Effect of Limiting Access to Drinking Water on Carcass Characteristics, Meat Quality and Kidneys of Rabbits

Effect of Limiting Access to Drinking Water on Carcass Characteristics, Meat Quality and Kidneys of Rabbits

Ben Rayana Aniss 1 , Ben Hamouda Mohamed 2 , Kaddech Anis 3 , Amara Abdelkader 4 and Bergaoui Ridha 1 1. Department of Animal Science, National Institute for Agronimic Studies, Tunis Mahrajène 1082, Tunisia 2. Institution for Agricultural Research and Higher Education, Tunis 1002, Tunisia 3. Centre de l’Agroalimentaire, Charguia II, Tunis 2035, Tunisia 4. Ecole Nationale de Médicine Vétérinaire, Sidi Thabet 2032, Tunisia

Received: January 09, 2012 / Accepted: April 07, 2012 / Published: January 30, 2013.

Abstract: Feed restriction is a common practice to reduce post weaning digestive disorders in rabbits and to get carcasses with less fat. Hydric restriction is commonly used to induce feed restriction in growing rabbits. Two hydric restriction times (2 h and 4 h per day, continuously) were used in our trial carried in good sanitary conditions. Thirty six rabbits were divided at weaning (28 days) into three groups: R2 with restricted access to drinking water from 35 days to 80 days age of 2 h per day (from 8 a.m. to 10 a.m.), R4: access to drinking water 4 h per day (from 8 a.m. to 12 a.m.) and T (control group) with unlimited access to drinking water. Six animals from each group were slaughtered at 80 days of age. Carcass measurements and meat quality were studied. Histological study for kidneys was carried out. Relative weight of gastrointestinal tract increases with the degree of restriction: 16.5, 18.4 and 18.9 for T, R4 and R2, respectively (P < 0.05). Perirenal fat was lower in restricted groups but differences were not statically significant. Scapular fat decreases of 2/3 or 1/3 respectively with limiting access to drinking water of 2 h or 4 h per day. Compared to control group (T), meat of restricted groups contains more water: 70.3%, 70% and 69.3% respectively for R4, R2 and T (P < 0.01). The same tendency was observed for proteins: 19.2%, 19.3% and 17.7% for groups R2, R4 and T. Fat decreases in meat of restricted animals: 8.4%, 8.2% and 8.1% respectively for T, R4 and R2 (P < 0.05). Water restriction induced decrease of carcass and meat adiposity. There were no differences between pH post mortem and pH ultimate (after 24 h) between the three groups. Hydric restriction does not effect on kidney histology.

Key words: Water restriction, growing rabbits, carcass, meat, kidneys.

1. Introduction  effects of limiting access to drinking water during the fattening period (35 days to 80 days) on carcass

Several authors studied the effects of food or water

characteristics and kidneys.

restriction on animal performance and the meat The objectives of our trial are to compare the quality in rabbits [1-6]. Effects on carcass yield, interest of two durations of access to drinking water muscle/bone ratio depends on the type of feed on carcass and meat quality and kidneys of rabbits in rationing [7]. Feed restriction tends to decrease lipid

good sanitary conditions.

content [5]. The effects on yields of carcass, muscle/bone ratio depends on the type of rationing

2. Materials and Methods

applied [8]. Food restriction tends to decrease the lipid The present study was carried out between April 23 content [5]. and June 14, 2007 at INAT (National Institute for The present work was undertaken to study the Agronomic studies of Tunis) research center in

Corresponding author: Ben Rayana Aniss, Ph.D., research

Mornag, Tunisia.

field: animal sciences. E-mail: benrayana.aniss@iresa.agrinet.tn.

Effect of Limiting Access to Drinking Water on Carcass

Characteristics, Meat Quality and Kidneys of Rabbits

2.1 Animals and Experimental Design

separated and weighed.

2.2.4 Adiposity Appreciation

Thirty six rabbit kits, from the rabbitry of INAT Scores from 1 to 5 were attributed to carcass [10]. (Californian grandparent stock) were randomly The score relates the general adiposity of commercial divided at weaning at 28 days of age into three groups:

carcass.

T, R2 and R4 of 12 individuals, homogenous in body weight, sex ratio and origin (mothers). The animals

2.3 Meat Quality

from each sex and each litter were allocated to The authors have measured pH in left biceps different lots. Rabbits from the three groups were femoris 15 min to 20 min after slaughter (pH PM) and placed in cages of four rabbits and received ad libitum

24 h at 4 °C (ultimate pH, pH u). The decrease of pH standard commercial pellet food (10.45 MJ DE/kg,

15.5% crude protein and 16% crude fibre). was calculated (pH PM －pH u). Analysis of chemical Group T had unrestricted access to drinking water

composition of meat (Longissimus dorsi muscle) was during the all fattening period (35 days to 80 days of carried out. Composition in water, proteins and lipids age). Group R2 had access to drinking water from 35

was determined [11-13].

days to 80 days of age for 2 h (continuously from 8 a.m. to 10 a.m.). Group R4 had access to drinking

2.4 Histology of Kidneys

water from 35 days to 80 days of age for 4 h Six rabbits from each group were used in the (continuously from 8 a.m. to 12 a.m.). Only one histological study of kidneys. Immediately after access to drinking water through a pipette in a 5 L slaughtering, kidneys (left and right) without perirenal tank was available per cage of four rabbits. fat depots were moved and placed in 10% (v/v)

2.2 Measurements formaldehyde solution, then transferred to the pathologic laboratory of veterinary school of Sidi

2.2.1 Slaughtering and Meat Quality & Carcass Thabet for histological examination. Six rabbits from each group were slaughtered at 80 Samples were fixed definitely in 10% (v/v) days of age. They were individually weighed before formaldehyde solution for 24 h. Then, they were slaughter. Carcasses were prepared as recommended included in and conditioned in paraffin blocs and by Blasco et al. [8]. finally debited with a microtome in thickness lamellas

2.2.2 Carcass Characteristics of 3 at 5 µ of width then placed on lames, before being

Body composition, proportions of all organs and stored at a temperature of 42 °C for at least 12 h.

carcass yields were determined as recommended by After dehydration, the coupes were stained with

many teams [8, 9]. After 24 h at 4 °C, commercial haematoxylin eosin which is a tinctorial stain. It stains

carcass was weighed. Scapular and perirenal fats were moved from the carcass and weighed. Then, each

the nucleus in blue and the cytoplasm and intercellular carcass was divided into three parts: hind part,

structures in red. It perms to study the general intermediate part and fore part. After one hour at

topography of tissues, the cytology and lesions. ambient temperature, one thigh (hind part) was

For each kidney (left and right), an appreciation of weighed and the muscle was removed from the bone

lesions was carried out at four levels: cortex, junction and muscle/bone ratio was determined.

between cortex and medulla, medulla and bassinet. A

2.2.3 Gastrointestinal Tract Characteristics score was attributed for each kidney. The defined

After slaughtering, gastrointestinal tract was scores are: 0: absence of lesions; +: very discreet weighed for each animal. Digestive organs were

cellular degeneration; ++: discreet cellular

28 Effect of Limiting Access to Drinking Water on Carcass Characteristics, Meat Quality and Kidneys of Rabbits

degeneration. have access to water for 4 h (0.32% of reference carcass) and decreases of 2/3 if access to water is for 2

2.5 Statistical Analysis

h per day (0.16% of RC).

Variance analyses were performed with SAS GLM

3.2 Adiposity Appreciation

procedure (version 9.1.3). Means were compared with the student t test.

General adiposity is better with restricted animals compared to group T (Table 2). The differences were

3. Results and Discussion

significant (P < 0.01). Limiting access to water

3.1 Carcass Characteristics engender feed restriction which induced a decrease in Water restriction had no effect on carcass yields

general adiposity.

(Table 1).

3.3 Meat Quality

Results in Table 2 show that there are no differences for weights of different parts of carcass.

There were no differences between pH PM and pH Gastrointestinal tract is more important for restricted

u (Table 3). The decrease of pH was the same for all groups. Feed restriction favourites systematically the

groups showing that muscles contain the same lipid increase of digestive content [14]. No significant

storage.

effects of restriction under the weight of Percentage of water was higher for groups R4 and gastrointestinal organs were illustrated.

R2 compared to group T (Table 4). The differences Perirenal fat is lower in restricted groups (R2 and

were highly significant (P < 0.01). Meat of restricted R4) than group T but the difference is not significant

groups (R2 and R4) contains more proteins and less (Table 2). Scapular fat decreases of 1/3 when animals

fat than group T. Restriction tends to decrease lipid

Table 1 Carcass yields of rabbits (80 days) having different times of access to drinking water.

Group MSE * T R4 R2 Probability

Rabbits, No.

6 6 6 Weight before slaughter (g) 2,195 2,180 2,135 0.89 245 Dressing percentage (DP)

59.53 58.35 57.84 0.35 2.30 Commercial carcass (CC)

57.52 56.89 56.09 0.63 6.14 Reference carcass (RC)

48.20 47.63 50.90 0.83 6.00 Means with different letters on the same row differ significantly at the 5% level (Duncan test). MSE * : means of standard error.

Table 2 Effect of water restriction (2 or 4 h/d) on gastrointestinal tract, organs proportions and fat.

Gastrointestinal tract (% of live weight) 16.5 b 18.4 a 18.9 a 0.03 1.97 In % of reference carcass (RC) Hind part

37.1 36.8 33.7 0.16 3.2 Intermediate part

25.9 26.5 25.4 0.70 2.35 Fore part

37.6 37.5 36.1 0.70 3.51 Perirenal fat

0.34 Scapular fat

0.48 a 0.32 ab 0.16 b 0.03 0.19 Muscle/bone ratio

5.7 5.0 5.74 0.13 0.67 Score of adiposity

3.8 A 2.8 B 2.5 B < 0.01 0.58 Means with different letters on the same row differ significantly at the 5% level (Duncan test). MSE * : means of standard error.

Effect of Limiting Access to Drinking Water on Carcass

Characteristics, Meat Quality and Kidneys of Rabbits

Table 3 Post-mortem changes of pH of the meat (rabbits aged 80 days).

Group MSE *

Decrease of pH

MSE * : means of standard error.

Table 4 Chemical composition (water, proteins and lipids) of meat (%).

Group MSE *

Water 69.3 B 70.3 A 70.0 A < 0.01 0.38 Proteins 17.7 B 19.3 A 19.2 A < 0.01 0.26

Lipids 8.4 a 8.2 ab 8.1 b < 0.05 0.19 Means with different letters on the same row differ significantly at the 5% level (Duncan test).

MSE * : means of standard error.

Table 5 Scores (0, + or ++) attributed to kidneys in function of lesions (frequency).

R L ** RLRL

Jonction between cortex and medulla

(images of cellular souffrance at the level of the loop of Henle)

Total score

R: right; L: left; * and ** : Absence of lesions in T group; R2 and R4: low at very low cellular degeneration only in tubular epithelium of the loop of Henle (cellular souffrance).

T: normal tubular epithelium R4: very discreet tubular degeneration R2: discreet tubular degeneration

Fig. 1 Photos of tubular epithelium (kidneys of rabbits aged 80 days).

content [2]. The values for group T (water, proteins and

3.3 Histology of Kidneys

fat) were almost similar to values mentioned by Lebas

Very low lesions (cellular degeneration) in tubular

[15]. epithelium at the level of the loop of Henle were

30 Effect of Limiting Access to Drinking Water on Carcass Characteristics, Meat Quality and Kidneys of Rabbits

observed (Table 5) on experimental groups (R2 and sans dégradation importante des performances de R4). They seem more pronounced in animals of group croissance: résultats d’une étude multi site. L’impact sanitaire d’une reproduction expérimentale de

R2 (Fig. 1). Hydric restriction does not effect on l’entéropathie épizootique (EEL) chez le lapin en kidney histology.

croissance, 10èmes Journées de la Recherche Cunicole, ITAVI Edition, Paris , Nov. 19-20, 2003. (in

French) [5] F. Gondret, F. Lebas, M. Bonneau, Restricted feed intake Limiting access to drink water for 2 h or 4 h per day

4. Conclusion

during fattening reduces intramuscular lipid deposition decrease adiposity of carcasses and meat, kidneys

without modifying muscle fiber characteristics in rabbits, were not affected by water restriction.

J. Nutr. 130 (2000) 228-233. [6] S. Verdelhan, A. Bourdillon, A. Morel-saives, Effect of a

Acknowledgments

limited access to water on water consumption, feed intake and growth of fattening rabbits, in: the 8th World Rabbit

This research was supported by the the laboratory

Congress, 2004.

of Agroeconomy of INAT. Thanks are extended to the [7] G. Perrier, J. Ouhayoun, Effet de différentes modalités de rationnement à l’engraissement sur les qualités bouchères

technical staff of INAT (Fadhila, Belgacem HEDFI) du lapin, Cuniculture 23 (1996) 147-154. (in French)

and to Mr. Mohamed Chokri REJEB, General [8] A. Blasco, J. Ouhayoun, G. Masoero, Harmonisation of Manager of the “Centre Technique de

criteria and terminology in rabbit meat research, World l’Agroalimentaire”.

Rabbit Sci. 1 (1993) 3-10. [9] A. Blasco, J. Ouhayoun, G. Masoero, Status of rabbit

References meat and carcass: Criteria and terminology, Options Méditerranéennes-Série Séminaires 17 (1992) 105-120.

[1] P. Boisot, D. Licois, T. Gidenne, Une restriction

(in French)

alimentaire réduit l’impact sanitaire d’une reproduction [10] AFNOR NF V47-001 (2004), Echelle d’adiposité des expérimentale de l’entéropathie épizootique (EEL) chez

carcasses de lapin. (in French)

le lapin en croissance, 10èmes Journées de la Recherche [11] AFNOR NF V 04-401 (1968), Viandes et produits à base Cunicole, ITAVI Edition, Paris, Nov. 19-20, 2003. (in

de viande, Détermination de l’humidité. (in French) French)

[12] AFNOR NF V 04-407 (1972), Viandes et produits à base [2] P. Boisot, J. Duperray, X. Dugenetais, A. Guyonvarch,

de viande, Dosage de l’azote. (in French) Interest of hydric restriction times of 2 and 3 hours per

[13] AFNOR ISO 1443-1973, Association Française de day to induce feed restriction in growing rabbits, in: the

normalisation. Informations provenant de la base «le 8th World Rabbit Congress, 2004.

référentiel», version septembre 1998, Viandes et produits [3] P. Boisot, J. Duperray, A. Guyonvarch, Intérêt d’une

à base de viande—Détermination de la teneur en matière restriction hydrique en comparaison au rationnement

grasse totale. (in French)

alimentaire en bonnes conditions sanitaires et lors d’une [14] F. Lebas, Cuniculture, Biologie du lapin 4.4 reproduction expérimentale de l’Entéropathie

Comportement Alimentaire [online], 2002, Epizootique du lapin (EEL), 11èmes Journées de la

http://www.cuniculture.info/Docs/Biologie/biologie-04-4. Recherche Cunicole, Paris, Nov. 29-30, 2005. (in French)

htm. (in French)

[4] T. Gidenne, A. Feugier, N. Jehl, P. Arveux, P. Boisot, C. [15] J. Ouhayoun, D. Delmas, La viande de lapin: composition Briens, et al., Un rationnement alimentaire quantitatif

de la fraction comestible de la carcasse et des morceaux post-servage permet de réduire la fréquence des diarrhées,

de découpe, Cuni-Sciences 5 (1989) 1-6. (in French)

Jan. 2013, Vol. 7, No. 1, pp. 31-41 Journal of Life Sciences, ISSN 1934-7391, USA

Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

Ana Filipa Ferreira , António Manuel de Frias Martins 1, 2 , Regina Tristão da Cunha 1, 2 , Paulo Jorge Melo 1, 2 1 and Armindo dos Santos Rodrigues 1, 3, 4

1. Department of Biology, University of the Azores, Ponta Delgada 9501-801, Azores, Portugal 2. Center for Research on Biodiversity and Genetic Resources (CIBIO), University of the Azores, Ponta Delgada 9501-801, Azores,

Portugal 3. Center of Research in Natural Resources (CIRN), University of the Azores, Ponta Delgada 9501-801, Azores, Portugal 4. Center of Volcanology and Geological Risks Assessment (CVARG), Ponta Delgada 9501-801, Azores, Portugal

Received: June 20, 2012 / Accepted: August 13, 2012 / Published: January 30, 2013.

Abstract: This study reports how the reproductive cycle of a land snail endemic from an oceanic island is shaped by abiotic factors over an altitudinal gradient of 800 m. In addition, the validity of two morphometric shell parameters (maximum diameter and total height) was assessed, as maturation diagnostic characteristics. The authors’ findings suggest that, at Piedade (200 m), individuals are active and mature from October/November to March, and they exhibit dormancy in the remaining months, as a consequence of soil dryness. Thus, at low altitude, gonadal maturation was considered to be primarily influenced by photoperiod, rainfall and temperature. At Cabeço da Bola (1000 m), in turn, individuals are mature from March to July/August and they seem to have minimal gametogenic conditions throughout the remaining months of the year to reproduce. As soil moisture is never a limiting factor, gonadal maturation is mainly shaped by photoperiod and temperature at high altitude. The morphometric shell parameters under analysis were unable to diagnose gonadal maturation, as recently described for related Azorean species.

Key words: Adaptation, land mollusk, climate, soil moisture.

1. Introduction  will affect physiological, biochemical and behavior mechanisms shown in pulmonates [2-5]. Several

Land snails (Mollusca, Pulmonata) are among the studies have attempted to establish latitudinal most important components of oceanic islands’ patterns in relation to environment factors (e.g. biodiversity due to their high rates of endemism. temperature, humidity and photoperiod) for a number However, many are under severe threats as a of species [6-8]; however, in regions with high relief,

consequence of changes in their habitats, of natural or altitudinal patterns are often examined as a good

anthropogenic origin [1]. The life cycles of pulmonates are shaped by

alternative, since climatic conditions within dozens several abiotic factors, such as temperature, rainfall,

of kilometers are similar to those seen in a latitudinal humidity, photoperiod and type of geological gradient [9]. According to Raut and Ghose [7], substrate. These factors often regulate the rate of

climate conditions and microhabitat become more species development by affecting reproduction and

distinct as altitudinal gradient increases thus leading survival; therefore, any change on the microhabitat

to different environments along this gradient; these differences may trigger different morphological and

 Corresponding author: Ana Filipa Ferreira, researcher,

behavioral adaptations.

research field: malacology. E-mail: anaferreira@uac.pt.

Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

Land snails, as many other species, developed a range of behavioral and physiological mechanisms to ensure their survival under adverse climatic conditions. In seasonally arid or semi-arid regions, snails are subject to an annual cycle of activity and aestivation. This state of aerobic torpor may persist for several months or even years and it is characterized mainly by a drop in the metabolic rate and decline in physiologic activities, which allow snails to survive during a long dry period, not compatible with active life [10]. The biochemical mechanisms that lead to metabolic suppression during aestivation need to be effective and easily reversible, so that snails can arise rapidly from dormancy when favorable conditions allow them to do that. In order to survive during a long-term aestivation, land snails, in particularly pulmonates, need to retard water loss and have sufficient metabolic fuel [11]. Several studies demonstrate that seasonal variations in land snail physiology and biochemical composition are related with reduced relative humidity, soil water availability, high temperatures, photoperiod, lack of food availability and reproduction [10-17].

Several studies on Azorean endemic land snails, e.g. Oxychilus (Drouetia) atlanticus (Morelet and Drouët 1857) [18], Leptaxis caldeirarum (Morelet and Drouët 1857) [19], Oxychilus (D.) brincki (Riedel 1964) [20] have been reporting the existence of extended reproductive cycles, although exhibiting minimum gametogenic conditions for reproduction throughout the year, as opposed to the existence of a well demarcated reproductive cycle observed by several authors in other terrestrial pulmonate snails from North and South America [21-23].

The aim of this work is (1) to describe the reproductive cycle of Oxychilus (Drouetia) minor (Riedel 1964) in two populations, sampled on sites with an altitudinal distance of 800 m, in Pico mountain, at Pico Island, relating its reproductive cycle with environmental parameters of the altitudinal

gradient, and (2) to assess the validity of two shell morphometric parameters, namely, maximum diameter and total height, as diagnostic maturation characters.

2. Materials and Methods

2.1 The Taxon The endemic, non-umbilicated Helix atlantica was

described by Morelet and Drouët [24] with the locality “Azores” mentioned only on the title of the publication; later Morelet [25] stated that was present in most Azorean islands; years later Gude [26] erected the monotypic genus Drouetia to accommodate that non-umbilicated taxon. The species from Pico Island was reported by Nobre [27] as Hyalinia atlantica. Later, Riedel [28] considered Drouetia a subgenus of Oxychilus (Fitzinger 1833) and described the specimens from Faial as a separate subspecies, which Riedel later raised to the specific rank as O. (D.) minor [29]. The taxon from Pico was considered conspecific to that of Faial by Martins et al. [30], who gave a detailed account of its morphological and anatomical variability. The wide range of variability of the anatomy of the reproductive system was quite apparent throughout the various demes studied, and three types were identified; nevertheless, taking into consideration that these types remained allotopic, the observed variation was interpreted as intraspecific; moreover, the population from Piedade was reported to exhibit some sort of allochrony relative to the maturation of the genitalia. After a more comprehensive anatomical study of the Drouetia from Pico and Faial, Martins [31] concluded that in both islands, there are two species involved: O. (D.) minor and an undescribed species. Both remain allotopic on Pico, but they are syntopic throughout Faial.

The specimens from Piedade and Cabeço da Bola, herein studied, were found to belong to O. (D.) minor.

2.2 The Study Area The climate in the archipelago of the Azores is

Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

considered as marine temperate, with low thermal distinguished two different seasons: a dry season from amplitude, high precipitation, high air humidity and

March to September and a rainy season from October persistent wind, as well as by a marked contrast

to February, even though in November rainfall was between a dryer season and a colder and wetter season.

less than twice the average air temperature. Soil pH Estimates from the monthly precipitation and soil temperature were measured with Hanna measurements show that about 75% of the annual

instruments (model HI 99121); soil pH ranged from precipitation occurs between October and March [32].

5.5 to 5.96 and soil temperature varied similarly with The photophase ranges from around 9 h in December

the profile exhibited by the average air temperature, and January, increasing after that, and reaching the

with the lowest values occurring also in January and maximum day length in June and July (14.5 h),

March (12.4 °C and 12.3 °C, respectively), and the followed afterwards by a decrease at the same rate

highest in September (20.8 °C).

[33]. Station 2: Cabeço da Bola, midway the northern Two sites were selected on Pico Island. Station 1:

slope of Pico mountain (latitude 38°28.654' N; Piedade, at the south-eastern tip of the island (latitude

longitude 28°26.156' W; altitude 1,044 m): this 38°25.874' N; longitude 28°04.222' W; altitude 205

sampling site is a small enclave of native forest with m): this sampling site is a secondary forest of

Laurus azoricus, Erica azorica, Viburnum treleasei, Pittosporum undulatum, Persea indica and sparse

Vaccinium cylindraceum and Euphorbia stygiana, an individuals of Laurus azorica, with the soil spotted by

understory of Woodwardia radicans, and the soil Pteridium aquilinum, Hedychium gardneranum,

covered with abundant leaf-litter. Considering the data Rubus sp and Selaginella kraussiana. Considering the

from the closest udometric station (Lagoa do Caiado, data from the closest meteorological station (altitude

altitude 805 m), the average annual precipitation

33 m), the average annual precipitation (1974-1996) in (1974-1996) for the study area is 4,547.2 mm/yr, the study area is 1,141.3 mm/yr, ranging from 31.2

ranging from 174.4 mm in July to 674.5 mm in mm in July to 152.8 mm in November, with 74.4% of

December. About 73% of the annual precipitation the annual precipitation occurring between September

occurs between September and March [37]. Annual and March. Annual average air temperature average air temperature (1974-1996), measured (1974-1996), measured at the same meteorological

according to Bettencourt’s [38] method and taking station, is 17.4 °C, ranging from 13.7 °C in February

into account the data from the meteorological station to 22.1 °C in August; in turn, actual annual

at the airport, is 12.4 °C, ranging from 8.7 °C in evapotranspiration (1974-1996) ranges from 726.5 to

February to 17.1 °C in August. Actual annual 814 mm/yr, following Thornthwaite [34], Coutagne

evapotranspiration (1974-1996) ranges from 673.4 to [35] and Turc [36] methods [37]. According to

690.9 mm/yr [37], according to Thornthwaite [34] and Bettencourt [38], in this area the climate may be

Turc [36] methods. According to Bettencourt [38], the classified as slightly humid. During the sampling

climate in high altitude is always considered as period (April 2009-March 2010), the average annual

extremely humid. During the sampling period (April precipitation from the closest meteorological station is

2009-March 2010), the annual average air temperature, 1,659.8 mm/yr, ranging from 34.4 mm in June to

measured according to Bettencourt’s [38] method and 308.2 mm in October; annual average air temperature

taking into account the data from meteorological is 17.1 °C, ranging from 12.9 °C in February to

station at the airport, is 12.1 °C, ranging from 8.1 °C

22.6 °C in August. The application of the Gaussen in February to 17.8 °C in August. Soil pH ranged index to the climate data for the sampling period

between 3.61 and 5.38 and soil temperature varied

34 Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

similarly with the profile exhibited by the average air classification used by Rodrigues and Medeiros [19], temperature, with the lowest values occurring in

Ferreira et al. [20] and Cúrdia et al. [43]. January (7.3 °C) and the highest in September

3. Results

(14.2 °C). Both sites have specific climate conditions and

3.1 Piedade

microhabitats, the main differences between them Among the specimens of O. (D.) minor under

being related to the existence of a dry season in analysis (N = 36), maximum shell diameter (ANOVA:

Piedade in contrast to what happens in Cabeço da

F = 3.280, P = 0.018) and total height of the shell (F = Bola. Our results are supported by Cruz [37], who

2.801, P = 0.034) showed significant differences reported the absence of a dry season at higher sites in

during the sampling period. In July the MD differed Pico Island since 1977/1978, contrary to what happens

statistically from the remaining sampling period (P < at lower sites.

0.05, LSD test), apart from May. The MD ranged

2.3 Sampling and Morphometrics from 5.1 mm to 5.7 mm, with the minimum values shown in July and the highest in January. In turn, the

The sampling period, chosen according to the new TH in May was significantly different from September

moon phase, started in May 2009 and continued for a and January (P < 0.05, LSD test) and in July, it full year, with bimonthly periodicity. Specimens were

differed statistically from the remaining sampling hand-picked, among leaves and under stones, during a

period (P < 0.05, LSD test), apart from May. The TH time effort of two hours; the individuals with the

of the shell ranged from 2.6 mm to 2.9 mm, with the largest shell diameter were selected at Piedade (May,

minimum values in May/July and the maximum n = 3; July, n = 7; September, n = 9; November, n = 9;

values in January.

January, n = 4; March, n = 4) and at Cabeço da Bola The proportion of immature/mature gametes in (May, n = 7; July, n = 5; September, n = 6; November,

specimens collected from Piedade in May and July n = 8; January, n = 9; March, n = 8). Sample size was

differed significantly from those collected during the conditioned by differential seasonal availability, as

remaining sampling period (nonparametric multiple already observed by Cunha [39] for O. (D.) atlanticus

comparison proportion test, P < 0.05) but did not in São Miguel Island. Prior to the preparation for

differ from each other; in September, this proportion histological studies, the shell of each specimen was

was significantly different from January and March (P measured for MD (maximum diameter) and TH (total

< 0.05) (Fig. 1a). The gonadal volume variation of the height), using a vernier caliper, according to the

early stages of spermatogenesis (Sg + Sc) and methodology used by Martins [40].

spermatozoa (Sz) was statistically significant (Kruskal-Wallis test: H = 32.446; P < 0.001 and H =

2.4 Gonadal Maturation State 28.059; P < 0.001, respectively). Post hoc Dunn’s test

Specimens were fixed in aqueous Bouin’s solution indicated that in May and July the relative volumetric for about 16 h, dehydrated and embedded in paraffin

density of Sg + Sc was significantly higher than those wax. Serial sections of 5 µm thick were obtained and

from November, January and March (P < 0.05) but stained with Mayer’s haemalum and eosin [41]. The

did not differ statistically from each other and from relative volumetric density of the gametes was

September (P > 0.05). In May and July the relative estimated using the M168 Weibel Multipurpose Test

volumetric density of Sz was significantly different System [42]. The stages of gametogenesis from November (P < 0.05) (Fig. 1b). In turn, the

development were distinguished according to the relative volumetric density of MO (mature oocytes) in

Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

July and September differed significantly from highest volume occupied by Sg+Sc (84.53% and January and March (P < 0.05) (Fig. 1c). Our data

66.61%, respectively) and the lowest volume occupied showed that in May and July the specimens had the

by Sz (3.48% and 17.09%, respectively). After that

Fig. 1 Gonadal maturation features of O. minor collected from May 2009 to March 2010; and temperatures (°C), precipitation (in mm) and EVR (evapotranspiration) (in mm) registered between 1974 to 1996 [37]. Both data were collected in two sites at Pico Island, a low altitude and high altitude. (a, e) Proportion of immature male (IMG) and female gametes (IFG)/mature gametes (Sz (spermatozoa) and MO (mature oocytes)); (b, f) Percentage (mean ± SE) of spermatogenesis stages: spermatogonia and spermatocytes (Sg+Sc), St (spermatids) and Sz (spermatozoa); (c, g) Percentage (mean ± SE) of oogenesis stages: PVO (pre vitellogenic), VO (vitellogenic) and MO (mature oocytes); (d, h) Month mean temperatures (°C), precipitation (in mm) and EVR (evapotranspiration) (in mm). The dashed rectangles indicate the dormancy period and the different lower cases above columns, within a stage of reproductive maturation between sampling dates, indicate significantly different values, Kruskall-Wallis test (post hoc Dunn’s test, P < 0.05).

36 Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

spermatozoa occupied more than 41% of the gonadal variation of the early stages of spermatogenesis volume throughout the year, reaching the highest

(Sg+Sc) and Sz (spermatozoa) was statistically value in November (67.01%) (Fig. 1b). The gonadal

significant (Kruskal-Wallis test: H = 24.168, P < volume of MO attained the highest values in January

0.001 and H = 14.309, P = 0.006, respectively). Post (23.04%) and March (36.68%) and the lowest values

hoc Dunn’s test indicated that in May the gonadal in July (2.11%) and September (3.20%) (Fig. 1c).

volume of Sg+Sc was significantly different from The morphometric variables of the shell, MD and

March (P < 0.05) while in July it differed significantly TH, showed a significant positive correlation from September, November and March (P < 0.05). (Spearman’s rho = 0.376, P = 0.024 and rho = 0.422,

The relative volumetric density of Sz in July was P = 0.010) with the gonadal volume occupied by

significantly different from March (P < 0.05), being mature gametes (Sz and MO, considered together).

statistically similar during the remaining sampling However, whereas the relative volumetric density of

period (Fig. 1f). In turn, the relative volumetric spermatozoa showed a significant positive correlation

density of MO (mature oocytes) in May and July was with MD (Spearman’s rho = 0.389, P = 0.019) and TH

significantly different from September and November of the shell (Spearman’s rho = 0.431, P = 0.009), the

(P < 0.05) (Fig. 1g). the data showed that gonadal mature oocytes did not show any significant volume occupied by Sg + Sc reached the lowest value correlation with those morphometric variables (P >

in July (5.96%) and the highest in March (33.16%). In 0.05).

turn, the gonadal volume occupied by Sz attained the highest value in July (71.66%) and the lowest in

3.2 Cabeço da Bola March (47.35%) and the highest in July (71.66%),

Among the specimens of O. (D.) minor under indicating that more than 47 % of the gonadal volume analysis (n = 43), maximum shell diameter (ANOVA:

was occupied by Sz throughout the year (Fig. 1f). The

F = 3.280, P = 0.018) showed significant differences gonadal volume occupied by MO attained the lowest during the sampling period contrary to total height of

values in September and November (0.72% and the shell (F = 1.462, P = 0.226). In July the MD

1.19%, respectively) and the highest values in May differed statistically from September and November

(8.35%) and July (11.32%) (Fig. 1g). (P < 0.05, LSD test) and in September it was

The shell morphometric variables (MD and TH) did statistically different from the remaining sampling

not show any significant correlation with the gonadal period (P < 0.05, LSD test), apart from November.

volume occupied by mature gametes (Sz and MO, The MD of the shell ranged from 4.9 mm to 5.8 mm,

considered together) and by spermatozoa (Pearson’s with the minimum values in September and the

(r), P > 0.05). However, the relative volumetric maximum values in July. In turn, the total height of

density of mature oocytes had a positively significant the shell ranged from 2.4 mm to 2.7 mm, with the

correlation with maximum diameter (Pearson’s (r) = minimum values in September and the maximum

0.394, P = 0.009).

values in July.

3.3 Comparison between Piedade and Cabeço da The proportion of immature/mature gametes in

Bola

specimens collected in May differed significantly from March (nonparametric multiple comparison

The specimens of O. (D.) minor collected at both proportion test, P < 0.05) and in July this proportion

stations during the sampling period did not show significant differences (X was significantly different from September, November 2 (1) = 3.841, P < 0.05) with

and March (P < 0.05) (Fig. 1e). The gonadal volume regard to the morphometric shell variables under

Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

analysis. However, the proportion of immature/mature

gametes was significantly different in May (X 2 (1) =

74.51, P < 0.05), July (X 2 (1) = 81.38, P < 0.05) and

March (X 2 (1) = 14.14, P < 0.05) (Figs. 1a and 1e).

4. Discussion

4.1 Piedade The morphometric data of O. (D.) minor indicate

that juveniles start growing after July, and later in September, they initiate their maturation development followed by reproduction during winter and early spring, and probably dying afterwards, since we observed a decrease in MD and TH of the shell.

Our observations on gonadal maturation showed that at low altitude they are mature from October/November to March, when spermatozoa and mature oocytes are both present in higher relative volumetric densities. The proportion of mature/immature gametes during the sampling period showed two distinctive reproductive phases: an immature phase from April/May to September and a mature and active reproductive phase from October/November to March, indicating that specimens of O. (D.) minor collected at Piedade followed an annual cycle of activity and aestivation. In fact, the occurrence of a strong overlap among an immature phase and a long-term dry season suggests that snails avoid a long period of dehydration by aestivating. These findings are supported by our data as well as by the literature: (1) the high percentage of immature stages between May and July/August suggests an accumulation of energy reserves in order to survive during a long dormancy period [11, 44, 45], through metabolic adjustments in the maturation of reproductive tissues [11], and (2) the proportion of immature stages started to increase before the end of the rainfall season (April), probably triggered by the increase of photophase, and did not cease before the actual start of the rainfall season (September) (Fig. 1a-1d). This result appears to be in agreement with Cook [17], who stated that the onset and termination

of aestivation is probably controlled by photoperiod and climate conditions. Although initially physiological preparations occur in response to a long-day length, after that this may be overridden by climate conditions, such as prolonged dehydration and high temperatures. The results also suggest that gonadal maturation shows a functional protandric tendency once spermatozoa occupied more than 50% of the gonadal volume in September while mature oocytes were still uncommon. These results can be explained by the size-advantage model of Ghiselin [46], which postulates that a small body size could be optimized for male function while a larger body size is adaptive for female function, since a greater amount of energy is necessary to produce eggs than sperm. These results are similar to those found for other land snails (e.g. Refs. [18-20, 47, 48]). On the other hand, the increase of gonadal volume of spermatozoa in September was probably linked to the substantial increase in rainfall after August. After September, as previously mentioned, snails arouse from dormancy in response to an increase in relative humidity and although gonadal maturation of mature oocytes started to increase after that it only reached the highest volumes in December/March. This last result is coincident with the occurrence of a humid season period where the average rainfall was twice as much as the average temperature.

There is a positive correlation between morphometric shell characters (MD and TH) and sexual maturity, as expressed by the gonadal volume of mature gametes, when combined together. A similar association between gonadal maturation and maximum diameter has been reported by other authors (e.g. Rodrigues et al. [18], Rodrigues and Medeiros [19], Ferreira et al. [20] and Cuezzo [49]).

4.2 Cabeço da Bola The morphometric data revealed a general decrease

in maximum shell diameter after July and a straight increase afterwards, reflecting the input of a new

38 Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

generation of adults into the samples. On the other and consequently to an high soil moisture content. hand, the general growth pattern exhibited may

Based on data reported for Pico by Cruz [37] (Fig. 1h), represent a response to the altitudinal gradient, as

we can assume that similar circumstances should be suggested by Baur [50], once snails living at high

observed at Cabeço da Bola. On the other hand, altitudes need more time to reach adult size, as they

during the low rainfall period that occurred between are exposed to lower temperatures.

May and September, we can assume that those The authors’ observations on gonadal maturation

environmental conditions associated with higher showed that at high altitude O. (D.) minor mature

altitudes will lead to a slow drying of the habitat. Like from March to July/August, when spermatozoa and

what happens with several other terrestrial snail mature oocytes are present in higher percentage.

species [20, 54-56], our data suggest that reproductive However, they seem to have minimal required

maturation was triggered by day length, while conditions to reproduce during all year, since gonadal

temperature acted as a regulatory factor, as the volume is occupied by more than 47% of spermatozoa

percentage of mature gametes began to increase with throughout the year and there are residual values of

photophase, while temperature was still low. mature oocytes during the period between September

Our results showed that contrary to what was and April. Our results indicated a functional reported by other authors [18-20, 49] as well as for O. protandric tendency since spermatozoa occupied more

(D.) minor collected at Piedade (present study) there is than 57% of the gonadal volume in specimens with

no correlation between morphometric characters (MD small body size whereas the mature oocytes were

and TH) and sexual maturity in specimens from uncommon. As observed above, these results are

Cabeço da Bola.

similar to those found for several other species (e.g.

5. Conclusion

[18-20, 47, 48]) as well as for O. (D.) minor collected at Piedade (present study).

This study allowed us to suggest that snails living in The results on the proportion of immature/mature

different altitudinal gradients develop several gametes and relative volumetric density of different

adaptations as a result of the environmental conditions stages of spermatogenesis and oogenesis were in

in those habitats. However, concerning morphometric accordance to the morphometric data, thus supporting

shell characters (MD and TH), the individuals the hypothesis of a new input of adults into the

collected at different altitudes did not show significant samples. In fact, an increase in the gonadal volume of

differences. On the other hand, some physiological the early stages of spermatogenesis after July, along

adaptations were observed. In specimens collected at with a decline in gonadal volume of mature oocytes,

low altitude (Piedade), we distinguished two well stands for that hypothesis.

demarcated phases of gametogenesis, each one Specimens living at higher altitudes are exposed to

occurring concurrently with one of the two existing higher relative humidity and also to horizontal

and demarcated seasons in the Azores—a summer precipitation due to the Foehn effect, a phenomenon

season between April and August, and a winter season particularly important in oceanic islands that provides

between September and March. These results reported, an important source of water to the vegetation [51, 52].

for the first time, the phenomenon of aestivation in a According to Mendes and Dias [53], high rainfall and

terrestrial mollusc living in an oceanic Island at this average temperatures relatively low, during the rainy

latitude. The findings showed that at low altitude, season, in addition to the factors mentioned above,

gonadal maturation was influenced by several factors, lead to low values of evapotranspiration of the soil

such as photoperiod, rainfall and temperature. On the

Reproductive Strategies of a Terrestrial Snail along an Altitudinal Gradient on an Oceanic Island

other hand, in specimens collected at high altitude, a influencing the distribution and life cycles of bilharsiasis intermediate host snails, Malacological Review, Ann

different scenario was observed, reflecting an

Arbor 11 (1978) 1-25.

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Macrochlamys indica, Malacological Review, Ann Arbor

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H. Burla, W. Stahel, Altitudinal variation in Arianta temperature.

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Jan. 2013, Vol. 7, No. 1, pp. 42-50 Journal of Life Sciences, ISSN 1934-7391, USA

Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm

1 1 1 1 1 Rolesu Sandro 2 , Aloi Daniela , Cappai Stefano , Mereu Piras Pierpaola , Fois Francesco , Satta Giuseppe ,

3 3 Palmas Costantino 4 , Ecca Anna Rita and Pulina Giuseppe

1. Osservatorio Epidemiologico Veterinario Regionale (OEVR.), Istituto Zooprofilattico della Sardegna “G. Pegreffi”, Cagliari 09100, Italy 2. Istituto Zooprofilattico della Sardegna “G .Pegreffi”, Sassari 07100, Italy 3. Sezione di Parassitologia-Laboratorio di Entomologia Medica, University of Cagliari, Cagliari 09100, Italy 4. Dipartimento di Scienze Zootecniche, Facoltà di Agraria, University of Sassari, Sassari 07100, Italy

Received: June 28, 2012 / Accepted: August 14, 2012 / Published: January 30, 2013.

Abstract: The main object of this research has been to apply the scientific assumption that the alkalization is able to make inhospitable the larval reproductive sites in the identified area (Sant’Antioco). The research has been conducted in the pilot farm into the selected area of Sant’Antioco where the first hypothesis has been tested and the information about larval development has been collected. During the whole experimental period have been collected all the data about captures in the black light traps and captures in the soile traps. Experimental data first of all have been emphasize the possibility of decrease the larval reproduction extremely in the alkalized sites then have allowed to study the Bluetongue vectors density closely and its relation with the disease prevalence in the heads presents in the near farms. The results revealed the different distribution of the disease prevalence in the several farms, referable to minute variation of the farm location. In the end it seems essential to improve the farm management through the widespread action of awakening to farmers.

Key words: Sardinia, vector density, Ceratopogonidae, larval habitat.

1. Introduction  catarrhal fever” or “epizootic catarrhal of sheep” in the original descriptions of investigators in South

BTV (Bluetongue virus) is a RNA-virus belonging Africa. The name “bluetongue” is the anglicized form to Orbivirus genus (Reoviridae family), transmitted by of the Afrikaans “bloutong”, which was coined by bite of Ceratopogonidae midges of the genus Boer farmers to describe the distinctive cyanotic Culicoides . BTV infects all ruminant species, however, tongue of some severely affected sheep [2, 3]. The clinical disease can be mainly observed in sheep and biological host range of this genus includes both goats. An exception to this rule has been represented insects and other arthropods. Vertebrate hosts include by the clinical figures seen in cattle of Central Europe man, horses, monkeys, rabbits, cattle, deer and (particularly in Belgium, Germany and the suckling mice. Transmission is through vectors such Netherlands) infected by serotype 8 in 2006 [1]. BT as Culicoides, mosquitoes, phlebotomines and ticks (Bluetongue) was first described as “fever”, “malarial [4]. After cutaneous instillation of BTV (by

inoculation or through the bite of a BTV-infected Corresponding author: Rolesu Sandro, research field:

epidemiology. E-mail: sandro.rolesu@izssardegna.it. Culicoides vector), the virus travels to the regional

Bluetongue: A Hypothesis of Control Strategy through Decrease

of Culicoides and Their Associated Damage in Farm

lymph node, where initial replication occurs [5]. In egg, four larval instars, pupa and adult stages. The 1944, Du Toit, in South Africa, proved that the virus

immatures require moisture and organic matter for is transmitted by Culicoides imicola, midge of the

development and breeding sites include damp or genus Culicoides. Among these, some are identified

saturated soils, bogs, marshes, swamps, tree holes, like disease vectors (C. imicola), others are only

animal dung and rotting fruits or other vegetation [16]. suspected or potential vectors. In North America, the

The duration of the life cycle depends on the species main vector is Culicoides sonorensis, while in Central

and climatic conditions, varying from seven days in and South America, C. insignis and C. pusillus are

the tropics to seven months in temperate regions, considered the principal vectors of the disease. In

where most species diapause as fourth instar larvae Australia, C. brevitarsis and C. fulvus are the species

during winter [17]. The life-span of the adults is involved in BTV transmission [1]. In Asia, several

usually short and is dependent on ambient conditions, species of Culicoides are considered infection vectors,

which will alter with climate change. Most adults while in Africa and in the Mediterranean basin, C.

survive less than 20 days, although occasionally they imicola is known as the major vector, although it is

live for up to 90 days [6]. Females feed on blood, possible that some species of Culicoides obsoletus

which provides protein for the development of eggs complex play the role as vector in some areas of

and one bloodmeal is usually required for each batch Bulgaria [6]. C. imicola is also the principal vector of

of eggs to mature. The frequency of feeding is, BTV to ruminant livestock in southern Europe. The

therefore, linked to the rate of egg development, other potential vectors are Culicoides obsoletus and C.

which is itself dependent on species and the ambient scoticus of the Culicoides obsoletus complex, C.

temperature. Increases in ambient temperature may pulicaris of the C. pulicaris complex and C. dewulfi.

lead to an increased feeding frequency. This is Within the Mediterranean C. imicola is the principal

relevant as virus transmission to susceptible hosts may vector accounting for as much as 90% of disease

occur at each feed. In the majority of species adult transmission. However, in the Balkans BT advanced

activity is crepuscular or nocturnal [18] and activity is northwards and eastwards into areas where C. imicola

greatest when evening and night-time conditions are is absent and also appeared unexpectedly in northern

warm, humid and calm [19]. First cases of BT start in Europe in August 2006 and again in 2007 in the

midsummer, the major prevalence is observed when absence of C. imicola. The appearance of the disease

the summer is ending then the disease disappears in areas where C. imicola is not present, leaves no

because night temperature decreases below 12 °C. doubt that species of Culicoides endemic to Europe

Cattle play an important role in the epidemiology of are transmitting BTV [7].

BT, because they generally keep the infection without The incriminated and potential palaearctic vectors

showing clinical symptoms. In fact, the endemization are C. obsoletus [8-11], C. pulicaris [12], C. of infection in temperate areas and its persistence from

dewulfi [13] and C. chiopterus [14]. In one of these one epidemic season to the following are sustained by studies, BTV was isolated from mixed Culicoides

the prolonged viremia in cattle. The infective period species pools which contained C. scoticus thereby

(the longest period during which an affected animal implicating it also in the transmission of BT disease

can be a source of infection) for cattle, in fact, is [9]. BT, being transmitted by vectors, is conditioned

considered usually to be equal to 60 days post by the environment and climate: it can be detected in

infection but it is possible to have a longer viremia the late summer. Culicoides biting midges are tiny

[20]. Climatic changes that alter these conditions may flies, 1-3 mm in length [15]. The life cycle consists of

affect the activity rates of Culicoides. For example,

Bluetongue: A Hypothesis of Control Strategy through Decrease

of Culicoides and Their Associated Damage in Farm

44 windier summer nights may suppress activity and

lessen the risk of virus transmission. As Culicoides can be carried on the wind for considerable distances, however, these same conditions may lead to more effective dispersal of infected midges to new areas [21]. Worldwide BTV has been estimated to cause direct (disease) and in direct (trade, vaccines, etc) losses of over $3 billion per year [22]. The disease has

a world-wide distribution in an area between 40-50° N and 35° S where climate and environment conditions are optimal for Culicoides survival. Actually drawing this line is a theoretical structure because particular environmental condition can go over these limits [23]. In fact, climate change is one of the most serious environmental issues of our day. In the last century, the global mean temperature has risen by 0.5 °C [24] and if no steps are taken to limit greenhouse gas emissions, temperatures could rise by a further 2 °C by 2100. This predicted rate of change is greater than global temperatures have changed at any time over the past 10,000 years. In addition to increases in temperature, changes in precipitation, wind patterns and climate variability are also predicted to occur [25]. There are several reasons to explain this dramatic change of BT epidemiology, but the most are mainly as follows [26]:

 the spread of the major BT vectors C. imicola everywhere;  the presence of new kind of vectors in Culicoides ;  the climate change. In August 2000, BTV-2 was reported for the first

time in Italy. First Sardinia was hit but the BT virus spread immediately in Sicily and in the south of Italy [27]. It seems definitely that BT epidemies in the south of Europe are determinated by climate change. Culicoides spp. are carried by wind certainly, the most reliable hypothesis to explain the appearance of BT in Sardinia is exactly that the vectors are carried by the winds which come from south and hit the region in 2000 between June and July; the next wind wave is recorded 25 days before the disease occurred [28].

2. Methods

The practical application of this observation was experienced in the pilot site of Sant’Antioco, and experimental deals with the “Department of Sciences Applied to Biosystems, University of Cagliari, Section of Parasitology—Entomology Laboratory Medicine”.

A procedure to “alkalize” the sites considered of high risk for the reproduction of the insect situated near the sheep and herds, through the use of certain substances (e.g. Lime milk), in amounts and arrangements evaluated according to the type of soil and the relative permeability of the same. The action was therefore directed only against pre-imaginal stages and was therefore reinforced the location of outbreaks of the insect larvae. In fact, it is well known that Culicoides are widespread and concentrated in all those muddy areas, even in modest extensions, which are created around the breedings. In particular, major outbreaks are shallow (less than 15 cm), rich in organic matter (with a concentration of 8% solids) determined from the effluent of animal excrements inside the farms. The larvae are found in shallow mud on the edge of the water line. The only sites capable of producing a sufficient quantity of insects to trigger the viral transmission of BT are, in fact, the large and small pools of water that are created by drinking troughs that leak, from washing water, water reserves as ponds used for watering livestock. The treatment consists in preparing a solution of lime in the ratio of 200-300 g/L per square meter, and then spread it in pond effluents containing predominant organics consisting of the cleaning of stables or animal shelters, etc. This treatment is hypothesized with these doses for wells with an average depth of about 10 cm, the quantities will be increased if the depths increase (for an average depth of 20 cm, the quantity of lime solution will be doubled).

The experiments carried out at Sant’ Antioco have already given more than favourable indications for the use of this effective and efficient “larvicide”. The correct georeferencing of the breedings (milking

Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm

45 rooms, stables, etc.) and structures in which these

animals have the opportunity to stay (pasture), summed of the layering of information elements such as hydrography, roads, land use, soil type, vegetation, etc., with the help of estimation models effectuated with satellite observation, offers a powerful evaluation-management tool of the territory, and through this, the identification of hazards (Fig. 1). This methodology made it possible to identify a set of correlation factors to the initial null hypothesis on the biology of the vector, in particular on the role that water has to influence the number of population of insect carriers, and in promoting the widespread even in wide fields. The techniques of spatial statistics, at this point, can be used with greater detail and with more efficiency.

2.1 Culicoides Data The choice of the territory of S. Antioco for the

experiment was dictates by:  the presence of a limited territory well-managed, where the influence over neighboring territories contained to a minimum by insularity;

 the presence within the territory of all susceptible species;  possession of the historical reference to the various epidemic waves and data on the damage they caused.

More specifically, the area (108 km 2 ) now includes

a total of 59 companies distributed in the two communities, Sant’Antioco and Calasetta.

It was also important to proceed immediately with the georeferencing of the breedings acquiring data from the national database, and positioning companies as graphically depicte.

In addition to the territory of the pilot site of S. Antioco, the trial took place in synchrony in other seven pilot sites (Iglesias, Orani, Posada, Cardedu, Villagrande—in three distinct locations), which has developed research through the planned activities of the various OU. For Iglesias (Baschieri Company), Sant’Antioco (Ghisu Company), and Posada (Marongiu Company), the companies host the traps of standard monitoring plan for the National Blue tongue, allow the comparative study with fixed traps positioned directly on the ground in larval outbreak (or supposed). The trap is developed using the life cycle of insects from larvae present in the first layers of soil and in the time of the flickering as adults, they head toward the only opening to any Erlenmeyer containing 70% ethanol. These traps allow the evaluation of the activity of single outbreak since catching the insects to the transition from larval to adult, before it can flicker in the immediate environment.

The farm chosen for pilot testing and breeding is placed 50 m above sea level, site also already used to the placement of a trap of national entomological

Fig. 1 Disposal of breedings on the island of Sant’Antioco.

46 Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm

surveillance system. After initial site inspections is

laminated mud.

defined as an area to be treated, near the main corporate body, where they assumed the presence of 2.2 Probing of the Soil

the general characteristics of the land suitable for On Apr. 6, 2007, there have been removed four breeding of the vector (Fig. 2). Therefore, the pH of a

cups of ground, two from the treated area and two in soil sample collected in the area chosen for treatment

the control area at four different points, the mean pH was measured:

value was 7.85. The measurement of pH was made pH of the mud = 7.45

with the pHmeter resulting in a volume of 50 mL with To quantities of lime milk have been added in the

distilled water, 15 mL of mud.

soil in proportion of the mud presence (1 L/m²):

2.3 Event History of Pilot Farm S. Antioco

A = 98 cm² Lime milk = 9.8 mL

The major tasks in the company were based on After the addition of lime milk, the pH was

periodic site inspections to review acts of the various immediately measured:

catches and the collection of the flasks containing the Surface pH = 12.25

insects to be classified with the department of pH at a depth of 3-4 cm = 8.52

parasitology at the IZS.

The pH was again re-measured on Feb. 23, 2007 (a From Mar. 23, 2006 to Apr. 03, 2006: Place 10 distance of 5 months after treatment) on the treated

traps (Fig. 3);

area, with the litmus test and obtaining a value around From May 05, 2006 to July 28, 2006: Reposit traps,

7.5. A sample of the same mud in stagnant water that protect of the trap 1a with 4 plates and move trap 3a; has been brought to the laboratory for pH Apr. 08, 2006: Treat with hydrated lime to 20% in an measurement with finding a pH value equals to 7.96.

area of 30 m around traps 1a, 4a and 5a and in an area

A month after the pH was re-measured in the treated

of 15 m around the trap 8a.

area and various points of the company, and value was From Aug. 25, 2006 to Sept. 15, 2006: Move and between 6.5 and 7 everywhere adjacent each trap. On

repositioning all traps to verify the sterilization and Mar. 30, 2007 in the area of treatment to a depth

re-colonization of soil treated and untreated area. of 2 to 4 cm are still visible traces of lime, which is From Oct. 01, 2006 to Apr. 06, 2007: Run and

reposit all traps,

From May 18, 2007 to Sept. 28, 2007: Reposit and verify all traps: the situation of the soil is almost identical to that of last autumn with all the treated waterlogged area.

2.4 Data Processing

For the acquisition of data, a database was created by Access 2000 (Microsoft) and subsequent statistical evaluation was performed using software SPSS for Windows ver 15.0. The evaluation of geographic data, the realization of thematic maps and spatial overall assessment was carried out with the aid of GIS Fig. 2 Photos of the grid treated and traps inside. software Mapinfo Professional ver 7.8 and the ArcGIS

Bluetongue: A Hypothesis of Control Strategy through Decrease

of Culicoides and Their Associated Damage in Farm

Fig. 3 Diagram of the grid and the arrangement of traps treated.

software. The maps were acquired at altitude Information System of BT, together with http://www.gdem.aster.ersdac.or.jp, while the entomological surveillance data on catches of insects mapping land use was acquired by the Autonomous

vectors. The experimental data on the catching of Region of Sardinia, Department of Defense insects using flicker traps were acquired by the Environment, map services. For the analysis of spatial

“Dipartimento di Scienze Applicate ai Biosistemi variables, the entire island of S. Antioco has been

dell’Università degli Studi di Cagliari, Sezione di divided into areas resulting from applying the theory

Parassitologia—Laboratorio Entomologia Medica”. of Voronoi. The partitioning of a plan with n points

3. Results and Discussion

into convex polygons such that each polygon contains exactly one generating point; every point in polygons

The comparative study of the fixed traps and is closer to its generating point than to any other. The

flickering traps concerned the classification of the cells are called Voronoi polygons (http://mathworld.

species C. imicola , C. obsoletus and C. pulicaris, all wolfram.com/VoronoiDiagram.html). With the basis

three possible vectors of BT virus. The entomological of the observation that the insect vector moves

survey provided a check that trends in populations of towards the nearest farm, we have therefore Culicoides spp. present in the different periods of the constructed the Voronoi polygons using geographical

year recorded by the two traps is similar, even for the referencing for each individual farmer, thus obtained a

different species, particularly C. imicola (Figs. 4 and breeder of reference for every single point of territory

5). Zeroing the larval development obtained in soil of the whole island. The data on the first wave of the

treated is considered to be the same on every part of epidemic BT, vintage 2000-2001, were acquired and

the alkaline surface, as during the experimental period processed by the Center veterinary epidemiology

the traps within the grid were constantly moved Regional (OEVR), which feeds the National and relocated in different areas of land to distribute the

48 Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm

Trend of Black light traps

N°

Date

Fig. 4 Culicoides trend of black light traps.

Trend of soil traps

N° N°

Culicoides spp. C. imicola C. obsoletus

C. pulicaris

Date

Fig. 5 Culicoides trend of soil traps.

Culicoides trend in the control area

TOTAL INSECT

TOTAL CULICOIDES

Other Ceratopogonidae Psychodidae Chironomidae Other Diptera

N°

Hemiptera Hymenoptera

Coleoptera Other orders

Date

Fig. 6 Catches in the control area.

Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm

49

Fig. 7 Catches in the treated area.

control over the entire area. The immediate slaughtering of the catch in the treated areas compared to untreated lasted for the period from 29/09/2006 to 27/10/2006, with an important permanency of the infertile soil treated (no catches) for long periods (about 5 months). It was also interesting to note the confirmation of the dependence in the presence of water by the larva, an aspect highlighted by the decrease of fish caught during the change of land by livestock farmers, a fact that prevented the normal influx of water to the area. The graphs of Figs. 6 and 7 show the evolution of catches on traps placed in the soil treated with flicker catches on traps placed of ground control (untreated). It is clear that the sharp decline to zero until the larval development during the period after the alkalinizzation of sites considered optimal for the vector cicle.

4. Conclusion

The preliminary findings are encouraging, even if limited to one pilot site, and worthy of consideration. Of particular interest:

 The immediate culling of the catch in the treated compared to untreated;  The permanence of infertility of the soil treated (no catches) for periods long (about 5 months). This important result led us to hypothesize a

treatment on a larger scale experiment. Also, because larval breeding sites are not normally used agronomically but for the most part are places of transit and parcels of land not cultivated, it might be thought that any secondary treatment does not create any damage to crops. This would ensure greater safety with a persistence of the basicity of the soil, keeping low the possible negative side effects. For the success of the intervention, it is very important to cooperate with the owner or manager of the company with the technical staff operating. This point varies from company to company, could be a source of confounding of final data. Diligent preparation and illustration of the type and arrangements need to be implemented in respect of the breeder, so that it can share the purposes of research and experiment. However, there would be other agronomic interventions, such as:

 any drainage of watercourses to drain stagnant and wells;

 remediation and cleaning of the marginal areas of cultivated fields;  changing the staging areas of livestock;  amendment of transit times to the admissions;  improvement of structural conditions of the

company and the milking parlor and resting;  possible use of insecticides.

N°

Date

Culicoides trend in the soil treated

TOTAL INSECT TOTAL CULICOIDES Other Ceratopogonidi

Psicodidi Chironomidi

Other Ditterri Emitteri

Imenotteri

Coleotteri Other Order

50 Bluetongue: A Hypothesis of Control Strategy through Decrease of Culicoides and Their Associated Damage in Farm

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Jan. 2013, Vol. 7, No. 1, pp. 51-56 Journal of Life Sciences, ISSN 1934-7391, USA

Removal of Different Dyes by Pseudomonas fluorescens

Sewgil Saaduldeen Anwer and Sawan Merkhan Department of Biology, Faculty of Health and Science, Koya University, Arbil 44001, Iraq

Received: July 05, 2012 / Accepted: October 10, 2012 / Published: January 30, 2013.

Abstract: The effluents of textile dyes are highly colored, and disposal of the wastes into receiving waters cause damage to the environment, therefore dyes removal is of great importance. In this research, removal of Orange II, Remazol Blue, Methyl red, Malachite green and Safranin dyes by Pseudomonas fluorescens was studied in batch system as function of temperature, pH and initial dye concentration. The rate of removal dyes was studied by using pectrophotometer. The optimum value was determined as pH 7 for all dyes tested about 87.8-72.7%, and optimum temperature for removal of all tested dyes was 30 °C about 86.6-60.8%. Higher removal of dyes observed at 25 mL for all tested dyes about 91.9-72.7%. In general, the increase in dye concentration inhibited the growth of bacteria. Pseudomonas fluorescens showed higher removal of Orange II among the dyes tested.

Key words: Textile dyes, Pseudomonas fluorescens, pH, temperature, initial dye concentration.

1. Introduction  experiment showed that at high concentrations all five strains of Pseudomonas fluorescens tested inhibit

Pseudomonas fluorescens is a common spore production by pathogenic plant fungus. Fungi Gram-negative, rod-shaped bacterium. It belongs to

such as Alternaria cajani and Curvularia lunata grow the Pseudomonas genus; 16S rRNA analysis has on plant surfaces causing disease and death of the placed P. fluorescens in the P. fluorescens group plant. Plant treatment with Pseudomonas fluorescens within the genus, to which it lends its name. Name can prevent these fungi from growing and spreading “fluorescens” is because it secretes a soluble

through spore production [8-10].

fluorescent pigment called pyoverdin (formerly called Numerous bacteria of dye decolorization have been fluorescein), which is a type of siderophore [1-4]. reported, efforts to isolate bacterial cultures of P. fluorescens has multiple flagella. It has an degrading azo dyes started in the 1970s with reports of extremely versatile metabolism, and can be found in Bacillus subtilis then Aeromonas hydrophila [11, 12], the soil and in water [5]. It is an obligate aerobe but the first two pseudomonas strains of dye certain strains are capable of using nitrate instead of decolorization [9]. An azoreductase enzyme was oxygen as a final electron acceptor during cellular responsible for the initiation of the degradation of the respiration. Optimal temperatures for growth of Orange I dye by these strains and substituting any of Pseudomonas fluorescens are 25-30 °C [6, 7]. It tests the groups near azo groups chemical structure positive for the oxidase test. Pseudomonas fluorescens hindered the degradation. Our purpose of this study is is also a non saccharolytic bacteria [8]. Studies done to determine ability of Pseudomonas fluorescens to on Pseudmonas fluorescens have shown the microbe’s remove five different dyes (Remazol Blue, Safranin, potential benefit in bioremediation against several Malachite green, Orange II and Methyle red) through strains of plant pathogens. The results of the studying three different parameters affection to dye

removal such as temperature (30, 35, 37 °C), pH (6, 7, Corresponding author: Sewgil Saaduldeen Anwer, Dr., associate professor, research field: industrial microbiology.

8), and initial Dye concentration (25, 50, 75 μL). E-mail: sevgil75@yahoo.com.

Removal of Different Dyes by Pseudomonas fuorescens

2. Materials and Methods

2.3 Decolorization Studies

2.1 Microorganism and Culture Conditions The tests were conducted in test tube containing Pseudomonas fluorescens which used for dyes

bacterial cultures (nutrient broth & bacteria) with removal study obtained from microbiology laboratory

50 μL of Orange II, Remazol Blue, Methyl red, of Koya University.

Malachite green and Safranin at different initial pH Biotypes characteristics were determined by using

7-8 and temperature 30-35 °C for 48 h. The dye the API 20 NE identification kit (BioMerieux,

concentration was determined after 48 h of Hazelwood, MO). Inoculums was prepared by sub

incubation by taking 3 mL of sample and centrifuged culturing from the stock culture into 10 mL culture

to remove suspended biomass.

broth and incubated at 30 ± 2 °C for 24 h. The concentration of dye in the supernatant was

2.2 Tested Dyes determined by reading the absorbance at nm: Orange Ability of bacterium to remove Orange II, Remazol

II 380 nm, Remazol Blue 600 nm, Methyl red 501 Blue, Methyl red, Malachite green and Safranin dyes

nm, Malachite green 621 nm and Safranin 521 nm by was studied. A stock solution of dyes were prepared at

using spectrophotometer [11, 12]. Dye containing

a concentration of 100 g/l and diluted as required. medium used as blank control medium contained Orange II and Remazol Blue were obtained from

both dye and medium without bacterial growth to AYTEMIZLER Textile Co., Methyl red, Malachite

observe any reaction of medium with dye [13, 14]. green and Safranin dyes were obtained from The rate of removal was obtained by using the microbiology lab.

following formula:

Initial dye concentrat ion (C o ) - final dye concentrat ion (C)

Removal % =  100 (1)

Initial dye concentrat ion (C o )

3. Results

Enterobacteriaceae (2201004) as shown in Table 1 and Fig. 1.

After obtaining of bacterial culture from microbiology lab, biochemical tests were performed to

3.1 Effect of Temperature on Dye Removal support the results using API20E test which is a rapid

accurate technique for the identification of the family The effect of different temperatures on the removal

Table 1 ApI 20 used for different biochemical test showing the result (2201004).

Tube 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 10 Oxidase Reaction

- - - + Point

Fig. 1 Pseudomonas fluorescens (a) macroscopic exam and (b) API test.

Removal of Different Dyes by Pseudomonas fuorescens

OrangeII Remazol Methyl Malachite Safranin

Fig. 2 Effect of different temperatures on the remove of dyes by Pseudomonas fluorescens (C o : 50 μL, pH 7.5).

Malachite Safranin

Fig. 3 Effect of different pH on remove of dyes by Pseudomonas fluorescens (C o : 50 μL, T: 30 °C).

OrangeII Remazol

Methyl Malachite Safranin

Fig. 4 Effect of initial dye concentration on removal of dyes by Pseudomonas fluorescens (T: 30 °C, pH: 7).

Removal of Different Dyes by Pseudomonas fuorescens

Test Control

(a) Orange II, blue and methyl red at 0 times (control and tested tube).

Test Control

(b) Orange II, blue and methyl red after 48 h (control and tested tube).

Fig. 5 Showing Dyes and removal of Dyes before and after 48 h.

of dye by pseudomonas sp. culture was followed at 3.3 Effect of Different Dye Concentration on Dye different incubation temperatures for tested dyes

Removal

control, the higher removal was at 30 °C (Fig. 2), and Different dyes concentration used for determine the there was no removal of dyes in medium without

ability of pseudomonas sp. to remove dyes. The strain culture (control).

shows ability to remove Orange II in all dye

3.2 Effect of Initial pH on Dye Removal concentrations while removal rate decreased by increasing concentration for other dyes (Fig. 4).

Three different initial pH values were tested in order to determine a suitable pH for the effect dye

4. Discussion

removal by pseudomonas sp. as shown in Fig. 3. The Dyes are extensively used in textile, paper, printing higher removal of all dyes was shown at pH 7. There

industries and dye houses, the effluents of these was no removal of dyes in medium without culture

industries are highly colored and the disposal of these (control).

wastes into receiving waters causes damage to the

Removal of Different Dyes by Pseudomonas fuorescens

environment. Physical and chemical methods used for

Merkhan.

removing dyes require long time and high cost, and

References

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5. Conclusion

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Removal of Different Dyes by Pseudomonas fuorescens

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Jan. 2013, Vol. 7, No. 1, pp. 57-62 Journal of Life Sciences, ISSN 1934-7391, USA

Preparation of Organic Selenocystine Using Locally Isolated Bread Yeast

Fouad Houssein Kamel Medical Technical Institute, University of Polytechnics, Erbil 44001, Iraq

Received: July 10, 2012 / Accepted: September 19, 2012 / Published: January 30, 2013.

Abstract: In contrast to the toxic inorganic forms of selenium to life at very low concentrations, the organic-selenium compounds are of considerable interest and several of them play essential roles in cell biochemistry and nutritional science. Se-yeast (Selenium-enriched yeast) is a common form of selenium used to supplement dietary intake of this important trace mineral. In the present study, we tried to prepare an organic selenocystine using locally isolated bread yeast (Saccharomyces cerevisiae). A novel locally prepared date extract media enriched by addition of 0.2% KH2PO4 (potassium phosphate), 0.6% ammonium sulfate was adopted as alternative culture media. Differences concentrations of selenium salt (30, 60, 120 and 240 μg/mL) were added to the yeast culture media. While the best concentration of selenium added was 30 μg/mL, it achieved optimal conditions for the growth of yeast and the production of red yeast growth identical to the standard. The products (organic selenocystine) were analyzed by HPLC (High Performance Liquid Chromatography) and AAS (Atomic Absorption Spectrophotometer) comparing with authentic standard obtained from Sigma. Results confirmed the formation of similar selenocystine products.

Key words: Organic selenocystine, selenium deficiency, bread yeast, selenium yeast.

1. Introduction  μg/kg dry weight), followed by liver (2.6-0.5 μg/kg),

while for the heart muscle they contain a higher Selenium is a trace element, with atomic weight of proportion of selenium from skeletal muscle as the

79 and is within the group in the periodic table of heart muscle was found to contain 1.8-0.3 μg/kg. In elements [1]. The selenium is essential trace elements the case of biological fluids which are the most in the human body as it is the most important nutrients changing factors in accordance with the activity need, necessary sources of aquatic organisms as it is present therefore, selenium is of essential nutrients [6]. There in the milk of animals, including cows, concentration are many factors affecting the balance of selenium in level in the milk is 110.9 μg/mL [2, 3] due to the the body of the organism such as the nature and importance of this element, selenium currently added quality of food [7] and the physiological status and to breast-milk factory of children to protect them from health of the body [7-10] in addition to alcohol and diseases, anemia [4]. Selenium exists in all cells and smoking [9, 11]. The use of high selenium levels tissues of the human and the biological fluid with (2.5-3 g/day) is a case of poison, at the time the rate of different concentrations, depending on the type of human intake (50-200 μg/day) was needed to ensure tissue and depending on the level of selenium. The safety for human health. Selenium was incorporated optimal concentration of selenium content in the tissue into molecules of an enzyme called GPX (glutathione is about 15 mg of normal adults [5]. The crust of peroxidase). This vital enzyme protects red blood cells, kidney was the richest of the body tissues selenium (4 cell membranes and sub-cellular components against

Corresponding author: Fouad Houssein Kamel, associate undesirable reactions with soluble peroxides. The professor, research field: microbiology. E-mail: discovery of GPX opened the door to understand how

fhkamel2013@yahoo.com.

58 Preparation of Organic Selenocystine Using Locally Isolated Bread Yeast

selenium is protective against cancer, heart disease,

2.2 Preparation of Culture Media arthritis and accelerated aging [12, 13]. Now more

Locally prepared liquid media contain dates juice scientific excitement is being generated with the (8%) instead of sugar (5.6%) was prepared. The media recent finding: selenium is also a vital component of were enriched by addition of 0.2% potassium other mammalian enzymes. Also selenium has e a bad phosphate (KH2PO4), 0.6% ammonium sulfate. The effect on the central nervous system, liver and spleen

media were inoculated by yeast and incubated at 30 o C [14]. Selenium was found within the protein content

for 24 h with shaking at speeds of 150 cycles/minute. of the tissues, therefore called Selenoproteins [15].

Selenium composition present in the active sites of

2.3 Addition of Inorganic Selenium Salts the enzyme Glutathione peroxidase, thioredoxin

Selenium salts were added to culture media in a reductase and mammalian Iodothyronine 5-deiodinase concentration extremely high (30, 60, 120 and 240 [16]. μg/mL) during the exponential growth phase, in the

Studies have confirmed the viability of selenium form of six doses with periods of one hour for each

cancer inhibition e.g. colon, cervical, breast, and liver dose continuously. The final product prepared after

cancer [17]. It was therefore highly recommended the incubation for a period of 24 h. Later the yeast growth

use of this organic component for the purpose of culture had been filtered and washed with distilled

cancer drugs, accompanied by the enzyme important water several times and then dried using acetone.

role in the transformation of selenium salts. Against this background and with new research

2.4 Examination of the Outputs

evidence regularly appearing for the role of selenium The concentration of the yeast output selenocystine in the reduction of viral virulence [18-28], it is not product was determined with the final estimation of surprising that many people are interested in ensuring organic transformation using HPLC, compared with they have adequate selenium status by supplementing authentic organic standard. It was dismissed on the their diets with selenium. Since the selenium linked type of reverse-phase column (C-8) dimensions (250 × to the amino acid Cystine that confirms the

4.6 mm Id), using mobile phase consisting of THF: importance of nutritional yeast since 1910 [29], and

phosphate buffer (95:5v/v) [31, 32]. yeast also contains 70 trace elements with organic Atomic analysis: In AAS, the sample is atomized forms [30]. into the vapor phase. A beam of electromagnetic The study aimed to produce organic selenocystine radiation passes through the vaporized sample. Some using locally bread yeast isolation and locally of the radiation is absorbed by the metal of interest. The

prepared liquid media from dates juice as alternative instrument measures the change in intensity, which is

culture media. then converted into an absorbance reading. After

2. Materials and Methods

calibration, the amount of absorption can be related to the concentrations of various metals through the use of

2.1 Micro-organism and the Material Basis

Beer-Lambert’s law [33].

The yeast strain used by the manufacturers to

3. Results and Discussion

produce selenium-enriched yeast in all cases was Saccharomyces cerevisiae. The selected strain of

In the preliminary production study of organic bread yeast was isolated locally and kept as a

selenocystine, a locally media of date juice has been reference source in the 4 o

prepared. The new novel culture media also contained activation.

C with a sequential

source of nitrogen and carbon nutrition as

Preparation of Organic Selenocystine Using Locally Isolated Bread Yeast

fundamental to the growth of the organism in the there may merely be a mixture of sodium selenite and media [34]. A novel locally date extract media was

yeast [32]. Therefore, filtering and washing of yeast adopted as an alternative used locally appropriate

several times finally with distilled water necessary is culture media by researchers [15]. Bread yeast

to separate free Se salt mixture media contains, then (Saccharomyces cerevisiae) was adopted in locally

drying using acetone.

date extract media and then developed. It was known that selenium can be included in the Different concentration of inorganic selenium

other biological molecules such as DNA carrier (sodium selenite) (30, 60, 120, 240 μg/mL) were

(t-RNA) producing (Seleno-tRNAs) [37] and this in dissolved in water and added to the growth culture.

turn leads to a change in the production of vital It is necessary to take consideration of the situation

proteins.

to avoid discouraging the growth of yeast, which can Fig. 1 confirms the results of the successful occur because of inorganic selenium salt work using HPLC. The formed selenocystine concentration [35, 36], in addition to the quality and

concentrations were measured qualitatively and quantity of organic selenocystine as an output. So the

quantitatively by comparison with the peak area of the inorganic selenium was added with each standard (Sigma). It shows various concentrations of concentration used in the experiment in form of six

the selenium salts noted in chromatograph salts added doses and interval with one hour to avoid the

to the culture media. While the best inorganic affection of the yeast growth. The inorganic

selenium concentration added was 30 μg/mL, which selenium salt were added during the process of stable

achieved optimal conditions for the growth of yeast, growth (exponential), where they are converted

and the production of red yeast grew identical to the inorganic selenium salts to the organic compounds

standard.

within the yeast cell, as a result of the replacement of AAS by flameless has confirmed existence of the sulfur atom of the amino acid (Cystine) by

produced from of yeast at various additions of selenium, so the composition of organic selenium in the organic product as compared with the (Selenocystine) was produced. Control of pH, standard. The data also confirmed the presence of temperature, selenium feeding profile and aeration

selenium in the required specifications. allows optimal growth of the yeast strain and

AAS was used to determine concentrations of Se maximum biomass production. Selenocystine compounds in plant extracts by Zhang and product could be investigated by altering the color of

Frankenberger [38]. They were able to separate Se into yeast cells to the red indicating the formation of the

non-amino acid organic Se, Se-amino acids, selenite organic Selenium, while the density of the color

and selenate by an anion-exchange method. The depended on concentration of organic selenocystine.

atomization efficiency is greatly increased after The ability of the yeast under appropriate hydride generation process. For selenium, HG-AAS conditions to accumulate quantities of trace elements

(hydride generation atomic absorption such as selenium, where the selenium salts dissolved

spectrophotometer) requires the Se (IV) oxidation state in water when added to the culture media, which is

(selenite), since Se (VI) is not reproducible [29]. required for the development of yeasts. Unfortunately,

Therefore, all selenium must be in the Se (IV) not all material sold as Se-yeast is produced according

oxidation state for analysis [33].

to these stringent criteria. Sometimes the percentage As final, the results of both technical (HPLC and of sodium selenite is more such that most of the

AAS) and morphological specifications of the product, selenium is clearly not bound to the yeast: at worst,

confirmed a successful process in the preparation of

60 Preparation of Organic Selenocystine Using Locally Isolated Bread Yeast

Fig. 1 Separation of Selenocystine salt result from different conc. of selenium added to the media, on reversed phase isocratic HPLC methods on the following separation condition for standard and prepared complex on Column: reversed

phase (50 × 4.6 mm under the same condition ID) 3 m particle size column, Mobile phase: 70:30 v/v 0.01 M potassium

phosphate buffer: THF tetrahydrofurn HPLC grade Merck Flow rate: 1 mL/min Detection: 254 nm,Temperature: 30 °C. (A: Standard, B: 30, C: 60 μg/mL).

organic selenocystine product using locally bread strain and maximum biomass production. As a result of yeast isolation, in addition to the efficiency of the

the fermentation in the selenium-enriched medium, the local media as alternative to the imported culture

selenium becomes organically bound to the yeast. media. It is an integral selenocystine substitute which

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Jan. 2013, Vol. 7, No. 1, pp. 63-68 Journal of Life Sciences, ISSN 1934-7391, USA

To a Question about Forecasting Number of Micromammalia (Rodentia)

Nadezhda Antonets 1 , Aleksandr Balalayev 2 and

Мargarita Shumkova 3

1. Dnipro-Orel’s Natural Reserve, Dnipropetrovs’k 52030, Ukraine 2. Dnipropetrovs’k National University, Dnipropetrovs’k 49050, Ukraine 3. Regional Sanitary-Epidemic’S Station, Dnipropetrovs’k 49006, Ukraine

Received: July 17, 2012 / Accepted: November 14, 2012 / Published: January 30, 2013.

Abstract: In the present study, the data for population dynamics of micromammalia in Dnipropetrovs’k Region in 1957-1999 and Dn іpro-Orel’s Natural Reserve at 1991-2009 are adduced. With the purpose to drawing up of the long-term forecast at number are analyzed long-term lines of number on background kind’s fine micromammals of dominant group. The analysis of long standing rows of number small mammals and comparison data with Wolf number were demonstration, which indicate that dynamic of number phone species (and dominant in particular) carry a cyclic character and contact with 11-year cycles of sun activity. Consequently, it was clearing up hidden periodical in dynamic of number mouse rodents. By that observation was dependence from cycles of sun activity. Probably, that in 2012 year, before peak of sun activity in 2013, next high increase about the number of small mammals for maxima (“outbreak” mass reproduction is a peak “big wave”) will take place.

Key words: Small mammals, number, forecasting.

1. Introduction  the presence of regular cyclic processes in the nature, which are connected with the sun activity. Zoologist

Zoologists are interested in the mass outbreak of the U.F. Z оlоtоv [4] associates the “big waves” of mass number of mouse rodent long time ago [1]. The reproduction of small mammals with the years of necessary condition for forecasting the number of minimal sun activity in the 11-year cycles. According small mammals is their registration. The long-term to the datum of the author, the 5-6-year cycles of sun forecasts are associated with attempts to forecast the activity and gelio-stipulated 5-6-year atmospheric changes of the number for many years forward. rhythms in their turn stipulate “small waves” about Consequently, the advance biological forecasting of mouse rodent number, and particularly 5-6-year the next increase of the number comes into question. rhythms of the fluctuations of small mammal number Such forecasts are not only of scientific interest, but in the south of former USSR. The researches have may be used in the corresponding branches of the

confirmed that hypothesis.

national economy (agriculture, medicine). The cyclical fluctuations of the animal number still haven’t

2. Materials and Methods

got a satisfactory explanation. The cosmophysical and For the analysis of number dynamics, the climatic factors is a basis of the cyclic fluctuations [2]. registrations of small mammals were used in the However, А.А. Маksimov [3] defends the view about regional sanitary-epidemiological station on the

territory of Dnipropetrovsk region (right and left

Corresponding author: Nadezhda Antonets, Ph.D., banks of Dnipro river). In general, from 1957 to 1999, research field: zoology (theriology). E-mail:

antonez_48@mail.ru. 630,615 traps/days were worked and 36,938

To a Question about Forecasting Number of Micromammalia (Rodentia)

individuals of micromammalia were got. By the way, the datum of monitoring researches in Dnipro-Orel’s Natural Reserve in 1991-2009 was used. The whole 56,200 traps/days were worked and 4,011 individuals of small mammals were caught. The number of mouse rodents was registered according to the standard methodology with Gero traps in the lines there were 100-50 traps with exposition 3 in twenty four hours. The bait was a crust of stale rye bread that was wetted with sunflower oil.

The data of climatic factors (precipitation and temperature) were taken at Ozernaya meteorological station in the town of Dniprodzershynsk (the right bank not far from Dnipro-Orel’s Natural Reserve). The materials of grain crops yield in Dnipropetrovsk region were taken in the regional statistics office. The various methods of time series analysis which were described in the literature [5-7] to find some regularity, periodicity and forecast the peaks moments. The long-term series of small mammal number were analyzed and the appropriate diagrams were built. Then the datum of sun activity (Wolf number), grain crops yield and climatic factors were analyzed and compared with the materials about number of small mammals. As a result, it made an attempt to forecast the number of background species of micromammalia, as climate changes (warm and moisture changes) are tightly connected with 11-year cycles of sun activity is 0.80-0.98 [8] and indirectly impact on the crop yield. The number changes of background species depend on climate and yield changes. Moreover, the food is a primary factor that regulates number and space distribution of animals [9].

3. Results and Discussion

The background species of micromammalia in Dnipropetrovsk region and particularly in Dnipro-Orel’s Natural Reserve, [1-3] are: mouse rodents (Sylvaemus uralensis Pallas, 1811), (S. sylvaticus Linnaeus, 1758), (Apodemus agrarius Pallas, 1778) and common shrew (Sorex araneus

Linnaeus, 1758). The fluctuations of the number of background species of small mammals which compose a dominant group are cyclic, synchronic [3,

10, 11] that allow analyzing the long-term time lines of number and perspective and retrospective forecasting. The dynamics number of background species has a conjugated bond with 11-year cycles of sun activity [10-13, 14, 15]. The following phases can

be observed from the changed number of small mammals: number development, “small wave” peak, number decrease to minimum, number depression, number rise, “big wave” peak and number decrease to minimum. The number rises in the years with relatively warm snowy winter, early spring and warm long autumn; the decrease is followed by a series of cold not snowy severe winters, early cold autumn and late spring. From Fig. 1, you can see that every 10-12 years the value of Wolf number is maximum, the crop yield increases and the number of micromammalia increases too (1961, 1971, 1981, 1990, 2001). The grain crop cultivated and wild cereals yield, as well as meadow grassed, berries and fruits yield increase accompany with the peak of number of small mammals. On the territory of Dnipro-Orel’s Natural Reserve, the number of peaks (mass reproduction in 11-year cycles) was registered in the year 1994, 2006-2007 (“small wave”), 1991 and 2001 (“big wave”), corresponding to the year of the minimum and sun activity decay [13]. So in the constant registration line (C.R.L.) No. 4 at the flood land in 1994 sum number of small mammals made up 29.7 individuals with 100 traps/day; in 2001, corresponding

34.8 individuals with 100 traps/day. Except for background species of mouse rodents in the year of “big wave” peak, “the explosion of number” was given by the species: (Microtus levis Miller, 1908) and (Mus spicilegus Petenyi, 1882). It can be explained by

a good grassed yield with an abundance of beans in the meadows and wild cereals on the steppe areas in 2001. In 1996 (Fig. 2) the number decreased to minimum was registered (depression at “small wave”).

To a Question about Forecasting Number of Micromammalia (Rodentia)

Since 2002 regular decrease of number of regularities, hidden periodicity, forecast the peak micromammalia began in the 11-year cycles that are

moments et cetera. Unfortunately, on the basis of confirmed with the registrations on the territory of

collected datum, it is impossible to make a reliable Dnipro-Orel’s Natural Reserve, and particularly in

mathematical model of forecasting of number of C.R.L. No. 4 is a 20 individuals on a 100 traps/day. In

mouse rodents with an effective method АRIМА 2003, the next decrease of number of small rodents to

(autoregressive model and moving average integrated minimum can be registered (depression) that was the

model) for evident non stationarity of time series. end of “high wave”. From Fig. 2, you can see that in

However, some conclusions can be made on the basis 1994 and 2001 the maximum of number of small

of the underhanded analyses that premise an mammals at the Dnipro-Orel’s Natural Reserve was

approximate long-term forecast.

accompanied by minimal rainfall. It is known that spectroscopic analysis is used to get On the one hand, the observed phenomenons have

hidden periodicity in the frequency domain of datum. an element of chance, and on the other hand, they vary

The results of number analysis and Wolf numbers are with time regularly. The various analysis methods of

represented in (Fig. 3, as spectral distribution time series were described in the literature [9, 10, 13],

smoothed by Nemming window. Maxima can aloe permit discovering at first sight invisible be clearly seen in the 4-5 year periods for number of

50 Region (right bank)

Region (left bank)

Reservation (phone species)

40 Reservation (dominant) 160

Crop capacity winter crops

35 Wolf number

30 120 W o

lf num

25 100 ber

40 Quantity persont (100 traps/day) Crop capacity (centner/hectare)