November 2007 Unpublished article S.A. van der Esch

Transcription

1 ANTI-FERTILITY EFFECT OF A NEEM (Azadirachta indica, A. Juss) DERIVED PRODUCT FOR LIMITING REPRODUCTIVE PERFORMANCE IN PIGEONS (Columba livia): AN ENVIRONMENTALLY FRIENDLY TOOL FOR PEST MANAGEMENT Carnevali Fiorella*, Pistidda Elisabetta** and van der Esch S. Andrew* * ENEA (Ente per le Nuove Tecnologie, l Energia e l Ambiente), Unità Tecnico Scientifica BIOTEC, Sez AGRO, Centro Ricerche Casaccia, Via Anguillarese.301, Santa Maria di Galeria ROMA; fiorella.carnevali@enea.it; stephen.vanderesch@enea.it, ** AUSL n 2, Dipartimento di Prevenzione e Sanità animale, Servizi Veterinari di Sanità Animale, Area dell Assisano, Piazza Moncada, 4, Bastia Umbra, Perugia, pistidda@libero.it. Introduction It can be said that there is no city or town, in Italy as well as in Europe, which doesn t have to face the problems caused by the presence of an excessive number of stray pigeons (Sbragia G. et al. 2001, Russo G., 2001). Recently Nomisma, an Italian Society for Economic Studies, published a report that evaluates the economic and social costs caused by urban pigeons (Rome, June 19 th 2003) based on a cost/benefit approach. They estimated the damage to be of Euro/year if one considers an average town with inhabitants. Indeed, high population densities of pigeons can lead to many different negative consequences like dirt, bad odours, damage to architectural elements -which in the case of monuments is often irreversible- and a generally degraded aspect of the urban environment. One must also add to this the public health problems that can arise when a high-density pigeon population exists. It is already recognised that pigeons (as other birds) can act as a vector for zoonotic diseases. Because of the high densities, the overall health of the pigeon population is generally precarious, which facilitates the presence of zoonotic diseases. Besides, new zoonotic diseases are slowly invading the northern hemisphere from the south. Many of those are a lot more lethal than the ones naturally present (for example West Nile Virus). Pigeons prefer to live inside the urban environment because they can easily find abundant food, sheltered spaces for nesting, absence of natural predators, many varieties of shelters according to the climatic conditions. Under these conditions, reproductive performances are exalted and the growth rate of the population becomes unacceptable with respect to urban life and a healthy and natural co-habitation with humans. The same report concluded that the only efficient and economically viable method for containing the number of pigeons is limiting fertility. All other approaches are either inefficient or the balance between costs and benefits is not favourable. Presently the tools for fertility control consist in using chemical substances that interfere at different levels on the reproductive activity (some at hormonal and others at not hormonal level). Recently, Nicarbazina received the authorization (August ) to be used as an anti-fertile in pigeons by the Italian Ministry of Health. At present, the effects of a large scale use of this chemical as for residuals, degradation rate in the environment, effects on non-target animals (both birds and mammals) are not well known. Independently of the side effects of the tools available for birthcontrol in vertebrate pests, such as pigeons, it is desirable to orient the research towards natural substances which can present less side effects, a reduced environmental impact, less problems on non-target animals. At the Enea we are studying a method for the fertility control of pigeons, based on natural substances derived from a tropical plant, named Neem tree (Azadirachta indica (A. Juss)), acting in a reversible manner (Shakti et al., 1993), inhibiting the reproductive performance without causing hormonal side effects (Mukherjee S. et al., 1996,1997,1999; Talwar G.P. at ali., 1997a,b; Upadhyay S. N.,et al., 1992; Prakash A.O.. et al., 1988). Being biodegradable and environmentally friendly (The Neem Tree, Mumbay ), neem derived products promise to be very useful for birth control in nuisance animals such as urban pigeons. ENEA 1

2 Materials and Methods The experiment was carried out on 72 adult domestic pigeons, Columba livia of proven fertility, divided into 36 stable couples and regularly bred on a farm, located in the centre of Italy (Bastia Umbra Perugia). The 36 stable couples of pigeons were divided into two homogeneous groups, namely the control group () and treated group (). The were fed with an isocaloric and isonitrogenuous diet containing 20% of soybeans. The were fed with the same food to which a neem derived product was added for the first 5 months of the experimental period (March August ). Each aviary of both groups was equipped with 18 double nests to permit each couple to brood the eggs in one nest and to feed the previously new born in the other. It was possible to inspect each double nest from the backside without stressing the inhabitants and the new born. The cage part of the nests was protected from the rain and sun; the rest of the aviary was included in a large meshed net and the basement raised from the floor to permit the fall of the dejections on the ground. These were removed once a day. Food was located in open boxes under the nests and was available ad libitum for the whole experimental period. Data were registered every ten days, through inspection of the nests of each experimental couple of both experimental groups. The number of eggs laid, their condition (broken, unfertile or fertile) and the number of born or dead chicks were registered. Statistical analysis was carried out using the SAS tm system. Results In order to represent the registered data, all the single observations (taken at ten days intervals) were grouped in 6 two-month periods, starting March till the end of February Medicated food was administered for the first five months of the twelve month experimental period. This means that during the third two-month period, received medicated food only during the first month of this period (July Aug. ). Observing the data over the entire trial period (12 months), three main functional periods (Table 1) have been identified: (i) a first period of two months before reaching the anti-fertility effect of the neem derived product (induction period); (ii) a second period of 4 months during which the antifertility effect is maintained (active period); (iii) a third and last period of 6 months, after the suspension of the neem treatment, during which the reproductive performance of the two groups becomes once more comparable (recovery period). Table 1: Timeline describing the relationship between the treatment period and the subsequently identified functional periods, over the whole observation period. Observation periods Treatment period Functional period March February 2003 March Apr. May June July Aug. Sept. Oct. Nov. Dec. Jan. Feb Treated group eating medicated food Both and are eating non-medicated food. Induction period Active period Recovery period Both in the control () and the treated group (), the number of eggs lain during the whole experimental period was similar, 329 and 318 respectively, and never varied between the two groups (see fig. 1). The percentage of fertile eggs on the total eggs lain and of dead chicks respectively produced by the and, varied according to the functional period considered (see fig. 2). ENEA 2

3 Figure 1: Egg production of both Control Group () and Treated Group () over the whole observation period Total n of Eggs laid March-April May-June July-August Sept.-Oct. Nov.-Dec. Jan.-Febr. Observation period Considering the data in more detail, one observes that during the induction period, the reproductive performance of the and was similar with 62% and 68% of live chicks produced, 17% and 17% of unfertile eggs and 21% and 15% of dead chicks, respectively (see first part of fig. 2). During the active period, significant differences between the and groups were found, with values of 45% and 41% (n.s.) of live chicks produced, 27% and 53% (P < 0.001) of unfertile eggs and 28% and 6% (P < 0.001) of dead chicks respectively (see second part of fig. 2). During the recovery period, the reproductive performance of both groups returned to similar values: 40% and 48 % of live chicks produced, 29% and 34% of unfertile eggs and 31% and 18% of dead chicks, with no statistical differences (see third part of fig. 2). ENEA 3

4 Figure 2: Percentages of, and of the total amount of eggs produced for Control group and () and Treated group () during the three functional periods. 17% 17% 21% 62% 15% First part of fig. 2 Induction period. (IP): March - April 68% 27% 45% 53% 41% 28% Second part of fig. 2 Active period. (AP): May - August. 6% Third part of fig. 2 Recovery period. (RP): September - February % 34% 40% 48% 31% 18% Considering only the egg fertility -determined by the sum of live and dead chicks- (see fig. 3), during the first two-month period, no effect on the percentage of fertile eggs was observed. During the second two-month period, 80% of the, but only 40% of the, were fertile (P < 0.001). ENEA 4

5 During the subsequent two-month periods, the egg fertility between the two experimental groups became comparable once more. Figure 3: Percentage of fertile eggs of total number of eggs laid for every two-month observation period in Control Group () and Treated Group () Percent fertile eggs of total n produced Start of feeding medicated food End of feeding medicated food 10 0 March-April May-June July-August Sept.-Oct. Nov.-Dec. Jan.-Febr. Observation period Considering only the post-natal mortality determined by the percentage of dead chicks over the total born- (see fig. 4), during the first two-month period, no difference was observed. During the second two-month period, post-natal mortality was 34% of the and 6% of the (P < 0.001). Figure 4: Percentage of dead chicks of the total number of eggs produced in Control Group () and Treated Group (). 50 Percent dead chicks of total n eggs produced March-April May-June July-August Sept.-Oct. Nov.-Dec. Jan.-Febr. ENEA 5 Observation period

6 During the subsequent two-month period this effect was still evident with a significant difference between the and (24% and 4% respectively (P < 0.001)). In the remaining two-month periods, post-natal mortality became comparable once more. Discussion During the active period, the neem derived product significantly enhanced the number of unfertile eggs, but did not alter the number of eggs laid. The reduction of egg fertility in the neem treated group was of 50% (80% and 40% of fertile eggs in the and respectively). Moreover, postnatal mortality was reduced by 83% in the neem treated group (34% and 6% of dead chicks in the and respectively) evidencing a health promoting effect. The data also indicate that the neem derived product needs almost a two-month period to reach an antifertility effect. An equivalent period is necessary for recovering normal fertility after the suspension of neem treatment. The health promoting effect lasts for a longer time with respect to the anti-fertility effect. Clearly two different effects are operating: First: antifertility effect. As the number of eggs laid in the and is equivalent, while the number of unfertile eggs is different, there is an anti-fertility effect on the male, as reported in male broilers fed with neem derived products (Mohan J. et al., 1997), but not on the avian female (Elangovan A.V., 2000). The antifertility effect of neem products has also been reported in mammal systems like rodents and primates (Talwar G.P. et al., 1997; Mukherjee S. et al., 1997, 1996; Sampathraj R: et al., Vijjan V.K. and Parihar N.S., 1983). The literature on the argument refers that the antispermatogenic effect of neem derived products seems to activate a local immune cell population which starts to produce unknown factors that possibly disturb, in a reversible manner (Upadhyay S. N., 1993a,b, 1992), the spermatogenic process (Mohan J. et al., 1997, Upadhyay S. N., 1992, 1993a,b). Our experiment confirms the reversible anti-fertility effect of the employed neem derived product, but in this preliminary experiment, it was not possible to verify its mode of action. However, extensive literature about this argument exists. In fact it is reported that neem derived products act by immunological and not by hormonal pathway (Prakash A.O. et al., 1998; Upadhyay S. N.,et al., 1993) as an immunomodulator mediated by the macrophages (Upadhyay S. N.,et al., 1993, 1992), without altering the presence of the primordial sperm cells (spermatogonia) and without inducing anti-sperm antibodies (Upadhyay S. N.,et al., 1993a,b). Therefore, being the spermatogenesis a continuous process, in order to obtain and mantain the antifertility effect, it is necessary to permanently feed the animal with medicated food. This means that if non-target animals should occasionally eat some doses of medicated food, that wouldn t entail any antifertility effect. Furthermore, Upadhyay S. N.,et al., et al. (1993a,b) reported that the neem treated male continues to mate normally as was observed during the whole experiment. Couples remained stable during the whole observation period, including the treatment period. This is very important for developing a useful method for limiting the fertility of the stray urban pigeons which does not disturb the sexual behavior of the population. Besides, the non-hormonal mode of action of neem derived products is very important in order to avoid side effects on human health. In the present experiment we observed that neem derived products need up to two months of application for reaching the anti-fertility effect, corresponding almost to two or three pigeon spermatogenetic cycles. The same period is necessary for recovering normal fertility after neem treatment suspension, even if this effect is masked by the two-month period data registration system used in this experiment. This means that, in order to obtain an efficient birth control, the administration of medicated food to stray pigeons should start during the low fertility period of the seasonal reproductive activity (autumn). Because of the high sensitivity of pigeons to stress by handling, it was not possible to determine the body weight of each experimental pigeon. Consequently we were not able to relate the antifertility effect to the individual body weight. Certainly a dose dependent effect exists, as reported by Mohan J. (1997), which will have to be further determined in future experimentations. ENEA 6

7 Second: health promoting effect. The reduction of post-natal mortality, observed in the is clearly related to the administered neem derived product. In fact, the presented a much higher mortality rate during the active period compared to the. It is necessary to report that during the second two-month period on the farm there was an intoxication due to a stock of aflatoxin contaminated corn used to feed all other aviaries except the two experimental aviaries. It is well known that aflatoxins can be inhaled and cause damage also without being eaten (Ueno Y., 1984; Placinta C.M. et al., 1999; Conkova E. et al., ). In fact, during this toxic period, in those avaries that received the contaminated corn, there was a high percentage of mortality in adults and new born. In the we registered a high mortality rate in new born but not in adults. This maybe due to the fact that mycotoxins inhalation is not lethal in adults but simply increases susceptibility to secondary diseases (Conkova E. et al., ). It is also recognized that young animals are more susceptible to the lethal effect of mycotoxin inhalation (Puschner B., ). During this toxic period, in the aviary the mortality in new born remained at 6%, while in the aviary it had reached 30%. The marked reduction of the post-natal mortality rate in neem treated chicks, with respect to the, in the presence of the toxic corn, shows that animals treated with neem derived product, benefited from a strong protection against the toxic effect of the mycotoxin inhalation. Anyway, even after the toxic episode, the health promoting effect of the neem derived product was still evident. In fact, also without the intoxication effect by the mycotoxin contaminated food, the mortality rate registered in the, was significantly inferior to the s (4% and 24% respectively). The effect is clearly due to the neem derived product, which could be responsible for a better health status or a better resistance to secondary diseases of the treated chicks. A possible explanation is that neem derived products act as immunomodulators, enhancing the immunological defences of the treated animals, as already reported elsewhere (Mukherjee S. et al., 1999, Mohan J. et al., 1997; Talwar G.P. et al.,1997a,b; Upadhyay S. N. et al., 1993a,b,1992). The health promoting effect lasts longer than the anti-fertility effect as shown in Figure 2 and 3. Only during the three final two-month periods the post-natal mortality rate of both the and the became equivalent once more. In terms of fertility reduction the experiment has demonstrated that the neem derived product is able to induce an anti-fertility effect (50% reduction). However, if one considers the amount of live chicks produced, i.e. still alive at the moment of weaning (30 days old), no difference is observed between the and. This is due to the health promoting effect of the neem derived product which, very strongly, reduces post-natal mortality in the (83% reduction). As in this experiment only one doses was applied, it is clearly necessary to carry out further experiments employing higher concentrations in order to verify if it is possible to reach a 100% reduction in fertility. Conclusions This preliminary study indicates that -in term of birth control- the employed neem derived product can effectively act as a tool for limiting the reproductive performance. However, it is necessary to determine the exact doses in relation to the body weight, which allows a complete anti-fertility effect on stray pigeons. Besides, it would be very interesting to further investigate the health promoting effect observed. Indeed, an improved health status in unfertile urban pigeons is very desirable. The reversible anti-fertility effect of neem derived products, together with the nonhormonal effect, should allow the control of nuisance animals (pigeons), without incurring in undesirable side effects, not only for the above-mentioned population, but also for non-target animals and humans. Finally, the complete biodegradability of neem derived products may avoid environmental pollution: a problem which may be encountered when using chemically derived products. ENEA 7

8 Bibliography Conkova E. et al.,. Fusarial Toxins and their Role in Animal Diseases. Veterinary Journal, 165, ; Elangovan A.V. et al. 2000: Laying performance of Japanese quail fed graded levels of neem (Azadirachta indica) kernel meal incorporated diets. Anim. Feed Sci. Technol.. 88 (1-2) ; Mohan J. et al., Antifertility effect of neem (Azadirachta indica) seed kernel meal in chichkens. Asian-Austral. J. of Anim. Sci, 10 (6), ; Mukherjee S. et al Early post implantation contraceptive effects of a purified fraction of neem (Azadirachta indica) seeds, given orally in rats: possible mechanism involved. J. of Ethnopharmac., 67, ; Mukherjee S. et al Purified extracts of neem (Azadirachta indica) seeds abrogate pregnancy in rodents and primates when given orally. J. of Reprod. Immun., 34, (1), 23-24; Mukherjee S. et al., Purified Neem (Azadirachta indica) seeds extracts (Praneem) abrogate pregnancy in Primates. Contraception,53, ; Nomisma Società di studi economici S.p.a. (Bologna) Valutazione dei costi economici e sociali dei colombi in ambiente urbano. Rome, 19th June; Placinta C.M. et al., 1999 A review of worldwide contamination of cereal grains and animal feed with Fusarium mycotoxin. Anim. Feed Sci and Techn, 78,21-37; Prakash A.O.. et al., Non-Hormonal post-coital contraceptive action of Neem Oil in Rats, J. of Ethnopharmac., 23, 53-59; Puschner B.,. Mycotoxins. Vet. Clinics of North America: Small Animal Practice. 32, ; Russo G., Dati preliminari sull attuale campagna di controlo numerico e sanitario del colombo urbano (Columba livia var. domestica) nel centro storico della città di Verona. Il Progresso Veterinario, Anno LVI, 3, ; Sampathraj R: et al., Effect of neemoil : structural and functional changes in the epididymis of rat. World Neem Conference, 24th to 28th Feb. 1993, Bangalore, India; SAS, SAS/STAT Users Guide (release 8.2). SAS Inst.Inc., Cary, NC.; Sbragia G. et al. 2001, Esplosione demografica del colombo di città: ruolo del veterinario nei piani di controllo delle popolazioni di uccelli sinantropi. Praxis veterinaria,xxii, 4, 15-21; Talwar G.P. et al., 1997a. Induced Termination of Pregnancy by Purified Extracts of Azadirachta indica (Neem): Mechanism Involved. Am. J. of Reprod. Immun., 37, ; Talwar G.P. et al., 1997b. Plant immunomodulators for termination of unwanted pregnancy and for contraception and reproductive health. Immunol. And Cell Biol. 75, ; The Neem Tree, Azadirachta indica A. Juss and other Meliaceous Plants. Sources of Unique Natural Products for integrated Pest Management, Medicine, Industry and Other purposes. 2ns Edition, Edited by H. Schmutterer,. Mumbay, Ueno Y., 1984; Toxicological features of T-2 toxin and related trichothecenes. Fund. And Appl. Toxic. 4, ; Upadhyay S. N. et al., Immunomodulatory effects of neem (Azadirachta indica) oil. Int. J. Immunopharmacol. 14, ; Upadhyay S. N. et al., 1993a. Antifertility effects of Neem (Azadirachta indica) oil in male rats by single intra-vas administration: an alternate approach to vasectomy. J. of Andrology, 14 (4), ; Upadhyay S. N. et al., 1993b. Long-term antifertility effect of neem seed extract following a single intra-uterineappliction and intra-vas administration in rats : World Neem Conference, 24th to 28th Feb. 1993, Bangalore, India; Vijjan V.K. and Parihar N.S., Toxic effects of neem (Azadirachta indica) seed cake feeding in rats. J. Environ. Biol. 4, ENEA 8