius (2%) containing meal mixtures by a sheepIntake of sceleratine nitrogen oxide containing mixture (%) Intake of S. latifolius containing mixture (%)0204060801001 3 5 7 9 11 13 15 17 19 21 23 25 27 29Intake (%)DayFigure 2. Effect of sceleratine nitrogen oxide ingested plus drenched (equivalent to 19 g S. latifolius) on intake of sceleratine nitrogen oxide (equivalent to 2% S. latifolius) and S. latifolius (2%) containing meal mixtures by a sheepIntake of sceleratine nitrogen oxide containing mixture (%) Intake of S. latifolius containing mixture (%)42Snyman: Sceleratine-NO for inducing aversion to S. latifoliusPublished by DigitalCommons@USU, 2023ascribed to temporary aversion induced by the preceding intake of S. latifolius. The results are shown in Fig. 2. Treatment with a dichloromethane extract. A dichloromethane extract equivalent to 1 g S. latifolius was mixed with 50 g maize meal (equivalent to 2% S. latifolius) and offered to a sheep. The sheep eagerly consumed the mixture. The sheep was subsequently dosed via a stomach tube with an additional amount of the dichloromethane extract (mixed with maize meal and shaken up with water) at the equivalent of 18 g S. latifolius. When offering the dichloromethane extract containing meal mixture the following day, the mixture was again completely consumed. Treatment with sceleratine-NO plus a dichloromethane extract. In subsequent trials the aversion inducing capability of sceleratine-NO with respect to S. latifolius was investigated when ingested together with a dichloromethaneextract of S. latifolius. In a first trial a sheep was offered sceleratine-NO plus adichloromethane extract of S. latifolius mixed with 50 g maize meal at theequivalent of 2% S. latifolius. The mixture was totally consumed. The sheep wassubsequently dosed via a stomach tube with sceleratine NO plus a dichloromethaneextract mixed with 50 g maize meal at the equivalent of 18 g S. latifolius. The mixture was shaken up with water before dosage. When offering the sceleratine-NO plus dichloromethane extract containing maize meal mixture (equivalent to 2% S. latifolius) the next day it was totally refused. The sheep also refused a 2% S.latifolius containing maize meal mixture (50 g) on days 4 and 5. The results areshown in Fig.3. In a following trial a steer was offered sceleratine-NO plus adichloromethane extract, mixed with 1 kg maize meal, both at the equivalent of0501001 2 3 4 5Intake (%)DayFigure 3. Effect of sceleratine nitrogen oxide plus a dichloromethane extract of S. latifolius (ingested plus dosed, equivalent to 19 g S. latifolius) on intake of a sceleratine nitrogen oxide plus dichloromethane extract containing meal mixture (equivaleIntake of sceleratine nitrogen oxide plus dichloromethane extract containing mixture (%)Intake of S. latifolius containing mixture (%)43Poisonous Plant Research (PPR), Vol. 6 [2023], Art. 4https://digitalcommons.usu.edu/poisonousplantresearch/vol6/iss1/40.6% S. latifolius. The mixture presented on Day 1 was totally consumed. The steer was subsequently dosed via a stomach tube with sceleratine-NO at the equivalent of 100 g S. latifolius in combination with a dichloromethane extract equivalent to 50 g S. latifolius. The sceleratine-NO and dichloromethane extract dosed were previously mixed with 100 g maize meal and shaken up with 1 L water. The steer was then challenged with 0.5% S. latifolius in 1 kg maize meal on Day 2, 0.5% S. latifolius in 1 kg forage meal on Day 3, and 0.5% S. latifolius in 1 kg maize meal again on Day 7. The steer totally refused each of the presentations. The results are shown in Fig. 4. Establishing aversion to S. latifolius grown in pots. Twenty-seven steers were treated for aversion to S. latifolius plants grown in pots. Each of the steers (individually penned) was dosed via a stomach tube with sceleratine-NO equivalent to 100 g S. latifolius (DM) plus a dichloromethane extract equivalent to 50 g S. latifolius (DM) after food and water had been withheld overnight. The sceleratine-NO and dichloromethane extract dosed were mixed with 100 g maize meal as earlier described and shaken up in 1 L water before dosage. Hay and water were made available 8 h. following treatment. Steers were exposed to a S. latifolius plant grown in a pot (Fig. 5) 24 h. after treatment. Three treated steers were simultaneously compared with 3 untreated control steers at a time. Each of the treated steers were housed in a pen next to a control steer. The steers were made 0501001 2 3 4 5 6 7Intake (%)DayFigure 4. Effect of sceleratine nitrogen oxide (equivalent to 100 g S. latifolius) plus dichloromethane extract (equivalent to 50 g S. latifolius) (ingested plus dosed) on intake of S. latifolius containing meal mixtures (0.5%) by a steerIntake of a sceleratine nitrogen oxide plus dichloromethane extract containing mixture (%) Intake of a S. latifolius maize meal mixture (%)Intake of a S. latifolius forage meal mixture (%)44Snyman: Sceleratine-NO for inducing aversion to S. latifoliusPublished by DigitalCommons@USU, 2023Figure 5. Steer exposed to a S. latifolius plant grown in a pot. accustomed to the environment by keeping them in the pens for a week prior to treatment. Two replications during the early part of the summer (October –April) were executed each year for 3 consecutive years. Total consumption of the plants was recorded after steers had been exposed to the plants for 24 h. and again after being exposed for an additional 48 h. (72 h. total). The results shown in Table 1 were statistically analyzed using Fisher’s Exact Test. Table 1. Number of treated and control steers that totally consumed a S. latifolius plant throughout different periods of exposure during early and late summer. Period of exposure (hours) Number of steers that totally consumed the S. latifolius plant Early summer (October – December) Late summer (January – April) Control (n=18) Treated (n=18) Significance of difference Control (n=9) Treated (n=9) Significance of difference 24 9 0 P<0.01 * * * 72 11 1 P<0.01 5 4 P>0.05* Not recordedDuring early summer significantly less treated steers totally consumed the plant during both 24 h. and 72 h. exposure periods compared with controls. Except for 45Poisonous Plant Research (PPR), Vol. 6 [2023], Art. 4https://digitalcommons.usu.edu/poisonousplantresearch/vol6/iss1/4one of the treated steers that partially consumed the plant, all treated and control steers either totally consumed or totally avoided the plant. Nine of the 11 control steers totally consumed the plant within the first 24 h. exposure period. No difference between treated and control steers could be detected during late summer. Challenging steers averted to S. Latifolius grown in pots with S. latifolius established on a pasture. Directly after termination of the pen trials executed during early summer of Year 3, three averted steers of each of the last two replications that refused eating S. latifolius grown in the pots were introduced to Figure 6. Averted steers on an established S. latifolius pasture S. latifolius established at a site on the premises of the institute (Fig. 6). Steers ofthe former replication were moved on to the established S. latifolius pasture for 3days followed by steers of the latter replication, also for 3 days. They receivedEragrostis curvula hay ad lib. No other plant material than S. latifolius wasavailable to eat. The steers were monitored for clinical signs of seneciosis. None ofthe steers exhibited clinical signs of S. latifolius poisoning after 3 days. Serumaspartate aminotransferase (AST) and gamma glutamyl transferase (GGT) -valuesalso remained within normal reference range.DISCUSSION The results show that treatment with sceleratine-NO as an aversive agent in combination with a dichloromethane extract of S. latifolius, containing the sensory characteristics of the plant, effectively conditioned steers to avoid S. latifolius grown in pots during early summer. Only one of 18 treated steers totally consumed the S. latifolius plant within the 72 h. period compared to 11 of the control steers. 46Snyman: Sceleratine-NO for inducing aversion to S. latifoliusPublished by DigitalCommons@USU, 2023The results are a positive indication that conditioned feed aversion might potentially be used as a means to prevent livestock from being poisoned on S. latifolius infested pastures during early summer. This possibility is supported by the observation that 6 steers averted to S. latifolius grown in pots also did not develop clinical signs of poisoning after being kept on an established S. latifolius pasture for 3 days. These results should be followed up by field trials proving the use of sceleratine-NO together with a dichloromethane extract as aversive agent in conditioning livestock to avoid S. latifolius. A possible explanation that no aversion to S. latifolius could be demonstrated for treated steers exposed to plants during late summer, might be ascribed to the fact that the sensory characteristics of the dichloromethane extract, obtained from S. latifolius collected during early summer, did not match the sensory characteristics of late summer S. latifolius. Further research needs to be done to clarify this matter. It is interesting to note that treated and control steers, with the exception of one treated animal (early summer), either totally consumed or totally refused the plant during a 72 h exposure period, indicating an all or nothing approach. Nine of 11 control steers exposed to the plant during early summer totally consumed the plant within 24 h. Total intake/refusal suggests that refusal of S. latifolius by both averted and control animals took place merely by smelling. The treatment regimen applied in these trials seemed to be safe as none of the animals exhibited clinical signs reminiscent of S. latifolius poisoning. Using pure sceleratine-NO as aversive agent ensures a controlled, safe and practical means of treatment. In contrast, using the plant material for establishing aversion might be risky as the toxicity of S. latifolius might vary under different conditions (Kellerman et al. 1988). REFERENCES Kellerman, T.S., Coetzer, J.A.W. and Naudѐ, T.W. 1988. In: Plant Poisonings and Mycotoxicosis of Livestock in Southern Africa. Capetown: Oxford University Press. Kellerman, T.S., Naudé, T.W. and Fourie, N. 1996. Distribution, diagnosis and estimated economic impact of plant poisoning. Onderstepoort Journal of Veterinary Research 63:65-90. Provenza, F.D., Pfister, J.A. and Cheney, C.D. 1992. Mechanisms of learning in diet selection with reference to phytotoxicosis in herbivores. Journal of Range Management 45:36-45. Snyman, L.D. 2023. Isolation of the Toxic Principle of Senecio latifolius by Means of the Sensory Receptors of Sheep. Poisonous Plant Research (PPR) 6 (3): 24-37. 47Poisonous Plant Research (PPR), Vol. 6 [2023], Art. 4https://digitalcommons.usu.edu/poisonousplantresearch/vol6/iss1/4

Sceleratin Nitrogen Oxide as Aversive Agent in Conditioning Livestock to Avoid \u3cem\u3eSenecio latifolius\u3c/em\u3e

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Sceleratin Nitrogen Oxide as Aversive Agent in Conditioning Livestock to Avoid \u3cem\u3eSenecio latifolius\u3c/em\u3e

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