Journal of Pharmacy and Pharmacology 6 (2018) 88-100 doi: 10.17265/2328-2150/2018.01.010 D DAVID PUBLISHING

Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from Mortalities Used in Traditional Breeding in Northern Togo

Lombo Yao1, 2, Tona Kokou2 and Bonfoh Bèdibètè1 1. Centre de Recherche Agronomique de la Savane Sèche (CRASS), Institut Togolais de Recherche Agronomique (ITRA), Lomé BP 1163, Togo. 2. Laboratoire des Sciences et Techniques de Production Avicoles, Centre d’Excellence Régionale des Sciences Aviaires (CERSA), Ecole Supérieure d’Agronomie (ESA), Université de Lomé, Lomé BP 1515, Togo.

Abstract: A study on the evaluation of the effectiveness of three traditional methods of protecting guinea fowl from mortality was carried out at CRA-SS (Centre de Recherche Agronomique de la Savane Sèche) in northern Togo. Guinea fowl were divided into 5 batches, of which 3 batches received prophylactic medical treatment including decoctions, the 4th batch was followed by a medical prophylaxis program as a control group and the 5th batch considered as a negative control, has not received any prophylactic treatment. The results showed that a mortality rate of 25% for the reference lot and two other batches which received plant decoctions ( poissonii or Khaya senegalensis) were recorded against a rate of 87.5% in the batch of guinea fowl which received the decoction of Pteleopsis suberosa. Meanwhile, regardless of weight growth, daily average gain (GMQ) or food consumption, no significant difference was observed (p > 0.05) between the guinea fowl that received a baseline health monitoring and those having taken the decoction of E. poissonii. On the other hand, the values recorded in these two batches of guinea fowl differed statistically from those in the guinea fowl that received the decoction of K. senegalensis. Although the decoction of K. senegalensis has a protective effect in guinea fowl, the growth of guinea fowl has been slowed down. On the basis of these results, it appears that two endogenous practices tested (K. senegalensis or E. poissonii) have positive effects. However, it would be desirable to carry out a toxicity test in order to determine the recommended doses.

Key words: Guinea fowl, protection, traditional methods, mortality, Savannah regions, Togo.

1. Introduction about 88% of the farmers use the traditional practices in the control of animal diseases [13]. For a long time, In the Savannah region, the breeding of the guinea poultry farmers have known different diseases of fowl has considerable potential for expansion in view poultry, and they develop expertise in the prevention of of the large number of rural households that practice it these diseases, based on the traditional pharmacopoeia, and the strong demand of the guinea fowl market [1-7]. in particular phytotherapy. With the advances in avian However, little attention is paid to this breeding science, we should expect the disappearance or at least characterized by the lack of habitats and the the decline of traditional veterinary medicine. However, contributions of food rations and inappropriate it must be noted that herbal medicine is still a very veterinary care [4, 7-12]. Faced with the problems of common practice in the prevention and control of decreasing productivity of this livestock and especially pathologies in general and avian diseases in particular the mortality of young subjects in the livestock farms, [14-16]. A study carried out by ITRA showed that, in the face of clinical signs, savannah farmers use several Corresponding author: Yao Lombo, Master in for the treatment of poultry [17]. Hien et al. [18] reported developmental biology, research fields: option nutrition and reproduction. the virtues diarrhea and deworming of Khaya

Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from 89 Mortalities Used in Traditional Breeding in Northern Togo senegalensis in drinking water on birds. The methanolic 2.2 Used Medicinal Plants extract of stem bark and the decoction of Pteleopsis 2.2.1 Pteleopsis suberosa Engl and Diels suberosa Engl and Diels were found to have moderate P. suberosa (Fig. 1a), of the family Combretaceae is antibacterial activity against Staphylococcus aureus in a shrub of 6 to 7 voire 10 m high, with erect more or vitro [19]. These endogenous veterinarians and less right, hail and cylindrical up to 35 cm in diameter. zootechnicians should use alternatives in avian control The bark is very characteristic, coarsely and densely programs because of the high cost or inaccessibility of covered with corky warts (which make it easy to certain veterinary products on the market. These recognize even when it is defoliated), it is traditional methods used in the protection of poultry blackish-gray to reddish-brown [19, 20]. against mortalities in traditional breeding deserve to be 2.2.2 Euphorbia poissonii Pax checked and known in order to optimize their use. Euphorbia poissonii Pax (Fig. 1b) is a plant of the The objective of this study was to evaluate the family and the order Euphorbiales. E. effectiveness of methods of protecting guinea fowl in poissonii is a succulent shrub, spiny or not (more or traditional breeding. More specifically, it is necessary less deciduous spines), 1 to 2 or even 3 m tall, with to determine the effects on mortality, growth, biochemical candelabra in the port, numerous branches branched parameters and bacterial flora of guinea fowl. and upright, full of caustic white latex power, irritating 2. Materials and Methods and purgative. The bark draws towards the color green to light gray on the old subjects, smooth and glabrous 2.1 Experimental Site [20]. The study was carried out at the “CRASS (Centre de 2.2.3 Khaya senegalensis (Desr.) A. Juss Recherche Agronomique de la Savane Sèche)” in K. senegalensis commonly (Fig. 1c) known as Dapaong in the Savannah Region of northern Togo. Caïlécédrat or mahogany of Senegal in French and This Region is located between 0° and 1° longitude and African mahogany in English, belongs to the family of 10° and 11° north latitude. The climate is marked by Meliaceae. It is an endemic species of many African the alternation of a long dry season from November to countries. It is a large tree, 25 to 35 m high, with a May and a short rainy season from June to October. barrel up to 10 m high and 2 m in diameter. The bark is Temperatures range from 20 °C to 35 °C. The rainfall gray and smooth, becoming more or less ferruginous of the region is between 800 mm and 1,200 mm. It took and scaly, with a pink slice more or less carmine, place from 20 September to 20 December 2016. exuding a little reddish liquid [14, 20].

a b c

Fig. 1 Medicinal plants used: a = Dried bark of Pteleopsis suberosa; b = Slices of Euphorbia poissonii; c = Dried bark of Khaya Senegalensis.

90 Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from Mortalities Used in Traditional Breeding in Northern Togo

2.3 Preparation of the Decoctions of the Bark and batches and were reared in starter cages with a capacity Slices of the Different Species of 10 subjects/m². The batch consists of 20 guinea fowl. Bark samples (K. senegalensis and P. suberosa) and  Batch 1 was subjected to drug with a decoction trunk slices (E. poissonii) were harvested in the Tone solution of prepared K. senegalensis barks (Table 1); Prefecture in the savanna region.  Batch 2 received drug with a decoction solution of These bark and harvested slices were dried under the P. suberosa bark (Table 2); hall in the open air at room temperature for one week.  Batch 3 had a watering solution with a decoction They are soaked in cold water before boiling for 10 solution of the E. poissonii slices prepared (Table 3); minutes. For one kilogram of bark or slices was added  5 liters of water. The solution thus obtained is Batch 4 or control group benefited from a medical administered to the guinea fowl as a drinking water prophylaxis based on conventional veterinary products after cooling, decanting and filtration with a clean cloth (Table 4); of cloth. Each morning, the decoction is reheated for 5  Batch 5 or negative control group received no min and the preparation is renewed weekly. prophylactic treatment. All batches were vaccinated against infectious 2.4 Conduct of Breeding brongitis, Newcastle disease and avian pox were fed At hatching, 100 guinea fowl were divided into five the same feed (Table 5) throughout the start-up period.

Table 1 Sanitary monitoring plan for guinea fowl that received the K. senegalensis decoction. Method of administration and Age Sanitary measures applied Products used dosage 1st day - Simple water Ad libitum Prevention against any non-viral disease Decoction of Khaya Senegalensis Oral use in drinking water : 1 kg in 2nd to 7th day Vaccination against infectious bronchitis BRON L 5 L of water disease 8th day - Simple water Ad libitum Prevention against any non-viral disease Decoction of Khaya Senegalensis Voie orale dans l’eau de boisson: 1 9th to 16th day Vaccination against Newcastle disease CEVAC UNI L ND kg in 5 L of water 17th day - Simple water Ad libitum 18th day to 50th Decoction of Khaya Senegalensis Oral use in drinking water : 1 kg in Prevention against any non-viral disease day CEVAC FPL 5 L of water 51st day - Simple water Ad libitum 52nd day to 56th Oral use in drinking water : 1 kg in Prevention against any non-viral disease Decoction of Khaya Senegalensis day 5 L of water

Table 2 Sanitary monitoring plan for guinea fowl that received the P. suberosa decoction. Age Mesures sanitaires appliquée Produits utilisés Mode d’administration et posologie 1st day - Simple water Ad libitum Prevention against any non-viral disease Decoction of P. suberosa Oral use in drinking water: 1 kg in 5 2nd to 7th day Vaccination against infectious bronchitis BRON L L of water disease 8th day - Simple water Ad libitum Prevention against any non-viral disease Decoction of P. suberosa Oral use in drinking water: 1 kg in 5 9th to 16th day Vaccination against Newcastle disease CEVAC UNI L L of water 17th day - Simple water Ad libitum 18th day to 50th Decoction of P. suberosa Oral use in drinking water: 1 kg in 5 Prevention against any non-viral disease day CEVAC FPL L of water 51st day - Simple water Ad libitum 52nd day to 56th Oral use in drinking water: 1 kg in 5 Prevention against any non-viral disease Decoction of P. suberosa day L of water

Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from 91 Mortalities Used in Traditional Breeding in Northern Togo

Table 3 Sanitary monitoring plan for guinea fowl that received the decoction of E. poissonii. Age Mesures sanitaires appliquée Produits utilisés Mode d’administration et posologie 1st day - Simple water Ad libitum Prevention against any non-viral disease Decoction of E. poissonnii Oral use in drinking water: 1 kg in 5 2nd to 7th day Vaccination against infectious bronchitis BRON L L of water disease 8th day - Simple water Ad libitum Prevention against any non-viral disease Decoction of E. poissonnii Oral use in drinking water: 1 kg in 5 9th to 16th day Vaccination against Newcastle disease CEVAC UNI L L of water 17th day - Simple water Ad libitum 18th day to 50th Decoction of E. poissonnii Oral use in drinking water: 1 kg in 5 Prevention against any non-viral disease day CEVAC FPL L of water 51st day - Simple water Ad libitum 52nd day to 56th Oral use in drinking water: 1 kg in 5 Prevention against any non-viral disease Decoction of E. poissonnii day L of water

Table 4 Monitoring Plan for Guinea Fowl of control group Age Mesures sanitaires appliquée Produits utilisés Mode d’administration et posologie 1st day - Simple water Ad libitum Infectious Prevention and Vaccination Képroceryl Oral use in drinking water: 0.5 g/L 2nd to 7th day against Infectious Bronchitis BRON L for 5 days 8th day - Simple water Ad libitum Anti-stress Vitaflash Oral use in drinking water: 0.5 g/L 9th to 16th day Vaccination against Newcastle disease CEVAC UNI L for 7 days 17th day - Simple water Ad libitum Amprolium Prevention against coccidiosis Powervit Anti-stress 18th day to 50th Pipérazine Oral use in drinking water: 0.5 g/L Worms day Tylo-Dox-Extra for 3 to 5 days infections Képroceryl Vaccination against avian pox CEVAC FPL 51st day - Simple water Ad libitum 52nd day to 56th Oral use in drinking water: 0.5 g/L Anti-stress Vitaflash day for 5 days

Table 5 Centesimal composition and nutritional value of diet. Raw material Incorporation rate (%) Corn 20 Millet 32 Fish flour 12 bran 20 Roasted soya 15 Shell 1 TOTAL 100 ME (kcal/kg) 2,900.20 BP (%) 21.32 Lys (%) 1.18 SAA (%) 0.77 Ca 1.06 P available (%) 0.74 Sodium chloride 0.41 ME/BP Ratio 136.03 ME: Metabolizable Energy; BP: brute protein; Lys: Lysine; SAA: Sulfur Amino Acids; Ca: Calcium; P: Phosphorus.

92 Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from Mortalities Used in Traditional Breeding in Northern Togo

The food was distributed in the morning at noon and in between the quantity served and the quantity refused. the afternoon. The DIC (daily individual food consumption) The watering was ad libitum. expressed in grams is determined by the following Food consumption was measured as the difference formula:

Amount of food distributed per day Amount of food refused per day DIC = number of subjects − Guinea fowl were weighed at hatching and weekly. and the internal organs were isolated. The lengths of The number of dead individuals was recorded for each the small intestine and the caeca were isolated and lot. taken. Similarly, the gizzard emptied of its contents, The guinea fowl were kept in cages maintained at an liver, lungs, heart, spleen and pancreas were weighed. ambient temperature of 25 and 38 °C. The humidity 2.6 Dosage of Plasma MDA (Malondialdehyde) ranged between 30.05 and 43.24%. And 75 WATT bulbs were used as heat sources. MDA (Malondialdehyde) is an indicator commonly used to estimate lipid peroxidation. The MDA level 2.5 Measurements and Weighing was evaluated in the blood plasma of guinea fowl The weight measurements were carried out using according to the method described by Agbonon and two electronic scales: the TH-5000 electronic scale, Gbéassor (2009) [21]. Indeed, during the final phases 5,000 g capacity and 2 g sensitivity used to determine of the lipid peroxidation reaction, the peroxides the weight of the guinea fowl and the quantity of feed decompose into volatile compounds responsible for the to be distributed to the guinea fowl and the IDS 833-2K rancid odor. There is in particular formation of electronic balance, with a capacity of 2,000 g and a aldehydes including malonic aldehyde which reacts precision sensitivity of 10 mg, used to determine the with thiobarbituric acid to form a pink complex which weight of the organs of the sacrificed guinea fowl. absorbs at wavelength λ = 530 nm. This complex For the climatic parameters, a thermo-hygrometer of accounts for the amount of malondialdehyde available mark JL 6886 (Photo 2), is with a range of 50 °C and a in the reaction medium [21-24]. sensitivity of 0.1 °C. And the temperature was used. Its The blood collected in tubes containing heparin was characteristics for RH (relative humidity) are 98% for homogenized and then was centrifuged at 3,000 range and 2% for sensitivity. turn/min for 10 min with the 80-2 Electronic Mortalities were recorded weekly. Individual live Scientifuge CE. The blood plasma collected is stored in weights of guinea fowl were tested at hatching and the freezer at -20 °C for the evaluation of MDA. In 200 every 7 days up to 8 weeks of age. ADG (average daily μL of the blood plasma each tube is successively added gain) was calculated by differentiating between two 0.6 μL of the 1% phosphoric acid solution (HPO3) and successive weighings (W1-W2) over the time interval 1 mL of the 1% thiobarbituric acid solution. The separating these two weighings. mixture is heated at 100 °C for 50 minutes in a water W2 W1 bath. It is then cooled with cold water for 10 minutes ADG = 7 and then 2 mL of n-butanol are added. The tubes are −  W1 = average weight of the previous weighing; vigorously stirred and centrifuged as before. The  W2 = average weight of the last weighing; supernatant was decanted and the OD (optical density)  7 = number of days between two weighings. was read at 530 nm against a blank using the Guinea fowl were sacrificed at two months of age UV-visible spectrophotometer. The standard curve is

Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from 93 Mortalities Used in Traditional Breeding in Northern Togo determined by 1,1,3,3-tetramethoxypropane or Equation of the linear regression line FRAP: malondialdehyde (Sigma-Aldrich Chemical, y = 0.0005268x + 0.0 Schnelldof, Germany) at concentrations of 0, 15.75, R2 = 0.9982 (good linearity) 31.5, 62.5, 125, 250, 500 nM [21, 23]. Y = Optical density; x = concentration Equivalent

If the OD is low, this means that the MDA is low. H2SO4 per μL of plasma. There is a decrease in the phenomenon of lipid 2.8 Determination of Biochemical Parameters peroxidation. Equation of Linear Regression Linear MDA: The blood was taken from dry tubes. The blood Y = 1.089 + 0.03860x samples in the tubes without anticoagulant were x is expressed as the concentration of MDA per μL centrifuged at 3,000 rpm for 10 min. The sera collected of plasma; were stored at -20 °C. And assayed for enzymes such as R2 = 0.9982 (good linearity). transaminases (ALAT (alanine aminotransferase), ASAT (aspartate aminotransferase), PAL (alkaline 2.7 Total Blood Antioxidant Capacity in Guinea Fowl phosphatase), total proteins), total cholesterol and The antioxidant power of a solution such as serum is triglycerides as biochemical indicators of damage determined by the FRAP (ferric reducing ability of and/or liver dysfunction. Kidney dysfunction was plasma) test. It consists of reducing the ferric ion (Fe3+) assessed at the blood level by the dosage of creatinine present in the tripyridytriazine-ferric complex and urea. The level was assayed for pancreatic (Fe3+-TPTZ) to ferrous ion (Fe2+). At low pH and 37 °C, function. Standard diagnostic kits from Human GmBH. the intense blue-colored tripyridytriazine-ferric D-65205 (Wiesbaden, Germany) were used for the (Fe3+-TPTZ) complex, added to a sample containing spectrophotometric determination of the biochemical antioxidants, is reduced to its dark Fe2+ (Fe2+-TPTZ) parameters [15, 23-25]. form. 2.9 Microbiological Analysis of the Intestines of the At two months of age, the guinea fowls were Guinea Fowl sacrificed and the collected blood was centrifuged at 3,000 rpm for 10 min. The collected serum was used Salmonella was examined in the intestines of the for the FRAP test and the method used is that described guinea-fowl in accordance with the method of analysis by Agbonon and Gbéassor (2009) [21]. In test tubes NF V08-052, May 1995. Salmonella research involves containing 300 μL of freshly prepared FRAP solution several steps: (25 mL acetate buffer, 2.5 mL mM Fe3+-TPTZ in 40 (a) Pre-enrichment: it consists of introducing 25 g of mM HCl and 2.5 mL 20 mM FeCl3-6H2O) were added the product to be analyzed into 225 mL of buffered added 30 μL of the distilled water and 10 μL of serum. peptone water. The broth thus obtained or the A blank without a sample was prepared under the same remainder of the stock solution was incubated at 37 °C. conditions. The reading was made at 593 nm using the for 24 h. UV-visible spectrophotometer. A calibration range was (b) The enrichment: the enrichment consisted in established (determined) from different concentrations adding, on the one hand, 0.1 mL of the pre-enrichment

(0-2,000 μM) of FeSO4-7H2O) dissolved in methanol. solution to 10 mL of the Rappaport broth medium and

The result was expressed in μmoleq Fe2SO4/mg protein also 2 mL to 10 mL of the sodium selenite broth. The [21-23]. tubes containing the seeded Rappaport and sodium The higher the optical density is, the higher the selenite media were incubated at 44 °C and 37 °C for antioxidant potential will be. 24 h, respectively.

94 Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from Mortalities Used in Traditional Breeding in Northern Togo

(c) Isolation: Striations were performed on the variance) followed by the Tukey HSV test for multiple Hektoen and SS (Salmonella/Schigella) media from the comparisons of the mean of the various parameters Rappaport and Selenite sodium tubes to obtain isolated studied. The Chi-2 test was used to compare mortality colonies. After incubation at 37 °C for 24 h, the rates. Values are considered to be significantly characteristic colonies (salmon on SS and blue gray on different when p < 0.05. Hektoen with or without black center) were 3. Results and Discussion transplanted onto Kligler medium and incubated at 37 °C for 24 h. Strains with the lactose (-), glucose (+), 3.1 Mortality Rates of Guinea Fowl H2S (-) or H2S (+) profile were subjected to the urease During the experimental period, a high mortality rate and indole tests. of 90% and 87.5% was observed respectively in guinea Germs without urease (or tryptophan deaminase) fowl that received no prophylactic traitment (negative and which do not produce indole after 24 h of control group) and in those the decoction of P. incubation at 37 °C were identified by the Api 20E suberosa with a mortality rate of 25% in the biochemical gallery (routine method, NFV 08-052 : guinea-fowl that took the decoctions of K. senegalensis May 1997) [18, 26, 27]. or E or in the reference lot (Fig. 2). This rate differs significantly from the other rates (p = 0.0211). The 2.10 Statistical Analyzes mortality rate of 25% in the other three lots is much The results are expressed on average more or less lower than that generally observed in traditional standard error on the mean (M ± E.S.M). Statistical breeding where the losses of guinea fowl can be up to analyzes were carried out using the R-Commander 90% or even 100% if the farmer does not carry out any software, version 2.15.2 and GraphPad Prism® prophylaxis plan [7, 11, 28, 29]. These results show Version 5.00. The results of the various tests are that these local practices are promising and their analyzed by single-factor ANOVA (analysis of efficiency in traditional breeding is conceivable.

100 90 90 87,5

80

70

60

50

40 Mortality rate (%) rate Mortality 30 25 25 25

20

10

0 Decoction of Khaya Decoction of Decoction of Control group negative control senegalensis Pteleopsis suberosaEuphorbia poissonii group Health monitoring method

Fig. 2 Guinea fowl mortality rates using health monitoring methods.

Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from 95 Mortalities Used in Traditional Breeding in Northern Togo

3.2 Effects of Decoctions on Growth Parameters The weight gain performance of the guinea fowls in the batch that received the decoction of K. senegalensis 3.2.1 Effects on Live Weight of Guinea Fowl was lower than that of the guinea fowl that had taken The Fig. 3 shows the evolution of weight growth of guinea fowl according to the methods of health the decoction of E. poissonii and those of the reference monitoring. The guinea fowl of the reference and those lot. Although elevated in the reference lot, weight of the lot having taken the decoction of E. poisonii growth is not statistically different from that of the showed a high growth compared to the bunch heads batch of guinea fowl that received E. poissonii decoction. with the use of K. senegalensis decoction. The analysis 3.2.2 Effect on Growth Rate and Food Consumption of variance revealed that the type of health monitoring In terms of growth rate (Fig. 4) and food has an effect on the weight growth of guinea fowl. consumption (Fig. 4), the same observations were Significant differences were observed in the 3rd and made according to the types of health monitoring. 4th week and the 7th and 8th week (p < 0.05). The Guinea fowl of control batch and those having received results of this study, in particular those of the guinea the decoction of E. fishes had higher growth rates and fowl that received the decoction of E. poissonii and the food consumption compared to the kidney-sized guinea reference lot, are comparable to those obtained by fowls that had taken the decoction of K. senegalensis. Ayanwale et al. (1998) and Halbouche et al. In Nigeria, The average daily gains and feed consumption of the which found values of 411 g to 516 g in the same age reference and decoction batches of fish were similar group, receiving a conventional ration and sanitary and differed significantly from those of the K. monitoring, and those of Hien et al. (2011) [18], in senegalensis decoction (p < 0.05) [3, 31, 32]. Burkina Faso who found a weight of 327 ± 7 g at 8th This growth of the guinea fowl of the reference lot, week [10, 30]. comparable to that of the guinea fowls subjected to the

600 575 550 Decoction of Khaya senegalensis 525 * 500 Decoction of Ptelopsis suberosa 475 450 Decoction of Euphorbia poissonii 425 * 400 Control group 375 350 Negative control group 325 300 275 weight (g) 250 225 200 175 *** 150 * 125 100 75 50 25 0 0 1 2 3 4 5 6 7 8 9 Age (week)

Fig. 3 Guinea fowl weight growth curves using health monitoring methods. * Significance at the 5% threshold; ** Significance at 1% threshold; *** Significance at the threshold of 1‰ compared to the control group (one-fold ANOVA followed by Tukey multiple comparison test).

96 Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from Mortalities Used in Traditional Breeding in Northern Togo

20 Decoction of Khaya senegalensis 80 18 Decoction of Euphorbia poissonii 75 * *** Control group 70 16 *** 65 ** 14 60 55 12 50 A 45 B 10 *** 40 8 35 * 30 Decoction of Khaya senegalensis 6 25 * Decoction of Pteleopsis suberosa 20 *** 4 15 Decoction of Euphorbia poissonii food consumption (g) 10 Control group average daily gain (g) 2 5 Negative control group 0 0 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Age (week) Age (week)

Fig. 4 Growth rate (A) and food consumption; (B) curves of guinea fowl following health monitoring methods. * Significance at the 5% threshold; ** Significance at 1% threshold; *** Significance at the threshold of 1‰ compared to the control group (ANOVA with a factor followed by Tukey multiple comparison test). decoction of E. poissonii shows the interesting effects range from 75.42 to 86.28 g/day [3, 26, 32]. of its use in reducing the mortality of guinea fowl in 3.3 Intestine Morphometry and Weight of Different rural areas and on improving growth performance. The Organs results of the batches of E. poissonii and reference data obtained during the 8th in this study are comparable to The morphometric data given in Table 6 show that those obtained in Benin by Houndonougbo et al. (2013) the lengths of the intestines, the weights of the empty [17] who found GMQs of 12.23 ± 0.6 g/day between gizzards, the livers, the hearts, the lungs, the spleens the 5th and 8th week of age with an incorporated food and the pancreas of the guinea fowls, having received of the imported amino acids. the decoction of E. poissonii were similar to those of The feed consumption of guinea fowl that received the guinea fowl of the reference lot. On the other hand, the decoction of E. poissonii being slightly higher than those of the group of guinea fowl that took the that of the other batches, this implies that the type of decoction of K. senegalensis were inferior and differ health monitoring influences the consumption of the statistically from the two previous batches. food. A significant difference was observed at the 6th 3.4 Effect of Decoctions on Blood Lipoperoxidation week when food consumption in the batch of guinea and Antioxidant Activity (Iron Reduction Test—FRAP) fowl which took the decoction of K. senegalensis (62.95 ± 7.13 g) became significantly higher than that Determination of blood MDA concentration of the other batches respectively 55.02 ± 7.78 g for the revealed no significant difference (p = 0.4550) in the lot of E. poissonii and 46.40 ± 3.74g for the reference concentration of MDA in the blood of guinea fowl lot. This sudden increase in consumption of the K. caught with decoctions of E. poissonii (29.75 ± 1.37 senegalensis batch would be due to abiotic stresses nM/mL) and K. senegalensis. (28.89 ± 0.58 nM/mL) (favorable temperature), thus requiring guinea fowl to compared to the control batch size (29.36 ± 0.57 consume more food. At week 8, no significant nM/mL) (Table 7). MDA is a specific marker for difference was observed between the different batches oxidative stress. Its dosage is used to estimate the and daily dietary consumption for the K. senegalensis, peroxidation of lipids in membranes and biological E. poissonii and reference batches were 63.01 ± 5.09 systems. It reflects the severity of the attack of cells by g/day, 66 ± 2.5 g/day and 66.21 ± 6.71 g/day free radicals. In this work, the almost identical respectively. These results are slightly lower than those concentration of MDA in blood plasma in guinea found by Halbouche et al. (2010) in Nigeria which fowl suggests that decoctions inhibit lipid peroxidation

Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from 97 Mortalities Used in Traditional Breeding in Northern Togo

Table 6 Morphometry of the intestine and weight of the internal organs of the guinea fowl according to the type of health monitoring. Batch Organ Decoction of Euphorbia Decoction of Khaya Probability Control poissonii senegalensis Intestine length (cm) 99.75 ± 12.15ab 58.75 ± 12.33bd 109.75 ± 7.42ac 0.0205* Heart (g) 2.24 ± 0.22ab 1.83 ± 0.07 bd 2.74 ± 0.08ac 0.0060** Liver (g) 10.75 ± 1.51 ac 6.59 ± 0.50bd 9.81 ± 0.40ab 0.0307* Gesier empty (g) 20.62 ± 1.36a 12.31 ± 0.95b 22.71 ± 0.85ac 0.0002*** Pancreas (g) 1.23 ± 0.13ab 0.85 ± 0.08bd 1.49 ± 0.12ac 0.0109* lungs 2.63 ± 0.22ab 1.79 ± 0.05 bd 3.52 ± 0.35ac 0.0024** Sleen 0.44 ± 0.04ac 0.27 ± 0.02bd 0.41 ± 0.04ab 0.0305* Head of guinea fowl 25.77 ± 1.08a 22.41 ± 0.94ab 21.98 ± 0.831b 0.0404* The values followed by different letters are significantly different (p < 0.05).* Significance at the 5% threshold; ** Significance at 1% threshold; *** Significance at the threshold of 1‰ compared to the control group (ANOVA with a factor followed by Tukey multiple comparison test).

Table 7 Effect of decoctions on FRAP and DMA values in guinea fowl blood at 2 months of age. batch Parameters Decoction of Euphorbia Decoction of Khaya Probability Control poissonii senegalensis Concentration of Fe2+ 1,545.40 ± 175.67 2,177.99 ± 842.24 1,921.73 ± 472.03 (µmol/L) à 10 min 0.7372 Concentration of MDA 29.75 ± 0.68 28.89 ± 0.29 29.36 ± 0.28 0.4550 (nM/mL) FRAP: Ferric Reducing Ability of Plasma; MDA: malondialdéhyde. and preserve the integrity of the lungs of guinea fowl. decoctions of K. senegalensis and of E. poissonii had a These results confirm the effect of decoctions on significant change in blood levels of ALT, creatinine, vascular permeability. Similarly, administration of triglycerides and total proteins (p < 0.05). On the other plant decoctions did not reveal any significant hand, in the blood level of ASAT, PAL, urea and total difference (p = 0.7320) in the concentration of Fe2+. cholesterol, the two decoctions caused no significant The concentrations of Fe2+ are respectively 1,545.40 ± change (p > 0.05). ALAT, ASAT, and PAL are 175.67 μmol/L, 2,177.99 ± 842.24 μmol/L for the enzymes commonly used as markers of hepatic toxicity decoctions of E. whereas it is 1,921.73 ± 472.03 [14]. ALT is a cytosolic enzyme secreted in hepatic μmol/L for the reference lot. This test carried out on the cells, from which it is released into the blood in case of serum in this study considers all the antioxidant hepatic cell necrosis. ASAT is also an indicator of the defenses of the body of the guinea pigs, since the blood destruction of hepatocytes. The PAL level rises in case contains various classes of antioxidants. The almost of obstruction of the bile duct. These results show that identical concentration of Fe2+ in the serum of guinea the decoctions are not toxic to the liver and could have fowl caught the decoctions compared to that of the a hepato-protective effect. Total protein levels were reference lot means that the decoctions reinforced the higher in guinea fowl that had taken the decoction of K. antioxidant potential of the plasma of the guinea fowl senegalensis indicating that the decoction had in the same way as those of the reference lot [21-23]. interfered with the mechanism of this parameter in the liver. This result would explain the low growth 3.5 Biochemical Parameters observed in the guinea fowl that had taken the

Table 8 shows that the daily administration of decoction of K. senegalensis. These results show that

98 Evaluation of the Effectiveness of Three Methods of Protecting Guinea Fowl from Mortalities Used in Traditional Breeding in Northern Togo

Table 8 Effect of decoctions on biochemical parameters of Guinea fowl. batch Parameters Decoction of Euphorbia Decoction of Khaya Probability Control poissonii senegalensis ASAT (UI/L) 262.00 ± 42.92 230.50 ± 46.97 243.00 ± 37.58 0.5937 ALAT (UI/L) 5.75 ± 0.95a 6.00 ± 0.81ab 13.25 ± 3.86c 0.0021** ALP (UI/L) 444.75 ± 148.09 295.75 ± 132.30 274.25 ± 129.51 0.2137 UREA (g/L) 0.19 ± 0.01 0.17 ± 0.01 0.17 ± 0.01 0.1706 CRAETININE (mg/L) 7.50 ± 1.29a 6.50 ± 0.57ab 10.75 ± 1.71c 0.0028** TRIGLYCERIDES (g/L) 0.69 ± 0.31a 0.40 ± 0.19ab 1.27 ± 0.27c 0.0036** Total protein 31.10 ± 5.27a 46.85 ± 9.85c 33.60 ± 2.66ab 0.0179* Total cholesterol 2.12 ± 0.47 1.45 ± 0.26 1.86 ± 0.35 0.0903 The values followed by different letters are significantly different (p < 0.05). * Significance at the 5% threshold; ** Significance at 1% threshold; *** Significantce at the threshold of 1‰ compared to the control group (one-fold ANOVA followed by Tukey multiple comparison test); ASAT: Aspartate Aminotransferase; ALAT: Alanine aminotransferase; ALP: Alkaline phosphatase.

Table 9 Results of salmonella sp research in the intestines of guinea fowl subjected to plant decoctions. batch Results criteria Decoction of Euphorbia poissonii absent Absence in 25 g Decoction of Khaya senegalensis absent Absence in 25 g Control group absent Absence in 25 g the triglyceride and total cholesterol levels in the level, the bark is used as a counter poison and against guinea fowl caught the two decoctions are similar and madness [20]. Work has shown that the latex of E. remain lower than those of the reference lot. An Poison is potent irritant and purgative. It is used for hair increase in the level of these substrates in the blood is removal, caries, dental extraction and trypanosomiasis. considered a sign of hepatic injury [23]. The decoctions Latex is a poison for hunting, fishing, for criminal use of K. senegalensis and E. poissonii had no adverse (the antidote would be cow's milk) glue to capture birds. effect on the concentration of creatinine and urea which Its root of Euphorbia sp. killed molluscs, particularly are markers of renal function. This result suggests that bulb vectors of bilharziasis [20]. the kidneys, like the liver, did not suffer any damage, 4. Conclusions especially at the level of the filtration mechanism [22, 23]. The use of decoctions of K. senegalensis and E. poissnii to protect guinea fowl from mortality has been 3.6 Searching for Salmonella sp. in the Intestines of the shown to be effective compared to the reference lot. Guinea Fowl The practice based on the decoction of P. suberosa The samples of the intestines of guinea fowl showed an unsatisfactory effect compared to the others. analyzed did not contain Salmonella sp (Table 9). In view of these results, it is important to note that Indeed, this work showed that extracts of K. some local knowledge holds promise in the fight senegalensis bark have important antiplasmodial against the mortalities and diseases of the guinea fowl activities, anti-inflammatory activity and antibacterial and deserve an improvement before their activities [15, 24, 27] with immunostimulatory, recommendation in rural environment. anthelmintic or deworming activity (Ademola et al. The two endogenous practices tested (K. Anti-tumor, antioxidant, anti-receptor confirming their senegalensis or E. poissonii) have indeed had positive ethnomedical use [14, 16, 23]. On the medico-magical effects. However, it would be desirable to carry out a

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