Effets de la dose de gonadotrophines sur l ’ œstrus et la production d ’ embryons chez les brebis Hamra et Ouled Djellal

L’application des techniques de superovulation et de production d’embryons est incontournable pour la conservation des ressources génétiques ovines. L’objectif de la présente étude a été d’évaluer l’effet de l’administration de deux doses d’hormone folliculostimulante d’origine porcine (pFSH) sur le délai d’apparition et la durée de l’œstrus, la réponse ovarienne, et la production d’embryons chez les brebis des races Ouled Djellal (OD) et Hamra (H). Les brebis OD (n = 15) et H (n = 14) ont été synchronisées par la pose d’éponges vaginales imprégnées avec 40 mg d’acétate de fluorogestone (FGA), et superovulées par l’administration de 16 ou 20 UA de pFSH durant les trois derniers jours du traitement progestagène. Le septième jour après œstrus et saillie naturelle, les embryons ont été collectés par laparotomie. Chez les brebis OD le début de l’œstrus a été plus précoce avec l’utilisation de 20 UA qu’avec celle de 16 UA (22,1 ± 4,5 vs 25,0 ± 3,5, p < 0,05), de même la durée de l’œstrus a été plus longue (34,0 ± 6,6 vs 40,0 ± 7,1, p < 0,05). Chez les brebis H l’augmentation de la dose de pFSH de 16 UA à 20 UA a induit une baisse de production d’embryons : taux de collecte 72,2 vs 47,1, p < 0,001 ; nombre de structures récoltées 5,9 ± 3,4 vs 3,6 ± 2,1, p < 0,05 ; nombre d’embryons récoltés 3,2 ± 1,9 vs 5,1 ± 2,6, p < 0,05 ; embryons de grade 1 et 2, 4,3 ± 2,4 vs 2,1 ± 1,7, p < 0,01. En revanche, chez les brebis OD le taux d’ovulation et la production d’embryons ont été nettement améliorés lors de l’utilisation d’une dose de 20 UA : nombre de corps jaunes 6,9 ± 3,9 vs 10,4 ± 5,4, p < 0,05 ; nombre d’embryons récoltés 3,2 ± 2,0 vs 5,1 ± 2,3, p < 0,05 ; embryons de grade 1 et 2, 2,2 ± 1,6 vs 4,3 ± 1,7, p < 0,05. Les résultats indiquent que la réponse ovulatoire, l’œstrus, et la production d’embryons sont influencés par la dose de la pFSH et par la race.


Summary
Gharbi I., Dechicha A.S., Ferrouk M., Baazize-Ammi D., Kebbal S., Guetarni D. Effects of the gonadotropin dose on estrus and embryo production in Hamra and Ouled Djellal ewes The application of superovulation and embryo production techniques is essential for the conservation of sheep genetic resources.The objective of this study was to evaluate the effect of two doses of porcine follicle-stimulating hormone (pFSH) on the onset and duration of estrus, the ovarian response and the production of embryos in sheep of Ouled Djellal (OD) and Hamra (H) breeds.OD ewes (n = 15) and H ewes (n = 14) were synchronized by the insertion of vaginal sponges impregnated with 40 mg of fluorogestone acetate (FGA), and superovulated by the administration of 16 or 20 AU of pFSH during the last three days of progestagen treatment.On day seven after estrus and natural mating the embryos were collected by laparotomy.In OD ewes treated with 20 AU the onset of estrus occurred earlier than in those treated with 16 AU (22.1 ± 4.5 vs 25.0 ± 3.5, p < 0.05), and the duration of estrus was longer (40.0 ± 7.1 vs 34.0 ± 6.6 , p < 0.05).In H ewes, the increase in the pFSH dose from 16 AU to 20 AU induced a decrease in embryo production: collection rate 72.2 vs. 47.1, p < 0.001; number of structures collected 5.9 ± 3.4 vs 3.6 ± 2.1, p < 0.05; number of embryos harvested 5.1 ± 2.6 vs 3.2 ± 1.9, p < 0.05; 1-and-2-grade embryos 4.3 ± 2.4 vs 2.1 ± 1.7, p < 0.01.On the other hand, in OD ewes treated with 20 AU the ovulation rate and embryo production were significantly improved: number of corpora lutea 6.9 ± 3.9 vs 10.4 ± 5.4, p < 0.05; number of embryos harvested 3.2 ± 2.0 vs 5.1 ± 2.3, p < 0.05; 1-and 2-grade embryos 2.2 ± 1.6 vs 4.3 ± 1.7, p < 0.05.The results indicate that the ovulatory response, estrus, and embryo production are influenced by the dose of pFSH and the breed.

■ INTRODUCTION
Intensive poultry production in developing countries could be further enhanced through feeding strategies that promote feed utilization in relation to bird performances.Before 2003 in the United Kingdom, the inclusion of antibiotics as growth promoters in animal feed was widely adopted following the reported growth stimulation observed (Bedford, 2000).Thus according to Falçao-e-Cunha et al. ( 2007), antibiotics have the ability to decrease feed usage per production unit with concomitant increase in production performance.This is in addition to the fact that antibiotics can help reduce the disease burden, inhibit pathogenic microorganisms, and reduce microbial toxic metabolites, epithelium turn over, intestinal motility and bacterial deconjugation of bile salt (Falçao-e-Cunha et al., 2007).However, the ban on the use of antibiotics in animal feed has fueled the search for alternative animal growth promoters in the form of enzyme supplementation (Garcia et al., 2008) and phytogenic feed additives (Oloruntola et al., 2018).Enzyme supplementation has been reported to improve changes in the intestinal environment, including the viscosity of the digesta, which may promote contact among nutrients, endogenous enzymes and absorptive mucosae, thus enhancing the usage of the diet by the concerned animal (Lázaro et al., 2003;Mateos et al., 2010).

Effect of pawpaw (Carica papaya) leaf meal and dietary enzymes on broiler performance, digestibility, carcass and blood composition
Olugbenga David Oloruntola

Summary
Exogenous enzymes and phytogenic feed additives are proposed as alternatives to antibiotic growth promoters in poultry production.This study assessed the effect of pawpaw leaf meal (PLM) inclusion and enzyme (E) supplementation in the diet of broiler chickens.In total, 288 Arbor-Acre day-old broiler chickens were used.Four diets were formulated to be isocaloric and isonitrogenous: diet 1, control (0% PLM, 0% E), diet 2 (0% PLM, 0.05% E), diet 3 (5% PLM, 0% E), and diet 4 (5% PLM, 0.05% E).Each diet was replicated six times with 12 chickens in each batch.E improved (p < 0.05) the body weight gain at three weeks.The dry matter (DM), crude protein (CP), ether extract and ash digestibility were improved (p < 0.05) with E, whereas PLM inclusion produced (p < 0.05) an increase in DM and CP digestibility.The E x PLM effect was significant (p < 0.05) for DM and CP digestibility.E improved (p < 0.05) the slaughter weight and reduced the liver weight.Platelets varied across diets and increased (p < 0.05) with enzyme supplementation.E reduced (p < 0.05) low-density lipoproteins (LDL), whereas PLM reduced (p < 0.05) cholesterol and LDL.In conclusion, the association of E and PLM improved chicken growth, and E or PLM inclusion should benefit chicken health.
Phytogenic feed additives are gradually gaining prominence in animal feeding to improve their performance and boost their immune response (Oloruntola et al., 2016b).These products include pawpaw whose leaf has been previously identified by Oloruntola et al. (2018) as a natural source of papain, chymopapain A and B, and papaya peptidase A. Papain is proteolytic and thus capable of enhancing protein digestion (Oloruntola et al., 2018).In addition, the papaya leaf contains broad-spectrum phytochemicals including alkaloids and phenols.Phenolic compounds have high antioxidant activity and free radical scavenging capacity, with the mechanism of inhibiting enzymes responsible for reactive oxygen species production (Kahkonen et al., 2001).
The antibacterial, antifungal, antiviral, neuroprotective and antifertility activities of papaya have also been documented (Kadiri et al., 2016).It is conceivable that the nutritive and phytogenic potentials of the papaya leaf could be used as a growth promoter in broiler chicken feed.Hematological and serum biochemistry assay of livestock can show the physiological disposition of animals to nutrition, meaning that they can be a reflection of the physiological responsiveness of animals to feed and feeding (Oloruntola et al., 2016b).In addition, deviation in the relative internal organ weight of animals has been reported to be indicative of a possible response of their internal organs to toxins in their feed (Ayodele et al., 2016).Therefore, the assessment of the effect of enzymes and pawpaw leaf meal on the performance, carcass, physiological status of the animals, especially the hematology and serum biochemistry, is important.The objective of this study was thus to assess the effect of exogenous enzymes and pawpaw leaf meal inclusion on the performance and physiological status of broiler chickens.

Experimental site, animal management and design
The experiment was carried out at the Teaching and Research Farm Department of the Agricultural Technology, The Federal Polytechnic, Ado Ekiti, Nigeria.In total, 288 Arbor-Acre one-day-old unsexed broiler chicks were randomly distributed into 24 pens of 12 birds each.The pens were allotted to four dietary treatment groups arranged in six replications.The experimental design was of a completely randomized design in a 2 x 2 factorial arrangement, i.e. diets with 0 and 50 g/ kg pawpaw leaf meal (PLM), and diets with 0 and 0.5 g/kg enzymes added after feed manufacturing.PLM was included in diets 3 and 4 by replacing palm kernel cake and soybean meal (Table I).
The birds were placed on wood shaving litter in controlled pens.The temperature was controlled within the range of 32 ± 2 o C on days 1 to 7, then reduced by 2 o C on each successive week and thereafter maintained at 28 ± 2 o C until the end of the experiment.In the first week, the birds were exposed daily to 23 hours of lighting and 1 hour of darkness, and thereafter to 18 hours of lighting and 6 hours of darkness until the end of the experiment.The birds were fed their respective experimental diets and water ad libitum for six weeks.

Experimental diets
The enzyme used was a commercial complex (Bioenzyme PH) whose composition is presented in Table II.Fresh pawpaw leaves were harvested from the Teaching and Research Farm at about 6:00 GMT, chopped into smaller pieces, air dried for 14 days, hammer milled and kept in an airtight container before use.The pawpaw leaf meal was analyzed in triplicate for proximate composition using an AOAC method (1990) (Table III).PLM was further analyzed for alkaloids (Harbone, 1973), saponins (Shad et al., 2013), tannins ( Van-Burden and Robinson, 1981) and flavonoids (Shad et al., 2013).Phytates and oxalates were determined according to Wheeler and Ferrel (1971), and Day and Underwood (1986), respectively.Four isocaloric and isoproteinacious experimental diets were formulated.They were designated as diet 1 or control (0% PLM, 0% E), diet 2 (0% PLM, 0.05% E), diet 3 (5% PLM, 0% E) and diet 4 (5% PLM, 0.05% E) (Table I).Diets 1 and 3 were not enzyme supplemented but had pawpaw leaf meal inclusion at 0% and 5% level, respectively, whereas diets 2 and 4 were enzyme supplemented at the rate of 0.05% but had PLM inclusion at 0% and 5% level, respectively.The calculated composition of experimental diets at starter (1 to 21 days of age) and grower-finisher (22 to 35 days of age) phases are shown in Tables I and IV, respectively, and were formulated to meet the nutritional requirements of broiler chickens.

Performance and apparent digestibility
The birds were weighed individually at the beginning of the trial and weekly.Feed consumption and mortality were recorded daily during the whole experiment.The weekly final weight gain was determined by subtracting the initial weight of birds from the final weight.The feed conversion ratio (FCR) was calculated as the ratio of feed consumed to weight gain, whereas the mortality was calculated as the number of deaths per treatment per day.Five birds were randomly selected from each replicate at 35 days of age, housed individually in a metabolism cage for fecal collection and apparent digestibility determination of dry matter (DM), crude protein (CP), ether extract (EE) and ash based on procedures described by Vogtman et al. (1975).Feed was supplied ad libitum; after the first three days of adaptation, feed intake and feces were recorded during four days.Droppings were collected twice daily, thoroughly mixed and weighed.Aliquots from each day samples were frozen at -20 o C and bulked at the end of the fourth day.Thereafter, the bulked samples were weighed and dried in a forced air circulation oven at 60 o C for 12 hours.The dried droppings were later ground (0.5 mm screen) and analyzed for proximate composition.Apparent digestibility were expressed as (feed intake -fecal) × 100 / feed intake.

Bird slaughtering, blood collection, carcass and organ evaluation
At 42 days old, three birds from each replicate were randomly selected, tagged, starved overnight, weighed and slaughtered by cutting the jugular veins, allowing blood to flow freely into a bottle containing ethylene diaminetetra acetic acid (EDTA), and a plain bottle for hematology and serum chemistry analysis, respectively.The serum was separated from the blood samples and frozen at -20ºC before analysis.Thereafter, the slaughtered chickens were defeathered after scalding in hot water (55-60°C) for 30 seconds, dressed and eviscerated.The dressed weights were estimated.The internal organs (liver, heart, lung and gizzard) were excised, weighed and expressed as a percentage of the slaughtered weight.

Blood parameter analysis
The packed cell volume, red blood cells, mean cell hemoglobin, mean corpuscular hemoglobin concentration, mean cell volume, hemoglobin concentration and white blood cells were determined according to Lambs (1981).Total protein, albumin, globulin, cholesterol, high-density lipoprotein, low-density lipoprotein (LDL), bilirubin, and alanine transaminase were determined with Reflectron Plus 8C79 kit (Roche Diagnostic, GonbH Mahnheim, Germany).

Data analysis
All data on performance, digestibility, blood parameters and carcass evaluation were subjected to analysis of variance from general linear model procedures for complete randomized design with two-enzyme

Chemical composition of pawpaw leaf meal, performance of broiler chicken and digestibility of nutrients
Pawpaw leaf meal contained high crude protein (26.14%), crude fiber (16.05%)contents and phytochemicals such as alkaloids, saponins, tannins, flavonoids, phytates and oxalates at reasonable levels (Table III).Table V shows that at three weeks old the body weight (BW) gain of broiler chickens in diet 4 was significantly (p < 0.05) higher than that in the other diets.At six weeks old, the BW gain of birds fed enzyme supplemented diets (diets 2 and 4) was similar but significantly (p < 0.05) higher than that with diets 1 and 3. Enzyme supplementation significantly (p < 0.05) improved the BW gain at three and six weeks, whereas PLM inclusion increased it (p < 0.05) at six weeks of age.The interaction of enzyme x PLM was not significant (p > 0.05) at three and six weeks of age.
Enzyme supplementation (E) numerically improved the feed intake and feed conversion ratio, whereas PLM inclusion numerically improved the feed conversion ratio but decreased the feed intake at both starter and finisher phases.Kadiri et al. (2016).These various reports may arise from the natural variability of plant secondary metabolite and chemical compositions (Nahak, 2014;Valenzuela-Grijalva et al., 2017).
The chemical composition of PLM in this study showed that it could be used as a plant protein source and phytogenic feed additive in monogastric nutrition.The presence of alkaloids and flavonoids in PLM indicates its antimicrobial and antioxidant potentials (Shad et al., 2013).Enzyme supplementation has been explored to improve the performance of broilers (Choct, 2006).In our study, multienzyme supplementation improved the BW gain at three and six weeks.This result agrees with that of Hossain et al. ( 2016) who reported significant growth of broilers when diets were supplemented with microbial enzymes.The improvement in BW gain due to enzyme supplementation in this study may be ascribed in part to the fact that exogenous enzymes could have helped to break down non-starch polysaccharides, antinutritional factors typified by phytates and oxalates with attendant improvement in the biological value of the diet (Choct, 2006).In addition, E could have changed the intestinal environmente.g.viscosity of digesta -leading to better contact between nutrients, endogenous enzymes and absorptive mucosa (Bedford, 1995).
In this study, although the multienzyme supplementation was able to improve the BW gain, it could only numerically improve the feed intake and conversion ratio.This numeric increase in feed intake due to enzyme supplementation may originate from the activity of supplemented enzymes that enhances the availability of certain trace minerals (e.g.Cu, Zn, Mn) known to stimulate a greater feed intake in broiler chickens (Larbier and Leclercq, 1994).The increased feed intake may also be the consequence of the faster growth of birds caused by E, which results in higher nutritional requirements (Hossain et al., 2016); chicks increase their feed intake in order to meet their nutritional requirements (Shrivastava et al., 1981).However, the present result disagrees in part with Olukosi et al. (2007) who reported an increase in the body weight gain, feed intake and feed efficiency in broilers fed rye-and wheat-based diets with xylanase supplementation.Variations may result in part from nutrient density reductions in control diets, and varied concentrations and compositions of exogenous enzymes used in these studies.
The influence of phytochemicals on the growth performance of farm animals has been linked to their antibacterial activity, stimulation of digestive fluid secretion and decreased intestinal PH, which result in enhanced nutrient utilization efficiency, reduced gut disorder and improved health (Ayodele et al., 2016).In addition, the enzyme (papain) present in PLM has been involved in enhancement of protein digestion and utilization, and as digestive tract cleanser (Oloruntola et al., 2018).According to Kadiri et al. (2016), papain also plays a crucial role in biological processes because of its ability to break down organic molecules made of amino acids (crucial nutrients for growth).This may explain the reasons behind the numeric increase in BW gain of broiler chicks with the 50 g/kg PLM diet.Furthermore, the nonsignificant interactive effect of E and PLM suggests that these two factors were independent from one another in broiler chicken nutrition: the presence or activity of one factor did not affect that of the other.
Enzymes are added to animal diets to make up some compounds in feed that cannot be broken down by the digestive enzymes.Enzyme supplementation in this study may have improved dry matter digestibility, crude protein, ether extract and ash digestibility.Protease, one of the components of the multienzymes used in this study might have played a major role in increasing crude protein digestibility by augmenting endogenous production of peptidase and thereby improving the digestibility of dietary protein.In addition, proteases may hydrolyze protein-based antinutrients (Ghazi et al., 2002), thus improving amino acid utilization efficiency and reducing protein turnover in Table VIII shows the effects of PLM and E on hematological indices of broiler chickens.These effects and their interaction were not significant (p > 0.05) for all the hematological parameters except the platelets.The levels of platelets in chickens fed E and/or PLM diets (diets 2, 3 and 4) were higher than those fed the control diet.Platelets significantly (p < 0.05) increased with E. Red blood cells tended to increase (p = 0.09) with E, and the interaction of E and PLM was significant (p < 0.05) for platelets.
The serum metabolites were similar in the experimental diets except for cholesterol and LDL that were significantly (p < 0.05) higher in the control diet than in the other diets.E significantly (p < 0.05) reduced LDL, whereas PLM significantly (p < 0.05) reduced cholesterol and LDL.The E and PLM interaction was significant (p < 0.05) for LDL (Table IX).
■ DISCUSSION Phytogenic feed additives or phytobiotics are used to boost livestock feed properties with the aim of improving performances.There were slight variations in the chemical composition of PLM in this study compared to values obtained by other authors.For instance in our study, the CP value of PLM (26.14%) was lower than that of 27.50% reported by Kadiri et al. (2016).The crude fiber value (16.05%) recorded for PLM was also higher than that of 5.60% reported by are herbal supplementation or antinutrients in the plant-based diet (Abou-Elkhair et al., 2014) as in this study.Therefore, in this study the breakage of antinutrients by exogenous enzymes might have normalized the activities of the liver.However, similar kidney, lung and gizzard weights across diets suggest that E, PLM and their interaction did not affect these organ weights.
Hematological indices are used to determine the health status and nutritional state of animals (Aletor and Egberongbe, 1992).The hematological indices determined in the present study were within the normal range reported by Mirtuka and Rawnsley (1997) and were not significantly affected by E and PLM in most cases.This indicates the nutritional adequacy of these factors.However, there was an increase in most erythrocytic indices in E diet.This suggests possible support of this main factor (enzyme) for adequate or normal hematopoiesis.E might enhance the utilization of minerals and vitamins (such as iron, copper, vitamin B, folic acids, among others) needed for blood formation.Several authors (Um and Palk, 1999;Hossain et al., 2016) have reported on improving the bioavailability of minerals (some of which play a significant role in blood formation) by phytase supplementation.White blood cells are part of the immune system which fights infections.Therefore, the tendency to increase in blood parameters was also related to the increase in digestibility in the present study.The increase in WBC and lymphocytes by enzyme supplementation and PLM inclusion in this study may imply that these factors produced an immunomodulatory effect in the birds, as reported by Oloruntola et al. (2016b).
Platelets, which are produced by the bone marrow, help in blood clotting and wound healing.The significant numerical increase in platelets in broiler chicks fed E and PLM diets, respectively, further birds.Phytase improves ileal fat digestibility by reducing endogenous loss and improving dietary nutrient solubility, thereby causing reduction in the undigested fraction (Selle et al., 2010).Supplementary microbial phytase has also been reported to improve bioavailability of dietary phosphorus and other minerals bound to phytates (Um and Palk, 1999).These may be responsible for the increase in ether extract and ash digestibility recorded in this study.The improvement in dry matter and crude protein digestibility due to PLM inclusion in this study may be in part due to the activities of a proteolytic enzyme (papain), an effective natural digestive aid, which aids break down proteins.Worthy of mention is the interaction effect of E and PLM on dry matter and crude protein digestibility in the study; the two factors could synergize one another to achieve optimal digestibility.
The increase in the slaughter weight obtained with E may be due to a better utilization of essential nutrients needed for growth in the birds; the enzymes present in multienzyme supplementation have been reported to enhance nutrient utilization by breaking down materials that interfere with digestion, absorption and utilization of nutrients.In addition, the improvement in the carcass trait due to E which translated to a mere increase in the dressed weight in this study agrees with a report by Ogunsipe et al. (2015) on broiler chickens fed roxazyme G2G supplemented cassava plant meals.Thus, apart from the diet, the carcass yields in broilers are affected by a number of factors, including slaughtering condition, sex, genetics and live weight (Havenstein et al., 2003;Bricket et al., 2007).Comparison of the relative organ weights between treated and untreated groups can be useful to predict the toxic effect of test materials.Liver hypotrophy as a result of enzyme supplementation in this study can be beneficial to health since cases of liver hypertrophy have been linked to increased liver activity through increased digestive enzyme production, when there confirmed the health protection effect against excessive blood loss in the birds.In addition, the significant interactive effect of E and PLM shows that combining these factors might promote healing and prevent blood loss.Some phytochemicals such as saponin present in leaf meals have been reported to exert a hypocholesterolemic effect and reduce gut cholesterol uptake through intralumenal physicochemical interaction (Yilkal, 2015).This may be responsible for the decrease in cholesterol in PLM inclusive diets, suggesting that PLM have hypocholesterolemic properties.In addition, the decrease in LDL due to the two main factors (E and PLM) and cholesterol level due to PLM inclusion further support the wholesomeness of the dietary treatment in promoting health in broiler chickens.

■ INTRODUCTION
Agriculture and most particularly extensive livestock production are vital for the livelihood of small landholders on the Southern Mediterranean shore.Sheep breeding is a major breeding activity in Tunisia.Its development is threatened by numerous constraints such as diseases, in particular tick-borne diseases.Ticks can cause hypersensitivity, inflammation and anemia impairing animal productivity.They also transmit several pathogens (parasites, bacteria and viruses), that can result in a high cost from loss of infected animals (Gharbi and Darghouth, 2014).In Tunisia, several of these ticks transmit pathogens to sheep with various prevalence and veterinary importance: Theileria ovis (Rjeibi et al., 2014), Theileria lestoquardi (Rjeibi et al., 2016b), Babesia ovis (Rjeibi et al., 2014) and Anaplasma ovis (Ben Said et al., 2015).
Climate change is an important emerging risk factor affecting agriculture, human and livestock health (Keleman Saxena et al., 2016).This is particularly true in North Africa considered as a "hot spot for climate change" (Giorgi, 2006).Increases in temperature cause heat stress in livestock species, hence provoking reductions in growth rate, milk yield and reproductive performance (Das et al., 2016).Furthermore, these changes affect the spread and abundance of several vector arthropods such as ticks, mosquitoes and flies, leading to a modified transmission of several pathogens (Elbers et al., 2015).The proliferation of vector ticks caused by the temperature increase promotes the emergence of various diseases such as Crimean-Congo hemorrhagic fever (Leblebicioglu et al., 2015).More precisely, increases in temperature might on one hand provide a suitable environment for the spread of thermophilic tick species such as Hyalomma marginatum and Rhipicephalus annulatus (Domșa et al., 2016), and, on the other hand, result in the population decrease of species which prefer more temperate conditions, e.g.Ixodes ricinus.
Under such epidemiological and changing climate conditions, the study of tick infestation dynamics in sheep is crucial.The implementation of any tick control and, subsequently, tick-borne pathogen control programs cannot succeed if no specific data are available regarding the seasonal prevalence and local phenology of the tick species concerned.This study reports, for the first time to our knowledge, tick infestation dynamics in sheep in arid Tunisian steppes.

■ MATERIALS AND METHODS
The current survey was carried out in Sned locality, Gafsa District (34° 25' N, 8° 47' E; mean altitude 313 meters) (Figure 1).The locality is typical of the arid steppes of Central Tunisia with a maximum annual rainfall of 200 millimeters.In this region, the dry period extends from April to September and temperatures can reach a maximum of 49°C in summer.The mean minimum temperature is recorded in January and February (0°C).
Each month, a sample of 57-64 sheep, randomly chosen within a flock comprising 400 animals, were included in the survey.Selected animals were classified as young (less than one year old) or adult (over a year old).The sheep grazed during the day on natural pastures and were kept under a semi-intensive breeding system.Ewes were supplemented before mating, at the end of pregnancy and during early suckling.Males were kept in barns during the low grazing period.Animals were treated against ectoparasites with a diazinon formulation at 2.5‰ once a year (in May).
The sheep were monitored for one year, from October 2013 to September 2014.All surveyed sheep were thoroughly examined and ticks were collected in labeled flasks containing 70% ethanol and 10% glycerine.They were then identified according to Walker et al. (2003).Because of morphological similarities between Rhipicephalus sanguineus and R. turanicus, these two species were pooled in the R. sanguineus sensu lato (s.l.) group (Nava et al., 2015;Hekimoğlu et al., 2016).Results were expressed using two parasitological indicators (Margolis et al., 1982): Infestation prevalence (%) = 100 × (num. of infested sheep / num. of examined sheep) Tick infestation intensity = num. of ticks / num. of infested sheep To compare infestation prevalence between age groups, gender and months, chi-square tests were performed with Epi-Info software (Dean et al., 2011).Comparison of tick infestation means were carried out with Student t test.Results were considered significant at 5% threshold.

■ RESULTS
No tick infestation was observed between November and March, whereas the infestation prevalence was maximal in August (83%).
The overall maximum infestation intensity (3.7) was also observed in August (Table I).
Except in July, the infestation prevalence of female sheep was significantly higher than that of males (p = 0.05).There was no significant difference between the infestation prevalence according to age group, although a higher infestation prevalence was observed in young sheep in April, August and September, and in adults in May, June, July and October (p = 0.6).No significant differences were observed between infestation intensities from April to September according to the age group (p = 0.16).

■ DISCUSSION
Two species were identified in the study: R. sanguineus s.l. and H. excavatum.In a similar survey, Rjeibi et al. (2015) showed the presence of five tick species in five districts of Tunisia (Jendouba, Ariana, Kairouan, Kebili and Tataouine) in different bioclimatic zones (humid, semiarid, arid and Saharan) (Figure 1): R. turanicus (45%), H. excavatum (41.8%) and R. sanguineus (4.6%) were present in all studied areas; H. dromedarii (7.8%) was collected in Tataouine District, and R. camicasi (0.7%) in Kebili District.Another survey carried out by Rjeibi et al. (2016b) in Kebili District, close to the location of the present study, revealed that H. excavatum was the most frequent tick in sheep, followed by Rhipicephalus spp.The variability among tick populations parasitizing sheep in different Tunisian areas may be related to both abiotic and biotic factors, i.e. the management systems, nature of pastures, cohabitation with other tick-host species (such as dromedaries in the South), and to the introduction of infested animals from other Tunisian regions and neighboring countries (Libya and Algeria).Climate conditions vary a lot from North (humid and subhumid) to South Tunisia (arid and Saharan).Several works show that some tick species are not adapted to the arid climate, e.g.Ixodes ticks; climate change could therefore lead to their disappearance and the appearance of other tick species (Estrada-Peña et al., 2013;Domșa, et al., 2016).
H. excavatum may be a two-or three-host tick.It is mainly present in Central and Southern Tunisia (Bouattour et al., 1999;Rjeibi et al., 2015;Rjeibi et al., 2016a) and transmits Theileria lestoquardi that has been reported in Kebili District (Rjeibi et al., 2016b).R. sanguineus s.l. is a three-host summer tick (Bouattour et al., 1999).R. turanicus is the most common sheep tick (Walker et al., 2003;Estrada-Peña et al., 2004).However, as in the present study, R. sanguineus has been reported as the most frequent tick species in sheep in North Central Spain (Estrada-Peña et al., 2004).On the other hand, Bouhous et al. (2011)  In the present study, the maximum infestation prevalence was recorded in August: any control program should thus be applied during this period.The tick infestation started in April and no infestation was observed during the cold months (from November to March) since the ticks were not active.Infestation onset is associated with the increase in temperature and photoperiod that promote the development and activity of ticks (Dantas- Torres and Otranto, 2013).
Global warming projections for the study area show that infestations in the future may start much earlier in spring and/or late winter, which would extend the tick activity period.
There was no significant difference between the infestation prevalence in adult and young sheep, both groups being alternately most infested, which could be due to the monthly fluctuation of the examined sample.This aleatory difference was unexpected because lambs are at first monogastric and do not graze, and thus release lower quantities of attractive gases for ticks (Bouattour et al., 1996).They should thus be less infested than adults.Rjeibi et al. (2016b) also observed no difference in the infestation prevalence between different age groups, which could be because, in North Tunisia where pastures are very rich, young sheep graze with adults.In Gafsa District, where our study was carried out, the management system was different: young sheep grazed near the farms with adults for a short period and were kept mostly inside.
Although the number of examined males was often, by far, lower than that of females, the results revealed that adult female (except in July) were more at risk than males, which were only infested between June and September.The difference may be due to the fact that males were often kept in the farm and had fewer grazing periods than females, as they were used for mating or fattening.Moreover, females expressed immunodepression during pregnancy and lactation (Rehman et al., 2017).
The number of female ticks was higher than that of male for both R. sanguineus s.l. and H. excavatum, but it was not significant for the latter.
Usually, the number of male ticks is higher because they spend a longer period attached to the animals, whereas female ticks engorge in more or less one week.This unusual observation could not be elucidated.
The identification of attachment sites increases the efficiency of tick control, whether it is by tick removal or acaricide application (Gharbi et al., 2013;Gharbi and Darghouth, 2014).A common attachment site for Rhipicephalus ticks was near the ear tag, which is consistent with the fact that questing Rhipicephalus ticks attach when sheep are headdown grazing.On the other hand, H. excavatum was mainly collected from the sternum.In Iran, Loui Monfared et al. (2015) showed that 42% of sheep ticks (comprising 42.2% of R. bursa and 17.3% of R. sanguineus) were present on the head, 30% on the tail and in anal region, 18% on the udder, 8% on the groin, and 2% elsewhere.In Iraq, 85% of ticks (mainly R. bursa) were observed on the ears, 9.8% on the udder and 5.1% on the hind legs and around the eyes (Omer et al., 2007).The preferential attachment sites are thus humid, warm, not accessible to grooming and with richly vascularized thin skin.However, these sites vary according to the species present in the area: farmers have thus to adapt their tick control practices.

■ CONCLUSION
Further studies are needed to determine the direct effect of ticks on sheep health and the pathogens they transmit to them.Studying tick species, their preferential hosts and phenology enables veterinary services to make recommendations to farmers that help them choose the suitable control period, thus reducing expenses for acaricides.In light of our results, we recommend the use of acaricides on the heads of sheep between April and October to eradicate the majority of tick populations.Revue d'élevage et de médecine vétérinaire des pays tropicaux, 2018, 71 (3) : 131-135

■ INTRODUCTION
The poultry industry in Nigeria has been rapidly expanding in past years despite having faced several challenges such as the avian influenza outbreak in 2006 (Fagbamila et al., 2017).However, prospects have been hampered by several factors such as morbidity and mortality caused by enteric bacterial diseases, which are on the increase in the poultry industry in Nigeria (Ojo et al., 2012).In addition, across the different regions of the country, the poultry sector is characterized by a low level of specialization and general weak level of biosecurity (Fagbamila et al., 2017).
Common bacterial diseases of poultry birds with huge impact on production and health in Nigeria include Escherichia coli, Salmonella serotypes and other enteric pathogens (Lutful Kabir, 2010).Apart from consequences on poultry production, many other species belonging to Enterobacteriaceae or non-Enterobacteriaceae have been implicated as opportunistic, nosocomial and foodborne pathogens responsible for a wide range of infections in humans, especially in critically ill or immunocompromised patients (Dowd et al., 2008;Gwida et al., 2014).Fecal contamination of poultry and poultry products with pathogens such as Salmonella, Escherichia coli, Proteus, Klebsiella, Enterobacter, Pseudomonas and others have shown to constitute problems in food hygiene and processing, with increasing poultry meat spoilage and pathogen dissemination to consumers Vibrio mimicus (1.4%), Pseudomonas aeruginosa (1.4%), Burkholderia pseudomallei (1.4%), Salmonella arizonae (0.7%), Klebsiella pneumonia (0.7%), Acinetobacter iwoffii (0.7%), Vibrio vulnificus (0.7%), Shewanella putrefaciens (0.7%), Proteus mirabilis (0.7%) and Proteus vulgaris (0.7%).There was 66.7% similarity between the bacterium profile of litters and that of feces; some additional strains were identified in the litters.No variation (p = 0.64) was observed in the number of isolated bacterium types from feces and litter samples.However, the number of bacterium types isolated from fecal samples differed (p = 0.002) between the two studied areas.Results suggest that there is a potential risk of wide-range bacterial transmission within poultry populations, and to humans in close contact with them.
Acquiring information on Gram-negative bacterium strains and populations (both Enterobacteriaceae and non-Enterobacteriaceae) circulating or present on poultry farms in Ogun State would be useful to develop efficient on-farm and food-safety mitigation strategies, conduct effective monitoring and surveillance of pathogens and antibiotic resistance, and, ultimately, reduce human exposure to zoonotic and opportunistic pathogens from poultry and poultry products.For these major concerns, we investigated and described diverse enteric bacterium types (Gram-negative bacilli) present in poultry feces and litters from commercial laying hen farms in Egba and Remo local government areas of Ogun State, Nigeria.

Study area
Ogun State has the highest concentration of poultry farms in Nigeria, particularly laying hen production; it is also known as the Gateway State and shares a border with Benin to the west, and interstate boundaries with Oyo to the north, Lagos to the south and Ondo to the east (Afolabi et al., 2013;Adebowale et al., 2016).The study area is located in the southwestern zone of the country between 6.20°-7.80°Nand 3.00-5.00°E,and covers 16,409.26square kilometers.

Survey design and farm recruitment
A survey was conducted to determine and characterize the prevalence of enteric bacterium isolates from poultry farms in Ogun State.All registered commercial laying hen farms in the State were included in the population target.In total 113 such farms from Egba (n = 72) and Remo zones (n = 41) were selected and sampled based on address accessibility and farmers' willingness to participate (Figure 1).When the farms could not be accessed, we replaced them with other farms from the farm list.
The sample size was estimated according to Thrusfield (2007); the formula used was: where Z is the degree of confidence (1.96), P the expected prevalence, and d the precision at 95% confidence level (5% in this study).
The adjusted sample size was estimated at 132 farms with expected frequency of at least 50%, and 5% precision at 95% confidence level: n adj = Nxn / N + n where n adj is the adjusted sample size, N the sample size of the study population, n the sample size based on infinite population.

Sample collection
From each farm studied, five pooled fresh fecal droppings and poultry litters were randomly collected with sterile disposable gloves.Samples were labeled and transported in a cold box to the Department of Microbiology Laboratory, College of Veterinary Medicine, Federal University of Agriculture, Abeokuta, for microbiological examination.All samples were processed using conventional methods of cultivation.

Bacterium isolation and characterization
In total 2.5 g each of feces and litter were pre-enriched in 22.5 ml buffered peptone water (CM0509, Oxoid, UK) and incubated at 37 o C for 18-24 hours.Thereafter, aliquots (a loop full) of enriched broth were inoculated on prepared sterile MacConkey agar plates (CM0115, Oxoid) and incubated at 37 o C for 18-24 hours.Plates were observed for both lactose and non-lactose fermenting bacteria, with pink and straw-colored colonies, respectively.Discrete colonies were recultured on MacConkey agar plates.Purified isolates were identified and confirmed by using 24 different biochemical tests distributed in microplates (Microbact GNB 24E, Oxoid) and Microbact 2000 computer software package (Oxoid).Bacterial identification by Microbact is based on pH changes in various dehydrated substrates and substrate utilization (O'Hara, 2005).Briefly, according to the manufacturer's procedure, 1-3 colonies picked from each purified isolate were resuspended in physiological saline solution (0.85% NaCl); four drops of bacterium suspension used to inoculate Microbact micro well plates were then added.Two drops of mineral oil (MB1093A) were added to black wells, seals were replaced over wells which were incubated at 35°C ± 2ºC for 18-24 hours.Subsequently, reagents such as indole Voges-Proskauer (V-P1, V-P11) and tryptophan deaminase (TDA) were added to corresponding wells as instructed by the manufacturer.

Statistical analysis
Prevalence with the 95% confidence interval was determined using Graphpad Quickcalcs online software package.Non-parametric Mann Whitney U test was used to compare variations in the number of bacterium types from different sample categories.All results generated with p value less than 0.05 were considered statistically significant.All graphic presentations and analyses were performed by Graphpad Prism 6.07, and Microsoft Excel 2013.

■ RESULTS
A wide range of different bacterium species were isolated including seventeen species of Enterobacteriaceae, and twelve of non-Enterobacteriaceae.The main Enterobacteriaceae isolated from poultry feces were, with 95% confidence interval  2, bottom).No variation (p = 0.64) was observed in the number of bacterium types isolated from poultry feces and litter samples.However, there was a difference (p = 0.002) between the two studied areas in the number of bacterium types isolated from fecal samples (Figure 2, bottom).

■ DISCUSSION
Poultry farming has contributed to the economy of many countries and accounts for 25% of local meat production in Nigeria.However, productivity of poultry is faced with various disease challenges especially caused by enteric pathogens (Mshelbwala et al., 2017).
There are limited data that show the distribution of various enteric bacterium types belonging to Enterobacteriaceae and non-Enterobacteriaceae in commercial laying hen farms in Ogun State, except in free-range local chickens (Ojo et al., 2012).
The present study was carried out to determine the distribution of various Gram-negative bacterium types of Enterobacteriaceae and non-Enterobacteriaceae in commercial laying hen farms in Ogun State.Microbact was used to group isolated bacteria because of its availability, cost (relatively cheap), and capacity to identify miscellaneous Enterobacteriaceae and other Gram-negative bacilli present in diverse biological samples.Microbact is reported to be very accurate and useful in routine flow work in microbiology, and a convenient means of identifying members of the family Enterobacteriaceae (Mugg and Hill, 1981).
In the study, most common Gram-negative rod-shaped bacteria identified belonged to the family Enterobacteriaceae or to non-Enterobacteriaceae -a few of which are highly pathogenic and associated with gastroenteritis and pneumonia in humans and animals.Others were opportunistic or nosocomial pathogens, which are clinically important and commonly found in the urinary or respiratory tract of critically ill or immunocompromised people.A study conducted by Kilonzo-Nthenge et al. ( 2013), which evaluated drug-resistant Enterobacteriaceae in retail poultry meat and beef showed that meat samples were contaminated with similar bacterium types isolated in our study.Such potential pathogenic bacterium types included Escherichia coli, Enterobacter aerogenes, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, Kluyvera sp., Morganella morganii, Vibrio parahaemolyticus, Yersinia enterocolitica and Salmonella sp. to mention a few.Klebsiella oxytoca was identified as the most commonly isolated pathogen in chicken and turkey meat (Kilonzo-Nthenge et al., 2013).
Poultry meat becomes contaminated with gastrointestinal flora during processing and may serve as a vehicle for foodborne pathogens.Sabarinath et al. ( 2009) described similar strains as major contaminants in commercial chicken in the West Indies.Interestingly, unlike these two studies, the present study showed that Providencia stuartii was the most commonly isolated Enterobacteriaceae in poultry feces.closely with poultry especially poultry workers or handlers or veterinarians, and associated food safety consequences would be reduced.

■ CONCLUSION
The present study reported the presence of various potentially pathogenic bacteria belonging to Enterobacteriaceae and non-Enterobacteriaceae from laying hen litters and feces in Ogun State commercial farms.It suggests a potential risk of wide-range bacterial transmission within poultry populations to humans in close contact with poultry, and to consumers.Data from this study aimed to provide information for monitoring and system surveillance of farms for pathogens, and formulation of operable guidance and policies for reducing on farm pathogen presence, transmission and risk of exposure to the wider community.Further evaluation and susceptibility pattern of these isolates to commonly used antibiotics in the poultry industry in Ogun State may be needed.
Providencia stuartii is reported to be isolated from human feces, burns, wounds, animals especially poultry, reptiles and the environment (Liu, 2011).Unlike the more pathogenic members of Enterobacteriaceae (Salmonella, Escherichia coli or Shigella) which have been widely studied, P. stuartii is a normal intestinal microflora of animals and humans but of clinical importance because of its implication in nosocomial infection outbreaks in communities and hospitals (Liu, 2011).It can cause severe infections in humans (especially in immunocompromised individuals), which range from diarrhea, urinary tract infections, endocarditis to septicemia (AlJehani et al., 2012).Infections are usually fatal because of its marked resistance to multiple antibiotics (Liu, 2011;AlJehani et al., 2012).This pathogen has been associated with poultry diseases and, like other Enterobacteriaceae, constitutes a major problem in meat hygiene and processing due to contamination and spoilage (Davies and Board, 1998).
Other similar intestinal microfloras isolated from this study were Proteus, Morganella, Citrobacter, Hafnia, and Serratia.Furthermore, the presence of E. coli, Salmonella, and Klebsiella pneumoniae in poultry samples poses great economic and public health concern in terms of high mortality rate in birds associated with colibacillosis, salmonellosis, respiratory tract infections, and food safety (Miranda et al., 2008).For instance, E. coli is associated with diarrhea caused by varying enteropathogenic, enterotoxigenic, enteroinvasive, enterohemorrhagic and enteroaggresive pathotypes.The presence of these highly pathogenic bacteria in feces and litter could prompt propagation within bird populations, and contamination of the environment and of the poultry food chain during processing, handling, or packaging of finished products (Miranda et al., 2008).Isolation of multiple pathogens on different farms in the studied areas also points to the poor sanitary conditions and biosecurity measures on many poultry farms in Nigeria.Improved hygiene primarily at farm level may be crucial to controlling the risk of bacterium propagation along the food chain.
With regard to non-Enterobacteriaceae from feces and litter samples Aeromonas hydrophila was the most isolated.This is not a surprise as Aeromonas have been reported particularly in poultry and isolated from sick and healthy commercial chickens in several studies conducted in Nigeria (Mailafia and Nok, 2009;Adeleke and Omafuvbe, 2009).A. hydrophila is considered as an opportunistic pathogen in poultry and incriminated in cases of fowl cholera outbreaks, a devastating disease in chickens in Nigeria (Mailafia and Nok, 2009).Furthermore, A. hydrophila has emerged as an important human pathogen associated with foodborne disease outbreaks and gastroenteritis ranging from mild diarrhea to cholera-like disease (Awaad et al., 2011;Evangelopoulou et al., 2015).Other than gastroenteritis, Aeromonas is responsible for meningitis, cellulitis, otitis, endocarditis, osteomyelitis, peritonitis, bacteremia, and septicemia, among other diseases (Albert et al., 2000).Considering in this study the high farm-level prevalence of A. hydrophila, people in close contact with birds especially poultry workers could be at high risk of exposure to infection, and dissemination.A study in Brazil reported a high prevalence of A. hydrophila in asymptomatic poultry farmers (18.5%), four of the farms studied had 50-75% of the workers contaminated, and 75% of farmers positive to the pathogen experienced frequent gastroenteric disorders (Zanella et al., 2012).In view of the present results, it is suggested that further work to investigate the prevalence of this pathogen in those closely in contact with poultry and reported cases of gastroenteritis should be conducted to evaluate public health and socioeconomic impacts.
Les ânes sont utilisés en milieu rural pour le labour, l'exhaure de l'eau, le transport des personnes et des biens, notamment les intrants et les produits agricoles.Ils jouent également un rôle important en ville pour le transport des marchandises ou des matériaux de construction, et la collecte des ordures ménagères.

Figure 1 :
Figure 1 : Geographical localization of Sned locality, Gafsa District (studied area) and other regions where previous studies on ticks were carried out.

Table II
Composition of Bioenzyme PH Source: Biomix, Sabaneta, Colombia 123 Revue d'élevage et de médecine vétérinaire des pays tropicaux, 2018, 71 (3) : 121-129 Proximate and phytochemical composition of pawpaw leaf meal using AOAC 1990's method digestibility of DM and CP was improved (p < 0.05) in enzyme and/or PLM supplemented diets (diets 2, 3 and 4) compared to control.The ether extract and ash digestibility in birds fed the control diet were similar to those of diet 3 with 5% PLM, but lower than those of diets 2 and 4. E significantly (p < 0.05) improved DM, CP, EE and ash digestibility, whereas PLM inclusion significantly (p < 0.05) increased DM and CP digestibility.The interaction effect of E and PLM was significant (p < 0.05) for DM and CP digestibility (TableVI).

Table I
Monthly infestation indicators of sheep according to age and gender and monthly population dynamics of ticks according to their species in Tunisia R. s.: Rhipicephalus sanguineus sensu lato; H. e: Hyalomma excavatum

Characterization of bacterium types isolated from commercial laying hen farms in Ogun State Nigeria
SummaryThis study investigated the distribution of bacterium categories isolated from poultry feces and litters on commercial laying hen farms in Remo and Egba local government areas, Ogun State, Nigeria.In total 29 species of lactose and non-lactose fermenters were recovered.Bacteria isolated from feces included Aeromonas hydrophila (27.5%),Providencia stuartii (15.5%),Actinobacillus sp.
■ SANTÉ ANIMALE ET ÉPIDÉMIOLOGIE 143 Revue d'élevage et de médecine vétérinaire des pays tropicaux, 2018, 71 (3) : 143-147 'étude a été conduite en septembre et octobre 2013 au Burkina Faso.Elle a été réalisée dans cinq des treize régions administratives, choisies aléatoirement et réparties dans trois zones agroclimatiques distinctes (figure1).Les régions du Nord et du Sahel se trouvent dans la zone sahélienne où il pleut moins de 600 mm par an.Les régions du Centre et de l'Est se situent dans la zone soudano-sahélienne caractérisée par une pluviométrie annuelle d'environ 900 mm.La région des Hauts-Bassins, au sud du pays, est caractérisée par un climat soudanien avec une pluviométrie de plus de 1000 mm par an.Dans ces cinq régions, les ânes jouent un rôle important dans les activités champêtres et le transport. L Revue d'élevage et de médecine vétérinaire des pays tropicaux, 2018, 71 (3) : 143-147 Epidemiological risk of African horse sickness in donkeys in Burkina Faso African horse sickness (AHS) virus causes severe epidemics when transmitted to horses by Culicoides vectors.A cross-sectional study was carried out in five areas of Burkina Faso to determine the seroprevalence of AHS virus in donkeys and to identify potential risk factors.A total of 460 sera were collected in 15 villages.A blocking enzyme-linked immunosorbent assay (ELISA) was carried out to detect AHS virus antibodies.The overall apparent seroprevalence was 72.6% indicating that the donkeys were infected with AHS virus.A multivariate logistic regression analysis was performed to determine the factors affecting the seroprevalence level.A significant effect between geographical areas was observed, confirming the role of the agroecological zone in the occurrence of AHS.Similarly, age and animal weight had significant effects on seroprevalence, whereas sex, coat color and herd size had not.This study highlighted the active circulation of AHS virus in donkeys in Burkina Faso.Riesgo epidemiológico de peste equina africana en burros en Burkina Faso SummarySavadogo M., Sow A., Dahourou L.D., Cailleau A., Kalandi M., Sawadogo G.J.ResumenSavadogo M., Sow A., Dahourou L.D., Cailleau A., Kalandi M., Sawadogo G.J.