Journal of Molluscan Research FARMING Implications for Industrialization

J. R. Cobbinah (Ph.D) CSIR-Forestry Research Institute of Ghana

BEING A KEYNOTE ADDRESS PRESENTED AT THE 3RD INTERNATIONAL CONFERENCE ON GIANT AFRICAN HELD ON 2ND JUNE, 2014 AT FEDERAL COLLEGE OF EDUCATION, ABEOKUTA, OGUN STATE. Snail Population under threat Snail meat has traditionally been major ingredient in the diet of communities in the forested areas of Africa However, wild snail populations have declined due to Unsustainable loggin slash and burn agriculture indiscriminate pesticides application bushfires collection of immature Heliciculture: Farming snails Snail have been farmed for human consumption since ancient times. Fattening snails in snail gardens dates back over 2000 years ago Successful snail farming (outdoors or indoors in controlled environment)however, requires knowledge of physiology, breeding and requirements and nutrition.

Different Types of Snails

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Distribution of Giant African Snails Distribution in Tropical Africa Distribution in West Africa

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Candidates for snail farming

General features

3 Journal of Molluscan Research Common Colouration Average name Weight (gm) & Species Size (cm Achatina ac The big Brown wit h 350gm hatina red/tiger snail black stripes 8cmx14cm Achatina fulica The foolish one Light brown 85gm / The garden with grey stripes 4.5cmx8cm snail Archachatina Brown with 250gm marginata The big black grey stripes 7cmx13cm Site selection for Ecological snail farming system

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Site Selection Dense vegetation cover and loamy are preferable Trees and shrubs will act as windbreaks, reduce drastic changes in , and provide shade, food and nutrients Soils with high organic matter support good growth (avoid clayey and pure sandy soils)

Housing Snails are adept in escaping from enclosures It is therefore necessary to construct escape-proof housing It is advisable to construct enclosures that allow you to take care of the snails without having to enter in habitual and regular fashion

5 Journal of Molluscan Research Hutch boxes

Mini-

paddock Pens

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Tr ench Pens

Snailery Snailery

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Food and Feeding Snails are vegetarians and accept a wide range of foods including leaves, fruits and nuts, tubers For good growth supplementary ingredients such as vitamins, minerals and feeding stimulants should be provided.  should be provided at all times in clean shallow bowls At all stages snails should be given variety of food, clean water and

Feed for snails

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Snails feeding on formulated diet

Breeding and Reproduction

Proportion of Ingredients for formulated diet

Snails re and therefore each snail has both male and female organ. The word is derived from a name of Greek god ermaphroditus believed to be part man and part woman. In A. achatina self-ertilisationprevails. However, in A. marginata crossfertilisation is the dominant feature Egg laying starts from April and usually ends in September Average period for incubation is 3 weeks

Species Cluthes/yr sexual maturity eggs laid A.Achatina 1-3 (18-24m) 50-400 9 Journal of Molluscan Research A.Fulica 1-3 (5-9m) 100- 600 A.Marginata 4-8 (12-15m) 4-12

Eggs in Chamber

Natural Enemies A range of natural enemies amongst them predators, parasites and diseases have been found on snails. These include; Predators – mice, rats, , , snakes, , Parasites - Without doubt the most important is the Allaudihella flavicornis Diseases - Agents that cause diseases in snails include Pseudomonas spp. Fusarium spp. Affected snails have a very bad odour

Life Cycle of Alluaudihella flavicornis

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Care of snails Newly hatched snails should be given tender foliage sprinkled with water and powdered egg shell Handling of newly hatched snails should be minimized to avoid damage to shell Leftover food, feaces and slime covered material should be removed daily Inactive snails should be moved from active ones and dead snails be burned to avoid disease transmission Once a month pens should be cleaned and soils changed. In pastures should be turned over. Overslimed soil and excess faeces is detrimental to reproduction and growth Snails require ample space. Too many snails in pen leads to high death rate and slow growth. About 1.25 kg body weight/m2 is recommended During dry season snails may be sprinkled with water. Too much water can ,however, be harmful

Snail farming as business As ecological farming system Commercial farming of snails in open pastures of living food is sustainable and can produce large numbers of quality snails 11 Journal of Molluscan Research Ecological farming system (Pasture )

Semi-ecological system

12 Journal of Molluscan Research Raised T r ench Pens Enclosed Pasture

Hi-tech Farming system

Farming Snails ( The 5-step Business Plan ) Step 1:  Marketing plan (Subsistence, LM, DM)  Production plan (Part /complete life cycle  Species to be farmed  Family operation/commercial venture Step 2:  Pilot Production (records of soft and hard inputs and records of growth performance) Step 3: Go or Not to Go Decision Determine profitability balancing production costs and sales revenue Step 4:Investment in Facilities and know-how Land, housing, equipment and tools  Labour in relation to size of operation  Capital (Private/microcredit/commercial)  know-how through appropriate training Step 5: upscaling

13 Journal of Molluscan Research Logistical Control (planning and control of movement of labour, materials, feed, medicines for operations) Quality control (delivering healthy snails)  Financial control (track progress towards financial goals)

Implications for Industrialization Potential for Commerce High protein, high Fe meat production Range of therapeutic products Cosmetic Products  Agricultural Products- Fertiliser, Nematicide, Feed  and ceramic biomaterials Waste water treatment

Therapeutic Properties From antiquity to now snails have been considered as a source of numerous therapeutic properties The great Roman naturalist Pliny(a.d 23-75) used snail secretions for treatment of coughs and skin diseases In 19 century Baron Berthelemy listed indications treated with snail secretions to include cough, bronchitis, catarrh, asthma,tonsilitis,sore throat, measles, small pox and scarlet. In the 20 century the therapeutic usage of snails was still alive Snail syrup was used for chronic bronchitis, whooping cough, anaemia. Concentrated snail mucus was demonstrated to have antibacteria effect. A recent study in 7 countries has shown that communities that eat large quantities of snails have high levels of a-linoleic acid reported to have protective effect against cardiovascular diseases Lectin extracted from snails is used as a prognastic indicator for some cancers- breast, stomach and colon

Cosmetic Products Snail secretions and extract contain allantoin, collagen, elastin ,glycolic acid, hyaluronic acid, vitamin E and protein that are natural activators for aging and damaged skin. Snail extract is also reported to contain unique enzymes that rearrange acne tissues and therefore effective for acne treatment. Commercial products include snail extract creams, cosmetic grade snail secretion powder, snail protein powder, snail shell powder, 100% snail cosmetic, snail extract skin care etc.

Other Products 14 Journal of Molluscan Research Snail shell powder as phosphate fortified fertilizer. Powdered shell as ingredient in formulation of feed Powdered chitin in snail shell showed nematicidal effect on root-knot of tomatoes (Meloidogyne spp) The shell has been used as partial replacement for fine aggregates in highway concrete The shell has been demonstrated to have potential for the fabrication of monoceramic biomaterials The high carbon in ash can play a vital role in removal of heavy metals, colour and other particles and substances that generate odour in waste water. The high Fe content has potential as coagulant in water treatment Snail shell powder has been used to bleach palm kernel oil.

Roadside Marketing of Snails

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Journal of Molluscan Research 1:13-17 (2015)

PERFORMANCE OF HATCHLING SNAILS (Archachatina marginata) FED VARYING DIETARY LEVELS OF KENAF GRAIN MEAL IN THE DIET

POPOOLA, Y.A1., OKPEZE, C.N 2., AYODELE, I.A3., OGUNLEKE, F.O2., 1FAYENUWO, J.O. AND 2TENIOLA,O.O,

1Obafemi Awolowo University, Institute Of Agricultural Research and Training Ibadan.. 2Federal College of Animal Health and Production Technology, Ibadan, Nigeria. 3 Department of Wildlife and Ecotourism, University of Ibadan ABSTRACT An experiment was conducted to determine the effect of varying levels of kenaf grain meal (KGM) as partial or total replacement for soybean meal in the diet of hatchling snails. A total number of 120 hatchling snails were randomly allotted to 4 dietary treatments in which soybean fraction of the diets was replaced at 0%, 50%, 75% and 100% in a completely randomized design with 4 treatments replicated 3 times with 10 snails per replicate. The parameters measured or calculated were feed intake, weight gain, feed conversion ratio, cost/kg feed, total feed cost and cost per weight gain. There was significant difference in the mean weight gain (P<0.05). The highest weight gain of 111.52g was recorded in snails fed Diet 1 which is the control diet while 110.72g, 109.57 and 103.85g were recorded in Diets 2, 3 and 4 containing 50%, 75% and 100% of KGM as replacement for soybean meal respectively. The total feed intake was affected by the dietary treatment (P<0.05). The lowest weight gain was recorded in Diet 4 while the highest feed intake was recorded in the control Diet 1 which was relatively similar to Diets 2, 3 and 4. The feed conversion ratio follows the same pattern with feed intake and weight gain. The feed cost/kg feed and total feed cost reduced as the level of KGM in the diet increased. The lowest cost per weight gain was recorded in the diet containing 100% KGM as replacement of soybean meal. It could be concluded that KGM could be used to replace soybean partially or wholly in the diet of hatchling snails.

Key words: Feed conversion ratio, feed cost, , kenaf grain meal, snails, soybean meal oil is an excellent source of edible oil for human consumption INTRODUCTION (Cross,2000; Swingle,2001). The grain is Kenaf grain is rich in oil and its content is also rich in minerals such as and similar to cotton seed oil suggest that the phosphorus. The protein content of KGM 16 POPOOLA ET AL: Performance of hatching of snails fed Kenaf grain meal is about 40% and has been used to replace Southern Farm of the Institute and used as soybean fraction of the diet of rabbit with bedding for impressive result, it was concluded that the snails inside the cage serum protein, packed cell volume, Design of Experiment A total of one haemoglobin, organ weights such as hundred and twenty hatchling snails of kidney, lungs, liver and heart were not mean weight significantly affected by different 31.46±2.11g were randomly inclusion of KGM as total replacement for allotted into four dietary treatments soybean in the diet of rabbit (Odetola replicated three times and each et.al., 2012). The cost of soy meal replicate contained ten snails per was fifty six naira six years ago, the price replicate in a completely randomized was around one hundred and twenty naira design. The snails were acclimatized during the period of conducting this for one week before the experiment. The cost is relatively high commencement of the feeding trial. compared to other sources of plant protein Kenaf grain meal (KGM) was hence there is need to look for alternative purchased from Institute of Agric. sources. Kenaf grain meal is another Research and Training (I.A.R&T), source of protein with high energy. The cost of KGM is relatively low compared to Ibadan, Oyo state, Nigeria. that of soybean meal. Kenaf grain meal Preparation of test ingredient has been included in the diet of broiler Kenaf grain was toasted and later which has resulted in reduction in the cost incorporated with other feedstuffs. of feed without any adverse effect. This Four diets were formulated to study was conducted to determine the contain KGM at 0% (H1 ) Control, effect of feeding hatchling snails with 50% (H2), 75% (H3) and 100% (H4) KGM as as replacement for soybean meal replacement for soybean meal fraction of fraction in the diet of growing snails. the diet on performance characteristics The diets were formulated to contain and cost benefits. about 26% crude protein and energy of 2400 kcal/kgME (Table 1). MATERIALS AND METHODS Data collection Experimental cage Feed intake and weight gain were The cage used for the experiment was built measured on daily and weekly basis of and wire netting. The cage was with the use of sensitive weighing demarcated into 12 compartments and balance. Feed intake was calculated each compartment had dimension of 0.4x by subtracting the left-over feed from 0.4 x 0.5m3. The bottom of the cages were the feed given while the weight gain perforated to allow free drain of water was calculated by deducting the when wetting the soil. The top was built initial weight from the final weight. of netting reinforced with wire Shell length and width were measured mesh. The stands of each cage were put on weekly basis with vernier caliper inside a container filled with used engine calibrated in millimeter. Micrometer oil to prevent soldier infestation. screw gauge was used to measure the Sandy loam soil was collected from the shell thickness on weekly basis. Feed 17 POPOOLA ET AL: Performance of hatching of snails fed Kenaf grain meal conversion ratio were calculated as Kenaf grain meal 0 the ratio of feed intake to weight gain. Fish meal 4.0 Feed cost and cost per weight gain shell 9.85 were also calculated. Carcass analysis Bone meal 2.1 was carried out at the end of the Palm kernel ca ke meal 5.0 feeding trial by randomly selecting Premix 0.2 5 eight snails from each treatment and Calculated Ch emical Com position Table 1 : Gross Composition of Experimental Diets for hatchling snails.

Ingredient (%) H1 (0% H2 (50% H3 (75% H4 (10 0% KGM) KGM) KGM) KGM)

19.5 19.5 19.5

10.0 10.0 10.0 8.5 8.5 8 .5

10.8 10.8 10 .8

15.0 7.5 0 .0

15.0 22.5 3 0.0 4. 0

4.0 4.0 9.85 9.85 9.85 2 .1 2.1 5.0 2.1 5 .0 0 . 0.25 .25 5 0 0.25 4.5 4

4 .54 0.64 4.54 0.64 0.64 25.93

2398.5 26.09 2404.3 25.74 2387. 7

weighed separately. Each snail was Calcium ( %) 4.5 4 killed by striking the shell with a club. Phosphorus (%) 0.64 The shell, foot and Maize 19.5 Protein (%) 26.13

Brewer dry grains. 10.0 Energy (Kcal ME /kg) 2408.4

Wheat offal 8.5 Groundnut cak e meal. 10.8 Soybean meal. 30.0 18 POPOOLA ET AL: Performance of hatching of snails fed Kenaf grain meal visceral were separated and been reported that the fibre content of weighed separately. KGM is highly digestive, rich in Chemical and statistical Analyses The minerals such as calcium and chemical composition of the phosphorus. There was no experimental diets and the foot were significant difference (P>0.05) in the done according to the method of mean initial weight of the snails as A.O.A.C. (1990). All data were Table 2 : Determined proximate composition of the KGM and the exper imental diets KGM H1 (0% H2 (50% H3 (75% H4 (100% Parameters KGM) KG M) KGM) KGM )

Dry Matter 96.47 93.87 93.78 94.15 93.90 39.75 25.45

Crude Protein 7.34 8.11 25.12 24.78 24.64 8.24 Crude Fibre 6.34 4.45 8.13 8 .34 4.84 Ether Extract 9.32 9.56 4.46 4 .92 9 .48 9.39 9. 23 Ash 37.35 53.43 Nitro gen Free 52.81 52 .87 52.7 Extract 5

KGM – Kenaf grain meal subjected analysis of variance and the shown in Table 3; the values ranged means were separated if they are between 30.69g and 32.44g. significantly different using Duncan There was significant difference (P<0.05) Multiple Range Test (SAS, 1999). in the mean weight gain as shown in Table 3. The highest weight gain of 111.52g was RESULTS AND DISCUSSION recorded in Diet 1 which is the control diet Table 2 shows the proximate composition while 110.72g, 109.57 and 103.85g were of kenaf grain meal recorded in Diets 2, 3 and 4 containing (KGM) and the experimental diets. The 50%, 75% and 100% of KGM as results show that KGM is high in replacement for SBM respectively. The protein and low in fibre content total feed intake was affected by the which dietary treatments as shown in Table 3. The lowest treatment was recorded in Diet

4 while the highest feed intake was implies that KGM could be used as a recorded in the control Diet 1 which was source of protein in the diet of snails. relatively similar to The crude protein of KGM reported in this trial was relatively similar to the Diets 2, 3 and 4. The feed conversion reports of Swingles, et. al., 2001. It has ratio follows the same pattern with feed intake and weight gain. The 19 POPOOLA ET AL: Performance of hatching of snails fed Kenaf grain meal lowest feed intake, weight gain reported in Diet 4 could be due to high inclusion levels of KGM in the diet. The relative similarity in the performance in Diets 1, 2 and 3 implied that KGM could be used to replace soybean meal up to 75% in the diet of hatchling snails. The low performance of snails in Diet 4 could be attributed to taste and effect of processing methods which has been reported to affect performance of livestock (Omole et al., 2012). as Tannin and Sapponin may also contribute to poor performance of snails at high inclusion levels of KGM Table 3: Summary of Growth performance of g rowing snails fed varying levels of KGM in the diet. Parameters (Means) H1 (0% H2 (50% H H4 ± SEM KGM) 3 (75% (100% KGM) KGM) KGM)

Initial weight (g) 31.76 30.69 31.07 32.44 2.45 Final weight (g) 143.28a 141.41 a 140.64 a 136.29 b 6.34

Total weight gain (g) 111.52 a 110.72 a 109.57 a 103.85 b 3.56 Total feed intake (g) 447.2 447.34 448.12 451.74 8.23 4.01 a 4.04 4.09 b 4.35 0.12 Feed conversion ratio a a Shell length increment (mm) 6.46 6.46 6.45 6.44 1.45

Shell width increment (mm) 4.78 4.77 4.77 4.76 0.33 Shell thickness increment (mm) 0.14 0.14 0.13 0.13 0.02

Mortality (Number) 0 0 0

0 Total feed cost (N/kg) 5.92 a 5.71 a 5.40 ab 4.61 b 1.08 Cost/weight gain (N/kg) 53.82 a 51.91 b 49.09 c 46.1 c 2.89

Means along rows with different superscript are significantly dif ferent from each other (P<0 .05) KGM – Kenaf grain meal such 20 Presence of antinutritional factors and 'saved nature' of kenaf grain meal at POPOOLA ET AL: Performance of hatching of snails fed Kenaf grain meal in the diets.(Hamzat,2004; Odetola et Haematology, Serum al.,2012) Shell length, width and Biochemistry and Organ thickness of the snails were not Histopathology of Rabbits Fed significantly (P>0.05) affected by Graded Levels of Whole Kenaf varying inclusion levels of KGM in the (Hibiscus canabinus) Seed diet of hatchling snails. The shell Meal. International Journal of length ranged between 6.44 and Agricultural Research, 7: 86-92 6.46mm, the shell width increment O m o l e , A . J . , O s u n k e y e , O . ranged between 4.76 and 4.78mm J . , while shell thickness ranged between Ojejide,J.O., Sodamola,M.O., 0.13 and 0.14 as shown in Table 3. and Poopola,Y.A.(2012).Back Good management practices the level To Agric.Series (10).The of inclusion could b e responsible for African Giant Land Snails. zero mortality recorded across the treatments. The lowest cost per weight Green choice publications. 76 gain was recorded in the diet containing pgs 100% KGM as replacement for SBM. S.A.S.(1999).S.A.S.User,s Guide. Statistical Analysis System CONCLUSION AND Institute,Inc.Cary,N.C: 7-78. RECOMMENDATION Swingle, R.S, A.R. Urias, J.C. Doyle and R.L. Voigt. (2001). It could be concluded that kenaf grain meal could be used to replace soybean Chemical composition of kenaf partially or wholly in the diet of forage and its digestibility by hatchling snails. lambs and in vitro. J. Anim. Sci., 46:1346-1350. REFERENCES Association of Official Analytical Chemist, A.O.A.C. (1990). Official Methods of Analysis, 13th Edition, Washington, D.C. 149 pgs Cross (2000) Kenaf -An Alternative C r o p f o r t h e T r o p i c s http://www.satglobal.com/about_ kenaf.htm Hamzat, R. A .(2004).Utilization of cocoa bean shell in the diet of growing snail(A. marginata). Phd Thesis, Department of Animal Science, University of Ibadan 232pgs Odetola, M. O., Ewuola, E. O and Adu, O.A. (2012). 21

Journal of Molluscan Research 1:18-23 (2015)

NUTRITIVE VALUES OF KENAF LEAF (Hibiscus cannabinus L) AS SUBSTITUTE FOR PAWPAW LEAF (Carica papaya) FOR GROWING SNAILS.

1POPOOLA Y.A , 2AYODELE, I.A., 1FAYENUWO, J.O., MAKANJUOLA2, B.A., 1OLORUNGBOHUNMI, T.O. AND 1BOLADURO, B.A.

1Obafemi Awolowo University, Institute Of Agricultural Research and Training Moor Plantation, Ibadan, Nigeria 2Department of Wildlife and Ecotourism, University of Ibadan ABSTRACT A total of one hundred and twenty growing snails (Archachatina marginata) of mean weight 56.98±1.6g were used for the feeding trial. The feeding trial consisted of 3 treatments and each treatment was replicated 4 times with 10 snails per replicate in a completely randomized design. Snails in treatment 1 (PL) were fed solely on leaf of pawpaw (control), snails in treatment 2 (KL) was fed kenaf leaf alone, in treatment 3 (KPL), snails were fed leaves pawpaw + kenaf in ratio 1:1. Parameters measured or calculated include weight gain, feed intake, feed conversion ratio and dressing percentage. Significant difference was reported in mean total feed intake (P<0.05). There was a significant difference in the mean total weight gain (P<0.05). No significant differences (P<0.05) were observed in the mean total feed intake and weight gain of snails fed KL and KPL. The mean shell length, width and thickness were not significantly influenced by the dietary treatments (P>0.05). Zero mortality was recorded in all the treatments. The dressing percentage of the snails in all the treatments was not significantly influenced (P<0.05) by the forages. Based on the results, kenaf leaf could be used to replace pawpaw leaf in feeding snails.

Keywords: Feed utilization, Hibiscus cannabinus, pawpaw leaf, snails INTRODUCTION cholesterol levels which make it a good The importance of snail meat cannot be antidote for related diseases such as over-emphasized; it is a good source of hypertension, etc. The meat is also rich animal protein, containing about 18% in calcium, , phosphorus and crude protein of high biological value potassium which are essential or (Kehinde, 2009). The meat contains all macrominerals needed for strong essential amino-acids such as lysine, bones, methionine, highly priced, and osmo-regulation and metabolic contains low fat content and low 22 POPOOLA ET AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf activities in the body of man and it is a amino acid composition of kenaf leaf is good source of vitamins A, B6, E and K similar to that of Alfalfa. The leaf is which are required for proper rich in carotene, vitamins and it is a utilization of primary nutrients such as good source of calcium and phosphorus , protein, fat and oil (Swingles et.al., 2001) . In Nigeria, (Kehinde, 2009 and Omole et.al., Kenaf leaf is considered as waste 2011). It has been established that during harvesting but it could be consumption of snail meat promotes utilized by livestock to improve growth fertility and make baby at early stage and reduce cost of feeding. The study stronger and healthier with strong was designed to determine the effects bones and teeth due to its high calcium of feeding kenaf leaf as substitute for and phosphorus content (Kehinde, pawpaw leaf on feed intake, weight 2009). The shell of snail is a good gain, feed source of calcium and could be used to conversion and carcass yield of replace bone meal and oyster shell growing snails fraction of diet of livestock without any adverse effect on growth and MATERIALS AND METHODS reproductive performance of the animal (Kehinde, 2009 and Omole et.al., Experimental site 2011) . The experiments were carried out at the Snailery Unit of the Institute of Snails could be fed with different leaf Agricultural Research and Training materials such as leaves of pawpaw, (I.A.R.& T.), Moor Plantation, Ibadan plantain, sweet potatoes to mention but which is located on Longitude 0351E, a few without any adverse effect on Latitude 0723N and Altitude 650” lies productivities authors (Omole et al., in the humid zone of the rainforest belt 2011; Adikwu, 2012; Omole et al., 0703.25 of Southwestern Nigeria with 2012). Kenaf is an annual or biennial mean annual rainfall of 1220 mm and herbaceous short-lived plant belongs to mean temperature of 26C. The family, malvaceae. It can be up to 3.5m experiment was carried out during in height when fully mature. Kenaf leaf rainy season (May and September). is used as food in and as Experimental design livestock feed in the United States of America. Kenaf leaf contains 8.7% A total of one hundred and twenty NDF, 3.5% growing snails of mean weight ADF and 15-34% crude protein 56.98±1.6g were used for the feeding trial. The snails were reared under (Webber, 1993; Swingles et.al., 2001. intensive system of production using The leaf and the petiole portion of the cages, the cage was demarcated into 12 plant contain the digestible nutrients. compartments and each compartment Kenaf leaf has been used to feed cattle, had dimension of 0.4x0.4x0.5cm3. The sheep and goat with an impressive cage used for the experiment was built result. It was established that dried of wood and wire netting. The bottom kenaf leaf ground into a meal had a of the cages were perforated to allow greater crude protein digestibility than free drain of water when wetting the that of Alfalfa meal (Cross, 2000). The soil. The feeding trial consisted of 3 23 POPOOLA ET AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf treatments and each treatment was range reported by (Swingles et.al., replicated 4 times with 10 snails per 2001, Cross, 2000). However the fibre replicate in a completely randomized content of kenaf leaf in this trial was design. Snails in treatment 1 (PL) were low compared to the reports of Cross, fed solely on leaf of pawpaw (Control), 2000 and this could be due to the snails in Treatment 2 (KL) was fed effects of cultivar planted, there is fresh kenaf leaf alone, in treatment 3 variation in the chemical composition (KPL), of cultivars as supported by (Webber, snails were fed fresh leaves of pawpaw 1993 ). Moreover, the variation in fibre + kenaf in ratio 1:1. The leaves of the levels could also be due to the seasonal plants used were fresh. The snails were effect, the fibre of the crop is always given 10g of grower mash as high during the dry season while the supplementary feed. fibre level is always low during the Data collection rainy season. The stage of harvesting of the crop could also affects the fibre Feed intake and weight gain were content of the leaf, kenaf harvested or measured daily and weekly with the use cut at early stage will have low fibre of electric weighing balance. Shell content compared to those that are length and width were measured on harvested at older age . Significant weekly basis with the use of vernier difference was reported in mean total caliper while shell thickness was feed intake (P<0.05) as observed in measured every week with micrometer Table 2. The highest total feed intake of screw gauge. Feed conversion ratio was 582.12g was recorded in snail fed both calculated as the ratio of feed intake to kenaf and pawpaw leaves (KPL) which weight gain. Twelve snails from each was relatively similar to those that fed treatment would be randomly selected kenaf leaf alone (KL). There was a at the end of the feeding trial for carcass significant difference in the mean total analysis. The snails were starved weight gain (P<0.05). No significant overnight and killed with Iron rod. The difference was observed between KL shell, foot and offals were then and KPL. The mean shell length, width separated and weighed separately. and thickness were not significantly Chemical and statistical Analyses The influenced by the dietary treatments chemical composition of the forages (P>0.05) as shown in Table 2. The and the meat were done according to better performance recorded in KL and the Method of A.O.A.C. (1990). All KPL implies that kenaf leaf could be data were subjected to analysis of used as substitute for pawpaw leaf and variance and where differences were could be used as alternative feed observed the means were further resource for snails. separated by Duncan Multiple Range Test (SAS, 1999). The results of improved performance of snail fed kenaf leaf was also in

agreement with what was observed RESULTS AND DISCUSSION when kenaf leaf was used as feed for The crude protein of 37.5% recorded for kenaf leaf (Table 1) fell within the Table 1: Chemical composition of PL, KL and KPL (% dry matter). 24 POPOOLA ET AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf PL KL KPL Dry matter 23.56 24.78 23.89 Crude protein 21.91 28.87 26.67

Crude fibre 16.28 14.36 15.53 2.89 Ether extract 2.41 3.54

Ash 9.23 10.79 9.89 Nitrogen free extract 50.17 42.44 45.02

PL- Pawpaw leaf, KL- Kenaf leaf, KPL- Kenaf -pawpaw leaf m ixtures

Table 2: Summary of growth performance of snails fed PL, KL and KPL Parameters PL KL KPL ? SEM Mean values

Means along rows with different superscript are significantly different from each other

(P<0.05) PL- Pawpaw leaf, KL - Kenaf leaf, KPL- Kenaf -pawpaw leaf mixtures.

25 Initial weight (g) 56.89 56.49 a 3.67 b 5.66 172.93 180.57 57.1 Final weight (g) 4 ab b 4.48 POPOOLA ET AL: Nutritive116.04 values of kenaf177.86 leaf as substitute124.08 for a pawpaw leaf b Total weight gain (g) 554.76 b ab 8.98 9.67 120.72 582.12 a Total feed intake (g) a b Weekly weight gain (g) 46.23 10.34 575.04 a 0.45 Weekly feed intake (g) 4.78 a ab Feed conversion ratio 48.51 11.56 10.06

Shell length increment (mm) ab 1.79 10.64 47.92 4.69 Shell width increment (mm) 0.12 Shell thickness increment (mm) 11.91 0.17 4.76 0.13 10.78

11.8 0.12 0.12 9 0.03 10.69 0.12 Mortality (Number) 0 0 2 lamb (Cross, 2000). It has also been The results of chemical composition of reported that kenaf leaf is nutritious, the meat as shown in table 4 shows that succulent and containing minerals and the protein content of the meat in all the vitamins with high protein content treatments were not affected by the (Webber, 1993; Swingles et.al., 2001). dietary treatment, the value varied The zero mortality recorded in all the between 19.21 and 20.26%, the protein treatments also buttress the fact that KL content reported in this study was in could be as substitute for pawpaw leaf agreement with the reports of Omole, et without any adverse effect. The al., 2011 and Ibom and Okon, 2012. The dressing percentage of the snails in the protein content recorded in the study treatments was not significantly compared favorably with other influenced by the forages as shown in livestock meat. The fat content also was table 3. The values ranged between not affected by dietary treatments, the 41.38 and 41.56% The dressing low fat content reported also buttress percentage reported in this trial was the fact that snail meat has low fat and relatively similar to the report of low cholesterol and this could also be (Kehinde, 2009 and Omole et.al., the reason why snail farming is 2011). becoming popular as reported by Table 3: Carcass analysis of growing snails fed PL, KL and KPL . Parameters (Means) PL KL KPL ?SEM

Final weight (g) 173.87 175.76 178.67 4.78 Shell weight (g) 47.20 46.46 44.76 3.45 Offal weight (g) 43.85 41.72 39.64 3.21 Foot weight (g) 77.26b 84.22a 85.95a 4.98 Dressing percent (%) 41.34 41.47 41.56 2.86 26 POPOOLA ET AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf Offal/live weight (%) 21.12 21.23 21.41 1.56 Shell/live weight (%) 22.78 22.91 22.96 1.98 Means along rows with different superscript are significantly different from each other (P<0.05) PL- Pawpaw leaf, KL- Kenaf leaf, KPL- Kenaf -pawpaw leaf mixtures

27 AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf

POPOOLA ET

POPOOLA ET AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf several authors (Omole et al., 2011; the meat supported the fact that kenaf Adikwu, 2012; Omole et al., 2012). leaf could be used as substitute for Similarly in protein and fat content of pawpaw leaf.

Table 4: Chemical co mposition of the meat of snails fed PL, KL and KPL (%

PL KL KPL Dry matter 23.56 24.78 23.89 Crude protein 19.81 20.26 Crude fibre 19.2 0.08 0.07 ct 1 0.96 0.98 Ether extra 0.06 11. 93 11.90 Ash 0.99 67.27 66.79 Nitrogen free extract 11. 89 67.85 dry matter).

PL- Pawpaw leaf, KL- Kenaf leaf, KPL- Kenaf -pawpaw leaf mixtures

CONCLUSION AND dressing percentage, chemical RECOMMENDATION composition of the meat hence kenaf leaf could be used as substitute for Based on the results of the study, it pawpaw leaf to feed snails. kenaf leaf could be concluded that feeding of should be used as alternative for feeding snails solely with kenaf leaf or in snails. combination with pawpaw leaf improved growth and feed efficiency with no adverse effect on mortality, REFERENCES

28 POPOOLA ET Adikwu, M.U. 2012 Snail Production Unpublished) 198 pgs for Sustainable Development and Omole, A.J., Popoola,Y.A.And Good Health Proceedings of the 1st Kolapo, B. 2012. Influence of International Conference on Giant Hamecozyne Enzyme on African Land Snails held at Federal Performance Characteristics of University of Agriculture, snails (Archachatina marginata) Abeokuta on 12 – 15 February, Fed Brewer Dry Grain Based Diet 2012. pp45-48 Proceedings of the 1st International Association of Official Analytical Conference on Giant African Land Chemist, A.O.A.C. (1990). Snails held at Federal University of Official Methods of Analysis, 13th Agriculture, Abeokuta on 12 – 15 Edition, Washington, D.C. February, 2012 .pp 89-92 149pgs Omole, A.J.; Osunkeye, O.J.; Odejide, AL: Nutritive values of kenaf leaf as substitute for pawpaw leaf Cross, I.J. (2000) Kenaf -An J.O.; Sodamola, M.O.; Popoola, Alternative Y.A (2011). The African Giant Crop for the Tropics http://www. Land Snails Green Choice Agric. satglobal. com/about_kenaf.htm Publications 56pp. Ibom, L.A. and Okon, B, 2012. S.A.S. User's Guide. Statistical Prospects of Snail for Animal Analysis System Institute, Protein Supply in Nigeria Inc.Cary, N.C. 1999 : 7-78 Proceedings of the 1st International Swingles, R.S. Urias, A.R, Doyle J.C Conference on Giant African Land And Voigt R.L. 2001 Chemical Snails held at Federal University of composition of kenaf forage and Agriculture, Abeokuta on 12 – 15 its digestibility by lamb and February, 2012. Webber, C.L. 1993 Crude protein Kehinde, A.J. (2009). Utilization of and yield components of six kenaf cassava peel products in the life cultivars as affected by crop cycle feeding of snails maturity. Ind. Crops Prod. J. (Archachatina marginata). Ph.D 2:27–31. Thesis ,Department of Animal Science, University of Ibadan.( invitro and digestibility. http://www.jas.fass.org

29

Journal of Molluscan Research 1:24-33 (2015)

EFFECT OF Mucuna pruriens ON GENE EXPRESSION OF PROGESTERONE AND FOLLICLE STIMULATING HORMONE RECEPTORS IN THE REPRODUCTIVE TRACT OF GIANT AFRICAN LAND SNAIL (Archachatina marginata)

ABIONA, J. A., ADELEKE, O. S., LADOKUN, A. O., ABIOJA, M. O., DARAMOLA, J. O., OSINOWO, O. A., AND ONAGBESAN, M. O

Department of Animal Physiology, College of Animal Science & Livestock Production, University of Agriculture, P.M.B. 2240 Abeokuta, Nigeria. Corresponding author: [email protected]

ABSTRACT A study on the effects of dietary treatment on gene expression of Progesterone and Follicle stimulating hormone Receptors in the reproductive tract of giant Africa land snail (A. marginata) was carried out. Thirty (30) snails with weight range of 100 g – 150 g were used. The snails were randomly allotted into 3 dietary treatments which included: layers mash (LMS), layers mash plus Mucuna seed meal in ratio of 1:1 (LMS + MP; 1:1) and Mucuna seed meal only (MP) and managed for eight weeks. Each treatment contained 10 replicates. After eight weeks, five snails were dissected, from each treatment and their complete reproductive tracts which consists of oviduct, albumen gland, ovo testis, spermatheca and ganglia were removed for DNA extraction. The parts were homogenized for the extraction of total RNA which was used for Reverse transcriptase – Polymerase chain reaction (RT-PCR). The PCR products were then subjected to electrophoresis. The sequence of the primers used for Progesterone receptor were Forward primer: 5' -GTC ATC ACT TTT TGT GAA AGA GGA GCC GC-3'; Reverse primer: 5' – CCC ACA GGA GTT TGT CAA ACT C-3'and for follicle stimulating hormone primers: Forward primer: AGA AGG CCA AGA ACC TCG TG; Reverse primer: ACA GCA ATG GCT AGG ATA G. The primers were designed to detect the expression of the gene encoding follicle stimulating hormone, FSH receptor mRNA and Progesterone receptor mRNA in the reproductive tract of A.marginata. The results showed that receptors for progesterone were most abundant in albumen gland, ovotestis and spermatheca, with few receptors in the oviduct. For FSH, receptors were present in medium form in both the Albumen and Ovo-tests. The snails fed Mucuna seed meal and mixture of Mucuna and layer mash in

30 POPOOLA ET ratio 1:1 had medium expression better than those fed layers mash for oviduct, albumen gland and ovo-testis with FSH receptors.This showed that these organs had receptors for follicle stimulating hormone and the receptors were stimulated by substance/substances found in Mucuna. Also, oviduct and spermatheca had higher expression in the snails fed Mucuna seed meal only, while albumen gland

31 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone and ovotestis recorded few expressions for Mucuna seed meal with progesterone receptors. These results showed that receptors for both progesterone and FSH were present and that Mucuna may have agent(s) which tends to up-regulate reproduction in some of organs and at the same time down-regulate reproductive activities in other organs. It is therefore recommended that Mucuna inclusion into the feed of snails needs to be monitored to favour reproduction.

Keyword: Reproductive tract, hormone receptor, gene expression, FSH, Progesterone, giant African land snail testosterone by leydig cells t o f u r t h e r m o r e p r o m o t e INTRODUCTION spermatogenesis. The role of Nutritional importance of Mucuna progesterone cannot be left out in seed has been well documented reproduction. It is not yet known (Panikkar et al., 1987; Mehta, whether this plant may also support 1994;Siddhuraju et al., 2000; its production. Siddhuraju and Becker, 2001; Prakash et al, 2001;Gilbert, 2002; Steroid hormones like progesterone and Ahmad et al., 2008; Singh et al., FSH are known to be key modulators of 2013). Mucuna pruriens are in great normal reproductive functions (Clark and demand in food and pharmaceutical Sutherland 1990; Nieschlag et al., 1999;). industries (Nwaoguikpe et al. 2011) Their role in the boost of reproduction is and are found to induce the activation only popular in mammals. The need to of sexual behaviour, reducing boost production of mollusc, especially reactive oxygen species (ROS) while giant African land snails has led to search also increasing plasma testosterone for artificial means of promoting level (Suresh et al., 2009; Suresh and production, especially during offseason of Prakash, 2010; Suresh et al,. this animal. Proper 2010;Singh et al, 2013). Mucuna evaluation of the presence of receptor for pruriens also known as velvet these two key hormones will justify the is an annual climbing vine that grows use of these steroids to manipulate 3-18 m in height. This plant is a rich reproduction in this animal. Therefore, it source of L-DOPA and has recently is important to locate feed that could been reported to restore enhance reproduction and at the same turn spermatogenic loss in rat mode on reproductive apparatus of this animal (Singh et al., 2013). This recent to favour production. The aim of this discovery is an indication that it has study is to determine the effect of Mucuna potential to stimulate the pruriens on gene expression of hypothalamus and forebrain to secrete progesterone and follicle stimulating gonadotropin releasing hormone hormone receptors in the reproductive (GnRH) which in turn up regulate the tract of giant African land snail anterior pituitary gland to secrete (Archachatina marginata). follicle stimulating hormone (FSH) and heuteinizing hormone (LH), MATERIALS AND METHODS causing increasing synthesis of 32 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone Location chemicals, primers, horizontal The research work was carried out at agarose gel for DNA analysis, digital Biotechnology Laboratory of the Federal camera for taking the photographs of University of Agriculture Abeokuta, dissected , gel, -70 ultra Ogun State, Nigeria. The Federal freezer to store RNA samples, plastic University of Agriculture is located on combs, and hand gloves to protect longitude 302'E and latitude 70 10' N, a against the biological chemicals location in the south western part of because some of them are cancerous. Nigeria with mean annual rainfall of Experimental Protocols about 1,037mm, average relative The snails were dissected and the selected humidity of about 82% . It is location tissues (Oviduct, albumen gland, ovo- between the tropical rain forest and the testis, spermatheca and ganglia) of the derived savannah region of Nigeria reproductive tract were collected. The (Google earth, 2006). tissues were stored in the freezer. RNA Experimental Animals was extracted with the use of a Thirty snails (Archachatina commercial kit (Norgen Biotek Corp; marginata) were purchased from a Ontario, Canada). The primers that were local farm in Abeokuta, Ogun State, used were designed using a commercial Nigeria. The snails weighed between program on the internet that could retrieve 100-150g. The snails were kept in the sequence of the target RNA. For this plastic baskets and fed for 8 weeks experiment, they were designed for after four weeks of acclimatization presence of period. progesterone and follicle stimulating Experimental procedure for hormone receptor in the reproductive tract rearing snails of A. marginata. Thirty snails were assigned into three Extraction of Rna dietary treatments with 10 snails each Sample Preparation and Cell Lysis A per treatment. These treatments are total of five parts of the reproductive tract shown as follows: (ovo-testis, oviduct, spermotheca and (i) Concentrate only albumin gland) of giant African and land snail (A. marginata) were removed after (ii) Concentrate + Mucuna seed dissection, 20-50mg of fresh tissues from meal; 1:1 different parts were collected and stored (iii) Mucuna seed meal only in a centrifuge tube and labelled for easy Feed and water were identification. Thereafter, 0.05g was provided ad libitum on weighed and transferred into mortal for daily basis. Daily cleaning grinding. During grinding, 600µL of lysis of left over feed and water solution was added to the tissues and were also carried out. homogenized using a mortar and pestle. Experimental Materials The lysate was spin for 2 minutes to Materials used for this experiment remove cell debris. Thereafter, were; dissecting instruments, supernatant/lysate was transferred into eppendoff tubes for storing samples, another RNase-free micro centrifuge PCR machine, molecular biology tube. An equal volume of 70% ethanol 33 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone was added to the lysate volume collected Primers (100µL of ethanol was added to every The primers were purchased from 100µL of lysate). The sample was vortex integrated DNA Technology (IDT) to make it mix together. Inc. Binding RNA to Column USA. The 600µL of lysate with 70% ethanol Progesterone receptor primers used was transferred into spin column tube for are: centrifuge at revolution of 1000 for 1 Forward primer: 5' -GTC ATC ACT minute. The spin column contained RNA TTT TGT GAA AGA GGA GCC binding column, which bound to the GC-3' column, thereby leaving the residues and then the flow-through was discarded. Reverse primer: 5' – CCC ACA GGA ' Column Washing GTT TGT CAA ACT C-3 Four hundred (400) micro-litre of Follicle stimulating hormone primers primary washing solution was used are: applied to the spin column. The spin Forward primer: AGA AGG CCA AGA column was then centrifuged at 1000 ACC TCG TG revolutions per minutes for 1 minute. Reverse primer: ACA GCA ATG GCT The flow- through was discarded and AGG ATA G the spin column was centrifuged Loading and Electrophoresis again at 1000 revolutions per minute The agarose gel was transferred into the for 1 minute to remove residual electrophoresis tank. The samples were ethanol. RNA Elution loaded in the wells after they have been The spin column was placed into an mixed with a loading dye. The loading elution tube, 50 microlitre elution dye allowed the samples to sink properly solution was added to the column. into the wells. The The samples were then centrifuge for electrophoresis chamber was then filled 1 minute to elute the RNA. The with TAE buffer; the electrophoresis was sample was then stored in the ultra- then connected and made to run for one freezer. hour. Agarose Gel Preparation Polymerase Chain Reaction A total of 1g of agarose powder was The extracted RNA was mixed with the weighed out and placed in a buffer. It primers, the master mix, which consisted was then mixed with 100ml of Tris of the buffers and other nucleotides. It Acetate EDTA (TAE) buffer . The was then subjected to Reverse mixture was well mixed and then Transcriptase Polymerase Chain Reaction placed in a microwave to melt at a (RT-PCR) in the PCR machine for about temperature range of 37-400. two hours. The PCR machine was set at Afterwards, the melted mixture was an annealing temperature of 94oC, which allowed to cool off and then mixed is the average of the stated with 5 µL of gel red. The mixture was on the primers. The PCR products were then poured into an agarose chamber. then subjected to Reverse Transcriptase Solid gel was formed after cooling, Polymerase Chain Reaction (RT-PCR) leaving wells in it. 34 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone

Plate 1: T otal mRNA extracted from dif ferent parts of the reproductive tract showing the expression of progesterone receptor gene of A. marginata snails fed different again. The samples were then made to Well 13: Ganglia Treatment 1 undergo electrophoresis again. The gel Well 14: Ganglia Treatment 2 was removed and viewed under the ultra- Well 15: Ganglia Treatment 3 violet light source. Data Collection And Analysis The of Archachatina marginata (oviduct, expression of the gene that encoded the albumen gland, ovotestis, protein of the receptors was obtained via spermatheca and ganglia) fed with PCR machine and gel electrophoresis. three different dietary treatments (Concentrate; Concentrate + Mucuna RESULTS AND DISCUSSION Plate seed meal, 1:1 and Mucuna seed meal 1 shows the expression of progesterone only. Influences of dietary treatments receptor gene in five different parts of the on levels of expressions are also reproductive tract diets. shown in Table 1. Well M: Marker The expression of progesterone Well 1: Oviduct Treatment 1 receptor gene was low in the oviduct Well 2: Oviduct Treatment 2 of snails fed concentrate when compared with those of snails fed Well 3: Oviduct Treatment 3 mixture of Mucuna seed meal plus Well 4: Albumen gland Treatment 1 concentrate and Mucuna seed meal Well 5: Albumen gland Treatment 2 only. It was also noticed that the Well 6: Albumen gland Treatment 3 expressions in the oviduct have two Well 7: Ovotestis Treatment 1 isotypes (1 and 2) (Plate 1). This Well 8: Ovotestis Treatment 2 observation indicated that Mucuna contain substance(s) that could Well 9: Ovotestis Treatment 3 influence this organ in terms of Well 10: Spermatheca Treatment 1 production or secretions. It also Well 11: Spermatheca Treatment 2 showed that Mucuna may influence Well 12: Spermatheca Treatment 3 egg production since oviduct is more

35 Table 1: D egree of expression of progesterone receptor gene in d ifferent reproductive par ts of A. marginata snai ls fed different diets/treatments Parts of the Expression of progesterone Receptor Gene ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone Reproductive tract Concentrate plus Mucuna Conce nt ra Mucuna seed meal meal seed te Oviduct (Control) + ++ +++ Albumen g land ++ _ + Ovo-testis ++ + _ Spermatheca ++ ++ ++ _ Ganglia _ + Foot note:

No visible ex pression - Low expressio n + Medium expression ++ H igh expression +++ relevant in egg production. Report of the three diets. This indicated that Omole et al. (2005) showed that egg receptors for progesterone were average in this organ and is not production in poultry was stimulated influenced by agent(s) in the feed by the use of Mucuna. given. This organ is known to be a The pattern of gene expression storage site for external spermatozoa obtained in this study was similar in since self–fertilize is not common the albumen gland and the ovotestis. among these breeds of snails. In the The ganglia, expression was only recorded by snails fed concentrate plus Mucuna expressions were better in snails fed seed meal. Such expression was not concentrate than those fed mixture of seen in snails fed sole concentrate and Mucuna and concentrate and Mucuna sole Mucuna diets. The reason for this only. This indicated that these two observation was not known, however organs are less stimulated by the this organ seem not to have larger agents present in Mucuna. The role of amount of receptors for progesterone. albumen gland is for albumin Although, it is expected to have deposition, while ovotestis is known similar role to mammalian brain since for both reproductive activities in mollusc are spermatozoa and ova production. It greatly influenced by the nerve centre. may also be possible that some The level of expression seen in antinutritional factors are present in ganglia in this study is in line with the Mucuna which may bind their report of Masaaki et al. (2006) who activity. Although the results in this confirmed presence of receptors for study contradicted the report of steroid in ganglia of marine snail Shukla et al.(2009) who reported that (Thais clavigera) Mucuna increased sperm count and Plate 2 shows the expression of follicle motility in mice. stimulating receptor gene in five parts of In the spermatheca, there was medium the reproductive tract of the giant African level of gene expression by snails fed land snail (Archachatina marginata) 36 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone

Tab le 2: De gre e o f e xp res sio n o f fo llic le s tim ula tin g h orm on e r ece pto r g ene in d ifferen t re pro du ctiv e p art s of A. ma rgin ata sna ils fed di f fer ent di ets / tre atm ent s. under three dietary treatments namely; influenced by the dietary treatments. Plate Concentrate, concentrate + Mucuna seed 2 and Table 2 revealed the expression of meal; 1:1 and Mucuna seed meal. Table 2 follicle stimulating also shows levels of expressions as Parts of the Expression of FSH Receptor Gene Reproductive tract Concentrate Concentrate plus Mucuna meal seed only Mucuna seed meal ;1:1 only Oviduct _ ++ ++

37 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone

Albumen g land ++ ++ ++ Ovo-testis ++ ++ +

Spermatheca _ _ _ _ Ganglia _ _ Foot note: No visible e xpression - Low exp res sion + Mediu m expression ++

hormone receptor gene in the oviduct. Catalan et al., (2002) also reproductive tract of giant African land implicate this organ in provision of egg snail. The results showed that better envelop which further confirms the expressions were seen in the albumen reason for the presence of receptor at gland and the ovo-testis, except for this cite. Influence of Mucuna on this those snails on Mucuna seed meal alone organ showed that substance(s) which which showed low expression. This turn on the secretion of this receptor was a clear indication that these two could be found in Mucuna. Various organs have receptors for FSH and their levels of expression were seen in these level of expressions could be organs, which confirmed the presence influenced by some agent(s) present in of receptor for FSH in them and the feed as well. This observation is in corroborated the earlier report by line with the report of Csaba and Fasoro (2011). None expression seen in Bierbauer (1975) who discover spermatheca and ganglia indicated that spermatogenic activity of FSH been they do not have receptor for FSH. In indicative of presence of receptors for term of functions, both organ do not FSH in Ovo-testis of pomatia . It have anything to do with oval is also not impossible that receptors for production as in the case of ovo-testis FSH are also present in Albumen gland and albumen gland whose primary role as shown in the result of this study. are to produce follicles and add Albumen gland is known to produce albumen to previously released oval and secretes the perivitelline fluid (albumen gland). Therefore absence of which coats fertilized eggs. This role receptors for FSH in both spermotheca was revealed by Egonmwan (2007) in and ganglia is a further confirmation of gross morphology and ultrastructural none participation in oval production. study of this organ. Interestingly, Mucuna also featured prominently in CONCLUSION the influence of expression in the

38 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone It was obvious from this study that Ovotestis of the snail Helix receptors for progesterone were present pomatia. in medium quantity in the albumen Acta Biologica 34(2):269-273 gland, Ovo-testis and spermatheca, Egonmwan, R. I. 2007. Gross while receptors for FSH were found in morphology and ultrastructural medium quantity in both the albumen study of albumen gland of the and the ovo-testis. This indicated that land snail Archachatina these organs could be targeted for marginata ovum (Pfeiffer) manipulation of reproduction using (: Achatinidae). synthetic hormones. Mucuna pruriens was also found to up-regulate some of Pakistan Journal of Biological these organs in various combinations Sciences. 10(2):322-325. while few were down-regulated, hence Fasoro P. M., 2011. Follicle the need for care in its usage to achieve stimulating hormone receptor better reproduction of A. marginata. in the reproductive system of Archachatina maginata. REFERENCES Department of Animal Ahmad, M. K., Mahdi, A. A., Shukla, Physiology, Federal K.K., Islam, N., Jaiswar, S. P. University of Agriculture, 2008. Effect of Mucuna Abeokuta, Ogun State, pruriens on semen profile and Nigeria. Bachelor of biochemical parameters in Agriculture Project. Pp. 55. seminal plasma of infertile men. Gilbert, R. 2002 Mucuna pruriens in Fertil Steril 90: 627–635. doi: Malawi: A promising legume 10.1016/j.fertnstert.2007.07.13 with a troubled history. In: 14 Food and Feed from Mucuna: Current Uses and the Way Catalan, N. M. Y., Fernandez, S. N. and Forward (Editors, Flores B M, Winik, B. C. 2002. Oviductal structure and provision of egg Eilittä M, Myhrman R, Carew envelops in the snail L B and Carsky R J), Workshop, CIDICCO, Pomacea canaliculata CIEPCA and World (, Prosobranchia, Hunger Research Center, ) BIOCELL Tegucigalpa, Honduras 26(1): 91-100 (April 26-29, 2000), 48-59. Clarke, C. L. and Sutherland, R. L. Google Earth, 2006. http:// 1990. Progestin regulation of www.google.earth cellular proliferation. Masaaki, K., Takako, K., Kenichi, K., Endocrine Reviews 11:266- Shingo, T., Hidenori, H., 300. Shigeru, T., Yasuyuki, S., Csaba, G., Bierbauer, J. 1975. Effect Taisen, I.e., Toshie, M., of Mammalian FSH on the Takashi, M., Takashi, M and

39 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone Yu j i , . T. 2 0 0 6 . Ti s s u e properties of the seeds of three preferential expression of Mucuna pruriens species. estrogen receptor gene in the International Journal of Food marine snail, Thais clavigera. Science and Nutrition 52: 7 9 – General and Comparative 8 2 . d o i : Endocrinology Volume 148 (3): 10.1080/09637480020027264 315–326 Shukla, K. K., Mahdi, A. A., Ahmad, Mehta, J. C. and Majumdar, D. N. 1994. M. K., Shankhwar, S. N., Indian Medicinal Plants-V. Rajender, S., . 2009. Mucuna I n d i a n J o u r n a l o f pruriens improves male fertility Pharmacology 6: 92–94. b y i t s a c t i o n o n t h e hypothalamus-pituitarygonadal Nieschlag, E., Simoni, M., Gromoll, J. axis. Fertility and Sterility 92: and Weinbauer, G. F. 1999. 1934–1940. doi: Role of FSH in the regulation of spermatogenesis: Clinical 10.1016/j.fertnstert.2008.09.04 aspect. 5 Edocrinology.,51:139146. Siddhuraju, P. and Becker, K. 2001. Nwaoguikpe, R. N., Braide, W and Preliminary evaluation of Ujowundu, C. O. 2011. The Mucuna seed meal (Mucuna effects of processing on the pruriens var. utilis) in common proximate and phytochemical carp (Cyprinus carpio L.): An compositions of Mucuna assessment by growth pruriense seeds (Velvet Beans). performance and feed Pakistan Journal of Nutrition utilization. Aquaculture 196: 10(10):947-951. 105-123. Omole A. J., Ogungbesan, A. M., Siddhuraju, P., Becker, K. and Fayenuwo, J. A. and Popoola, Makkar, H. P. S. 2000. Studies Y. A. 2005 Comparative on the nutritional composition utilization of Centrosema and antinutritional factors of pubescens or Mucuna pruriens three different germplasm seed as a substitute for pawpaw leaf materials of an underutilized by growing snails. Obafemi tropical legume. Mucuna Awolowo University, Ile Ife, pruriens var. utilis. Journal of Nigeria. Agricultural Food Chemistry Panikkar, K. R, Majella, V.L. and 48: 6048-6060. Pillai, P. M . 1987. Lecithin Singh, A. P., Sarkar, S., Tripathi, M. from Mucuna pruriens. Planta and Med Rajender, S. 2013. Mucuna 53: 503–507. doi: 10.1055/s- pruriens and its major 2006-962789 constituent L-DOPA recover Prakash, D., Niranjan, A., Tewari, S. K. spermatogenic loss by 2001. Some nutritional combating ROS, loss of mitochondrial membrane 40 ABIONA ET AL: Effect of Mucuna pruriens on Gene expression of progesterone and follicle hormone potential and Apoptosis. PL0S ONE 8(1):e54655. Suresh, S. and Prakash, S. (2010) Effect of Mucuna pruriens (Linn.) on Sexual Behavior and Sperm Parameters in StreptozotocinInduced Diabetic Male Rat. Journal of Sex Medicine. Suresh, S., Prithiviraj, E., Prakash, S. 2009. Dose- and timedependent effects of ethanolic extract of Mucuna pruriens Linn. seed on sexual behaviour of normal male rats. Journal of Ethnopharmacology 122: 4 9 7 – 5 0 1 . d o i : 10.1016/j.jep.2009.01.032 Suresh, S., Prithiviraj, E. and Prakash, S. 2010. Effect of Mucuna pruriens on oxidative stress mediated damage in aged rat sperm. International Journal of Andrology 33: 22–32. doi: 1 0 . 1 1 1 1 / j . 1 3 6 5 - 2605.2008.00949 .x

41

Journal of Molluscan Research 1:34-38 (2015)

EVALUATION OF VARYING LEVELS OF SESAME SEED MEAL (Sesamum indicum) IN THE DIET OF GROWING SNAILS (Archachatina marginata)

OMOLE1, A.J., EBENSO2 I.E., ADETOLA3,O.O., ADEKUNLE3,O.F., ONONOGBU3, C. AND OWOSIBO1,O.T.

1Obafemi Awolowo University, Institute Of Agricultural Research and Training Moor Plantation, Ibadan, Nigeria 2Dept. of Animal Science, University of Uyo, Nigeria 3Federal College of Animal Health and Production Technology, Ibadan, Nigeria.

ABSTRACT This study was conducted to determine the effect of feeding snail with sesame seed meal as replacement for groundnut cake meal fraction of the diet on performance characteristics, carcass analysis and cost benefits. A total of one hundred and twenty snails of mean weight 175±2.7g were randomly allotted into four dietary treatments replicated three times and each replicate contained ten snails per replicate in a completely randomized design. Four diets were formulated to contain SSM at 0% (SSM1) Control, 50% (SSM2), 75% (SSM 3), 100% (SSM 4) as replacement for groundnut cake meal fraction in the diet of growing snails. The diets were formulated to contain about 24% crude protein and energy of 2400 kcal/kgME. Feed intake and weight gain were measured on daily and weekly basis with the use of sensitive weighing balance. Feed intake and weight gain was calculated. Shell length, thickness and width were measured Feed conversion ratio were calculated as the ratio of feed intake to weight gain. Feed cost and cost per weight gain were also calculated. The results shows that the mean total feed intake was significantly influenced by the varying inclusion of SSM in the diet (P<0.05) The highest feed intake of 709.69g was recorded in SSM 4 while the lowest feed intake of 690.61g was recorded in the control (SSM 4). The mean total weight gain followed the same pattern with the feed intake. The highest mean weight gain (P<0.05) of 176.98g was recorded in SSM 4. The feed conversion ratio (FCR) which is the ratio of feed intake to weight gain was relatively the same in all the treatments. The lowest cost per weight gain (CPW) of N170.61 was recorded in the diet containing high level of SSM in the diet while the highest CPW of N221.88 was recorded in the control diet containing 0% SSM.

Keywords: Sesame seed meal, Growing snails (Archachatina marginata) 42

INTRODUCTION protein in Africa and Nigeria in Beef, pork, mutton and poultry meat particular. Recently, there is increase in have been the major source of animal production of snail and grass-cutter as OMOLE ET AL: Varying levels of sesame seed meal (Sesamum indicum) in the diet of growing snails alternative sources of animal protein groundnut cake meal fraction of the (Omole, et.al., 2012; Hamzat, and diet on performance characteristics, Longe 2014; Odeyinka, 2014;. carcass analysis and cost benefits. Omoyinmi,et al., 2014). Snail meat MATERIALS AND METHODS contains low fat and low cholesterol level Site of the experiment which makes the meat a good antidote for The experiment was carried out at the fat related diseases. The cost of Snailery Unit of the Institute of production in terms of housing and feed Agricultural Research and Training is relatively low compared to other (I.A.R.& T.), Moor Plantation, Ibadan. conventional livestock (Hamzat and The cage was demarcated into 12 Longe 2014; Odeyinka, 2014). One of the compartments and each compartment major limitations to the efficient snail had dimension of 0.4x 0.4 x 0.5m3. The rearing is the availability of quality feed bottom of the cages were perforated to at affordable price Omole, et.al., 2012. allow free drainage of water when The protein feed stuff mainly used in wetting the soil. The top was built of livestock production in Nigeria are mosquito netting reinforced with wire groundnut cake, soybean meal and fish mesh. The stands of each cage were put meal etc. (Omole, et.,al.,2012; Odeyinka, inside a container filled with used 2014). The high cost of groundnut cake engine oil to prevent soldier ant meal as a source of animal protein infestation. Sandy loam soil was necessitate looking for alternative source collected from the Southern Farm of of animal protein which is affordable and the Institute and used as bedding for the available. Sesame seed meal is nutritious snails inside the cage. and contain high protein level of about 46% crude protein. Sesame seed meal has Experimental design been used in the diet of broilers and layers A total of one hundred and twenty with impressive results on growth snails (Achachatina marginata) of performance and carcass yield (Farran, et. mean weight 175±2.7g were randomly al., 2000, AlHarthi and allotted into four dietary treatments replicated three times and each D e e k , 2 0 0 9 ; A g b u l u e t replicate contained ten snails per al.,2010).Groundnut cake meal is replicate in a completely randomized expensive than SSM, the prevailing cost design. The snails were acclimatized of GNC/kg. is N110 while the cost of for one week before the SSM/kg is N85 during the course of the commencement of the feeding trial. experiment. There is paucity of Four diets were formulated to contain information in the use of SSM in the diet SSM at 0% (SSM1 ) Control, 50% of snails hence this study was conducted (SSM2), 75% (SSM 3), 100% (SSM 4) as to determine the effect of feeding snail replacement for soybean meal fraction with sesame seed meal as replacement for 43

OMOLE ET AL: Varying levels of sesame seed meal ( Sesamum indicum ) in the diet of growing snails

Table 1: Gros s Com position of Ex perim ental Diet .

Ingredient (% SSM 1 (0% SSM 2 (50% SSM 3 (75% SSM 4 (100% ) SSM ) SMM) SSM) SSM)

e Maiz 22.00 22.00 22 22

Groundnut cake meal 24 12 6 0.00

Sesame seed meal 0.00 12 16.5 22.00 54.0 *Others 54.0 54.0 54.0

T otal 100.0 100.0 100.0 100.0 Cost/kg feed (N /kg) 54.67 50.89 46.67 43.25 Calcu lated anal ysis Crude protein (%) 23.63 24.02 24.32 24.47 Metabolizable energy 2438.1 2411.6 2401.3 2383.3

(kcal/KgME)

*Other fixed ingredients : B.D.G.- 12.8, Rice - 15, Fish meal-4, Soy bean meal-10, Bone in the diet of growing snails. The diets weekly basis. Feed conversion ratio were formulated to contain about 24% were calculated as the ratio of feed crude protein and energy of 2400 intake to weight gain. Feed cost and kcal/kgME (Table 1). cost per weight gain (total feed cost Collection of data divided by total weight gain) were also Feed intake and weight gain were calculated. measured on daily and weekly basis with Chemical and data analyses the use of sensitive weighing All data were subjected to statistical

meal- 2.15,Oyster shell-9.8, Premix-0.25 analysis using analysis of variance and SSM- Sesame seed meal balance the means were separated if they are respectively. Feed intake was calculated significantly different using Duncan by subtracting the left-over feed from the Multiple Range Test (SAS, 1999). feed given while the weight gain was calculated by RESULTS AND DISCUSSION deducting the initial weight from the final The results of growth performance of weight. Shell length and width were the snails fed varying levels of SSM in measured on weekly basis with vernier the diet are shown in Table 2.The mean caliper. Micrometer screw gauge was total feed intake was significantly used to measure the shell thickness on influenced by the varying inclusion of

44

SSM in the diet (P<0.05). The highest pattern with the feed intake. The feed intake of 709.69g was recorded in highest mean weight gain (P<0.05) of SSM 4 while the lowest feed intake of 176.98g was recorded in SSM 4.The 690.61g was recorded in the control feed conversion ratio (FCR) which is (SSM 4). The increased feed intake from the ratio of feed intake to weight gain SSM1 to SSM4 could be due to was relatively the same in all the differences in protein level in the diet. treatments. The relatively the same Snails tend to eat more when the protein FCR reported in the study is an level of the diet increases (Omole, et.al., indication that SSM could be used to 2012; Hamzat, and Longe 2014). The replace GNC in the diet of growing mean total weight gain followed the same snails. The FCR reported in this study pattern with the feed intake. The highest was in agreement with observation of mean weight gain was (P<0.05) was several authors who reported FCR recorded in the diet containing 100% ranging between 3.78 and 4.21 (Omole SSM as replacement for et. al., 2007; Hamzat and Longe et.al., groundnut cake meal (GNC). The mean 2014;). There was no total weight gain followed the same

OMOLE ET AL: Varying levels of sesame seed meal ( Sesamum indicum) in the diet of growing snails

Table 2: Summary of Growth performance of growing snails fed varying levels of SSM in the diet . Parameters (Means) SSM1 (0% SSM2 SSM3 SSM4 ± SEM

SSM ) (50% (75% SMM) SSM ) (100% SSM ) Initial weight (g) 5.89 176.23 175.38 177.12 175.12

45

Final weight (g) 344.67 b 345.69 b 349.33 351.5 a 6.45

ab otal weight gain (g) b ab T 168.44 170.31 172.21 176.98 5.39 ab a Total feed intake (g) 690.61 c 699.97 b 706.06 a 709.69 a 8.72

4.10 4.10 0.14 Feed conversion ratio 4.11 4.01 Shell length increment (mm ) 9.78 9.78 9.79 9.81 1.02

Shell width increment (m 8.34 8.35 8.35 8.38 0.49 m) Shell thickness increment (mm) 0.11 0.11 0.13 0.13 0.01 Mortality (Number) 0.00 2 1 0 Means along rows with different superscript are significantly different from each other (P<0.05) SSM- Sesame seed meal significant difference in the mean shell snails. (Farran, et. al., 2000;Agbulu et length and values ranged between 9.78 al.,2010). The report of zero mortality and 9.79mm as shown in Table 3. Also was an indication that sesame seed meal the mean shell width thickness also could be used as feed resource for snail. were not significantly influenced by the The result of cost analysis is shown in dietary treatments (P>0.05). This Table 3, the cost/kg feed reduced from observation also buttressed the fact that N54.67 in SSM1, to N43.25 in SSM4, SSM could be used as replacement for also the total feed cost reduced as the GNC. Also there was no case of shell level of SSM in the diet increased from deformity in all the treatments. The zero 0% in SSM1 to 100% in SSM4. The mortality recorded could be due to lowest cost per weight gain (CPW) of proper management practice such N170.61 was recorded in the diet cleaning of the environment, regular containing high level of SSM in the diet washing of feeding and water troughs, while the highest CPW of N221.88 was proper shading and others as recorded in the control diet containing recommended by different authors 0% SSM. The lowest cost/kg feed, total (Omole et al., 2007; Hamzat and Longe feed cost and cost per weight gain et.al., 2014). The zero mortality reported in this study could due to high reported could also be due to fact that price of GNC compared to SSM. The the test ingredient used in this study was prevailing market price of GNC during well processed and preserved and did course of the feeding trials was N110 not have any deleterious effect on the while the

Table 3: Cost analysis of growing snails fed varying levels of SSM in the diet. Parameters (Means) SSM1 SSM2 SSM3 SSM4 ± SEM (75% (100% (0% (50% SSM) SSM) SSM) SMM) 46

Total weight gain (kg) 0.17 0.17 - 0.17 0.18 Total feed intake (kg) 0.69 0.70 0.71 0.71 - Cost/kg feed (N) 54.67 a 50.89 b 46.67 c 43.25 d 2.02 a ab b Total feed cost (N/kg) 37.72 35.64 ab 33.13 30.71 2.12

221.88 a 209.65 b Cost/weight gain (N/kg) 194.82c 170.61 d 4,78 SSM- Sesame seed meal OMOLE ET AL: Varying levels of sesame seed meal (Sesamum indicum) in the diet of growing snails cost of SSM was N85. The reduced diets with phytase and probiotic cost reported in this study was in supplementation. Egypt. Poult. agreement with several authors that Sci. J . Vol., 29 No. 1 pp. 99- replaced conventional feed stuffs, 125 such as maize, soya bean meal, Association of Official Analytical groundnut cake meal with Chemist, A.O.A.C. (1990). unconventional ones such as noodle Official Methods of Analysis, waste meal, chicken offal meal, kenaf 13th Edition, Washington, D. grain meal, kola testa meal e.t.c ( C. 149pgs Odeyinka, 2014). The lowest cost/ weight gain reported is an indication Dan, B., 2005. Sesame Profile. http, that sesame seed meal could be used www.Agmrc. org. to replace groundnut cake at reduced Farran.M.T.G.,Uwayjan, A.M., cost. Ashkarian, V.M., 2000. Performance of broilers and CONCLUSION AND layers fed graded level of RECOMMENDATION sesame hull. J. Appl. Poul. Res., 9, 453-459. It could be concluded that sesame seed meal could be used as replacement for Hamzat, R. A. and O. G., Longe (2014) groundnut cake meal in the diet of Performance characteristics and growing snails. economics analysis of snailets (Achahatina marginata) fed varying dietary inclusion of Kola REFERENCES Testa. Proceedings of the 3rd Agbulu, O., Gyau, A.M., Abakura, International Conference on J.B., 2010. Effect of the Giant African Land Snails held Replacement of Sesame Seed at Federal College of for Methionine in Broiler Education Osiele, Abeokuta on Production in Middle Belt 1st 4th June, 2014. Pp.87-90 Region , Nigeria. J. Emerg. Odeyinka S. M., (2014) Animal feed Trends Educat. Res. Pol. Stud., industries and snail farming (1), 16-21. lecture delivered at the 3rd Al Harthi, M.A. and El Deek, A.A., International Conference on 2009. Evaluation of sesame Giant Land Snail (GALS) meal replacement in broiler

47

Proceedings of the 3 r d Omoyinmi, G.A.K., A. B., Gbadegesin, International Conference on T. T., Bello, and FAO Akinnusi Giant African Land Snails held (2014) Utilization of Agrowaste at Federal College of in the culturing of Garden snail Education Osiele, Abeokuta on (Limcolaria auroria). r d 1st 4th June, 2014. Pp.vii-xxx Proceedings of the 3 International Conference on O m o l e , A . J . , O s u n k e y e , O . J Giant African Land Snails held . , at Federal College of Ojejide,J.O., Sodamola,M.O., Education Osiele, Abeokuta on and Poopola,Y.A.(2012).Back 1st 4th June, 2014. Pp 114-116 To Agric.Series (10).The African Giant Land Snails. S.A.S. (1999). S.A.S. User's Guide. Greenchoice publications Statistical Analysis System 76 pgs Institute, Inc.Cary, N.C. 78 pgs

Journal of Molluscan Research 1:39-49 (2015)

QUANTITATIVE AND QUALITATIVE EVALUATION OF THE HAEMOCYTES IN THE HAEMOLYMPH OF Achatina achatina AND Archachatina marginata

SODIPE1, O.G., OSINOWO1, O. A., ONAGBESAN1, O. M. AND

BANKOLE 2, O. M. 1Department of Animal Physiology, 2Department of Microbiological Sciences, Federal University of Agriculture, Abeokuta. P.M.B. 2240, Abeokuta, Nigeria. Email: [email protected]

ABSTRACT The haemocyte content of the haemolymph of plays key roles in both cellular and humoral immune reactions by phagocytosis or delivering immune factors such as lectin and antimicrobial peptides. There is no literature on the haemacytes content in the haemolymph of the Giant African Land Snails hence the need to investigate the haemacytes content in the haemolymph of the Giant African Land Snails. An experiment was conducted to examine haemocytes composition of the haemolymph of two Giant African Land Snails, A. marginata and A. achatina. Haemolymph from ten (10) active 48

mature snails for each species was collected to evaluate the concentration and type of haemocytes in the haemolymph. The haemocytes concentrations of both species were determined using inverted phase contrast microscope and haemocytometer. The experiment was a 2 x 2 factorial arrangement, in a completely randomized design. The results of this study showed that the haemocytes concentration in haemolymph of A. achatina (8.16 x 106 ± 1.465 x 106 per ml) was significantly higher (P<0.001) than that of the A. marginata (4.58 x 106 ± 1.465 x 106 per ml). Further, the haemocytes concentration in the mantle cavity haemolymph (8.11 x 106 ± 1.465 x 106 per ml) was significantly higher (P<0.001) than that of the foot sinuses haemolymph (4.64 x 106 ± 1.465 x 106 per ml). The haemocytes concentration in the A. achatina haemolymph (8.16 x 106 ± 1.465 x 106 per ml) was significantly higher (P<0.001) than that of the A. marginata haemolymph (4.58 x 106 ± 1.465 x 106 per ml). The morphology of the haemocytes was similar in both the A. achatina and A. marginata, with the presence of spiny processes on the cell membranes which gave a hint that there might be phagocytosis in progress. These results of haemocytes concentrations indicated that the two species of snail were not similar. Contrarily, the results of haemocytes morphology showed similarity between the two species of snails.

Key words: Haemolymph, haemocyte, haemocytes count and type, foot sinuses, mantle cavity.

49 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata INTRODUCTION (Cheng, 1984; Cheng and Bayne, 1995; Snails are able to survive various Mount et al., 2004). The knowledge of microbial by a special the types and functions of haemocytes mechanism in the wild. Sufficient in molluscs under natural environment evidence have been accumulated to is essentially helpful in studying basic indicate that the phagocytic cells of cell responses of the molluscs (Burge et many invertebrates recognize foreign al., 2002; Pamparinin et al., 2003; materials by a system analogous to that Cannesi et al., 2002). observed in the vertebrates, i.e. by the Studies on some haemolymph interaction of specific humoral proteins biochemical parameters of Giant with foreign materials (Schluter et al., African Land Snail (A. marginata) 1980). have been reported (Afolayan et al., Haemolymph is the blood analogue 1997; found in all and most Akinloye and Olorode, 2000; molluscs have open circulatory system. Ogunsanmi et al., 2003; Ademolu and It is composed of water, inorganic salts, Idowu, 2005; Abdussamad, 2009). mostly Na+, Cl-, Mg2+ and organic Comprehensive cellular components of compounds, mostly , the Giant African Land Snails are proteins and lipids. Muscular scarcely reported in literature. movement by the animal during Considerable interest had been in the locomotion can facilitate haemolymph nutrition, growth, stocking density, movement, but diverting flow from one housing systems, reproduction, effect area to another is limited (Alexander of and photoperiod, and Gary, 2012). The haemolymph is biophysical and biochemical an important medium for the transport parameters of the haemolymph of Giant of nutrients to, and waste from various African Land Snails (GALS). The organs of the snails. types of haemocytes in the GALS have Haemolymph also contains cells called not been studied. Most studies have haemocytes. The haemocyte content of been carried out on the bivalves the haemolymph of invertebrates plays (Cheng, 1984), while relative work has key roles in both cellular and humoral been carried out on the gastropods immune reactions by phagocytosis or (Malham et al., 2003). This study was delivering immune factors such as therefore aimed at examining the lectin and antimicrobial peptides. haemocyte contents of the GALS Bacterial causes changes in haemolymph from the mantle cavity components such as lectin and and the foot sinues. antibacterial peptides and lysosomal enzymes of plasma or haemolymph in MATERIALS AND METHODS molluscs (Kang et al., 2006). The experiment was carried out at the Haemocytes are important component Snail Research Unit and the involved in the crucial physiological Department Animal Physiology processes which includes the nutrient Laboratory of the distribution and excretion, wound College of Animal Science and repair, shell repair and phagocytosis Livestock Production, University of 50 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata Agriculture, Abeokuta, Nigeria. The results of the Least Square means Abeokuta lies within the rain forest showing the effect of species and vegetation zone of Western Nigeria on haemolymph type on haemocyte latitude 70 10' N and longitude 30 2' E, concentration per ml are presented in and 76 m above sea level. The climate Tables 1 and 2. The haemocyte count is humid with a mean annual rainfall of per ml of haemolymph of A. achatina 1,037 mm, average temperature of 34.7 (8.16 x 106 ± 1.465 x 106 per ml) was 0C and average relative humidity of 82 significantly higher (P<0.001) than that % (60 % in January and 94 % from July of the A. marginata (4.58 x 106 ± 1.465 to x 106 per ml) (Table 2). There was also September) (Google earth, 2010). The a highly significant (P<0.001) experiment was a 2 x 2 factorial difference in haemocyte count per ml arrangement, including the type of between haemolymph types (Table 1). haemolymph (Mantle cavity vs. Foot The haemocytes concentration per ml sinuses haemolymph) and Species (A. haemolymph of mantle cavity marginata and A. achatina). haemolymph (8.11 x 106 ± 1.465 x 106 Haemolymph of thirty (30) active per ml) was significantly higher mature and apparently healthy snails (P<0.001) than that of the foot sinuses for each species were reared for two haemolymph (4.64 x 106 ± 1.465 x 106 months weighing between 150g and per ml). There was no significant 200g. They were fed with fresh (P>0.05) interaction between species pawpaw leaves and unripe pawpaw and haemolymph type effect on fruits. From each sample, 10 µl was haemocyte concentration per ml. taken by micropipette (under gentle Photomicrographs of representative mixing for homogeneous haemocyte haemocytes found in the haemolymph suspension) and total cell counts were determined in a Neubauer counting chamber. Snail haemolymph were placed on 2.2 x 2.2 inches slides and covered with a glass cover slip and examined under Observer A1. Axio, a Zeiss inverted microscope. Different types of haemocytes were differentiated and counted. Statistical analysis Data collected were subjected to analysis of variance (ANOVA) procedure in 2 x 2 factorial (species x haemolymph type) design in 3 replicates using the Systat Analytical Computer Package version 5.02 (Systat, 1993).

RESULTS AND DISCUSSION 51 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata

Table 1. Comparative effect of haemolymph type on haemocyte count/ ml of haemolymph of giant African land snails Variables Haemoly m ph Least Square means ± SE Ty pe H aemocyte count/ ml haemolymph Mantle cavity 6 6a 8.11 x 10 ± 1.465 x 10 Foot sinues 4.64 x 1014 ± 1.465 x 10 6b

abMeans with different superscripts within the same column are significan tly different (P<0.001)

Table 2. Comparative effect of species on haemocyte count/ ml of haemolymp h of Giant African land snails

Variables Species Least Square means ± SE 6 6b Haemocyte count/ ml haemolymph A. marginata 4.58 x 10 ± 1.465 x 10

A. ac hatina 8.16 x 10 6 ± 1.465 x 106a ab Means with different superscripts within the same column ar e sig nificantly different (P<0.001)

52 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata of A. achatina and A. marginata are phagocytosis of foreign materials shown in Plate 1. The haemocytes are might be in progress as shown in Plate similar in both in A. achatina and A. 2. This is in agreement with results marginata. They are either large or obtained by Cannsei et al., 2002 who small with a relatively larger nucleus. explained that haemolymph possess The presence of spiny processes on the materials that facilitate phagocytosis. haemocytes with spiny process are the p h o t o m i c r o g r a p h s u g g e s t i n g t h a t most common type of haemocyte in

1 a 1 d

1 b 1 e

1 c 1 f cell membrane was observed in some The Plate 1. Photomicrograph of the types of the haemocyte in t he haemolymph of A. marginata (1a

53 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata

- c) and A. achatina (1d – f).

Plate 2. Photomicrograph of the haemocytes in the haemolymph showing evident of phagocytosis CONCLUSION AND RECOMMENDATION both species, they might be found to In conclusion, our results indicated be immunologically active. From this that haemocytes of different types study, it was also observed that the were present in the haemolymph of haemolymph from the GALS does not Giant African Land Snails. Further clot. research on snail haemocytes It was observed that A. achatina and morphological differentiation and A. marginata have morphologically immunological functions using and functionally specialized Scanning Electron microscope could haemocytes. offer novel physiological and This may be associated with economic benefits. immunological functions which we cannot explain in this study. REFERENCES

54 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata Abdussamad, A. M. 2009. Effect of gigas. Journal of duration of aestivation on Research 22:601. haemolymph biochemical and Burton, R. F. 1965. Na, K, Mg, in the biophysical parameters of two l i blood of snail . v e w e i g h t g r o u p s o f Physiological Zoology 38: 335 Archachatina marginata – 342 [cited by Langley, C.K. Swainson, 1821. Masters 1979]. Thermal acclimation of a Thesis, Department of Animal central neurone of Helix aspera. Physiology, University of I. Effect of temperature on Agriculture, Abeokuta. 216 pp electrolyte level in the Ademolu, A. O. and Idowu, A. B. 2005. haemolymph. Journal of The use of poultry dropping as snail Experimental Biology 78: 181 - feed. Journal of Animal 186 and Veterinary Advances, 4(6): Cannsei, L.; Gallo, G.; Gavioli, M. and 566 – 569. Pruzzo, C. 2002. Afolayan, A., Agboola, F. K and Bacteriahaemocyte interactions Akinloye, A. O. 1997. Some and phagocytosis in marine properties of beta – glucosidase bivalves. Microscopy Research from the haemolymph of the and Techiniques 57: 469- 476. giant African land snail Cheng, T. C. 1984. A classification s c (Archachatina marginata). h e m e o f m o l l u s c a n h a e Nigeria Journal of Biochemical m o c y t e s b a s e d o n f u n c t and Molecular Biology, 12: 43 – i o n a l e v i d e n c e s . 51. Comparative pathology Vol. 6: Akinloye, A. O. and Olorode, B. R. 111-146. 2000. Effect of different Cheng, J. H. and Bayne, C. L., 1995. conditions on performance and Bivalves mollusk haemocytes haemolymph biochemical and behaviors: characterization of mineral values of Giant African haemocyte aggregation and S n a i l ( A r c h a c h a t i n a adhesion and their inhibition in marginata). Journal of the California (Mytilus californianus). Biological Agriculture and Environment, Bulletin 188:255-266. 1(1): 143 – 147. Alexander, F. and Gary, F. (2012). Goggle Earth, (2010). http:// Plasma Medicine. John Wiley www.google.earth and Sons pp 592. Kang, Y. S.; Kim, Y. M.; Park, K. I.; Choi, K. S. and Cho, M. 2006. Burge, C. A., Saito, Y. E., and Analysis of EST and lectin Friedman, C. S. 2002. expressions in hemocytes of Relationships between summer Manila (Ruditapes mortality and immune responses philippinarum) (: in the ) infected with P e r k i n s u s O l s e n i . 55 SODIPE ET AL: Evaluation of the haemocytes in the haemolymph of Arhatina achatina and Archachatina marginata D e v e l o p m e n t a l Mount A. S.; Wheeler, A. P.; Paradkar, Complementary Immunology, R. P. and Snider, D. 2004. 30(12): 1119 –1131. Malham, Hemocyte-mediated shell S.; Lacoste, A.; Gelebart, F.; mineralization in the eastern Cueff, A. and Poulet, S.(2003). Oyster. Science 304:297-300. Evidence for a direct link Ogunsanmi, A. O.; Taiwo, V. O. and between stress and immunity in t Akintomide, T.O. 2003. h e m o l l u s c H a l i o t i s Haemolymph biochemical tuberculata. J Exp Zool parameters of African Giant 295A:136e44 Snail (Achatina) and the big Schluter, S. F.; Deindre, P. L. E. Y.; black snail (Archachatina Keough, R. and Jenkin, C. R. m a r g i n a t a ) . Tr o p i c a l 1980. Identification of two Veterinarian, 21(2):43-48 c a r b o h y d r a t e - s p e c i f i c Pamparinin D. M.; Marin, M. G. and erythrocyte agglutinins in the B a l l a r i n , L . 2 0 0 2 . h a e m o l y m p h o f t h e M o r p h o l o g i c a l a n d Protochordate Botrylloides cytoenzymatic characterization leachii. Immunology. 42: 241 – of haemocytes of the venus 250. . Chamelea gallina. Systat, 1993. Systat Computer Diseases of Aquatic Organisms Package, version 5.02 Systat, 49:227-234. Inc. 1800 Sherman Ave., Evanston IL USA. 6021, 708, 864, 5670.

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Journal of Molluscan Research 1:46-51 (2015)

CONCENTRATIONS OF STEROIDS IN THE GIANT AFRICAN LAND SNAIL (Archahatina marginata) AT DIFFERENT REPRODUCTIVE PHASES

*BAMIDELE, J.A., IDOWU, A.B., ADEMOLU, K.O., AGBAJE, A.A. AND AJAYI, O.A

Department of Pure and Applied Zoology, Federal University of Agriculture, Abeokuta. P.M.B 2240, Abeokuta, Nigeria *Corresponding author: [email protected]

ABSTRACT Steroid hormones play important roles in snail reproduction, however their roles have not been properly understood in the Giant African Land Snails. Progesterone and oestrogen concentrations in the Giant African Land Snail (Archahatina marginata) were therefore investigated at different reproductive stages. Twenty (20) matured giant African land Snails obtained and divided into two groups, A (ten snails placed singly in plastic baskets for pre-mating stage) and B (ten snails paired into plastic baskets for mating and post-laying stages). Samples of the ovo-testis, albumen gland and haemolymph were obtained from pre-mating, mating and post-laying snails and analysed for oestrogen and progesterone levels using standard methods. Concentration of progesterone was higher in the haemolymph of pre-mating, mating and post- laying snails than in the albumen gland and the ovo-testis. Progesterone increased in the albumen gland and haemolymph from the pre-mating (albumen gland – 0.363, haemolymph – 0.697) to the mating snails (albumen gland – 0.469, haemolymph – 0.779). Oestrogen was higher in the ovo-testis than all the reproductive organs tested in all the snails' reproductive stages. Oestrogen increased in the albumen gland, ovo-testis and the haemolymph from the pre-mating to the post-laying stage. Steroid hormones, most especially progesterone and oestrogen are therefore responsible for the reproductive cycle of the giant African land snails, just like the vertebrates.

57 BAMIDELE ET AL: Steroids concentration in the Giant African Land snail at different reproductive phases

Keywords: Steroids, Progesterone, Oestrogen, Archahatina marginata, Reproductive stages. INTRODUCTION West Africa (Akinnusi, 2004). They The giant African land snail are hermaphrodites and therefore have (Archahatina marginata) belongs to the both male and female reproductive Class Gastropoda, a member of the organs and develop both eggs and Phylum Mollusca. Land snails are air sperm (Idowu and Akinnusi, 2006). breathing snails and are found mostly in The hermaphroditic duct from the ovo- testis coils to a junction between a large reproductive cycle, there has been no albumen gland and a small seminal information on the steroids receptacle where the ovum awaits concentrations of the giant African land fertilization which occurs at the mouth snail (Archahatina marginata) at of the receptacle by sperm from another different reproductive phases. snail, then the ovum receives a coat of Knowledge of their presence and albumen from the albumen gland. The pattern in various organs during the albumen gland is an elongated, tongue reproductive cycle will aid the – like shaped organ that contains the understanding of their biology and chamber where the ova are provide clues as to their reproductive fertilised (Odaibo, 1997). abilities. Sex steroids have significant physiological roles in reproduction in MATERIALS AND METHODS invertebrates as they have long been Experimental Procedures known to have in vertebrates (Dixson, Twenty (20) matured Giant African 2011). The biosynthesis and Land Snails were purchased from Itoku metabolism of steroid hormones were market in Abeokuta, Ogun State, reported to occur in the ovo-testis of Nigeria. The snails were divided into snails (Carreau, 1997). Vertebrate-like two groups, A (ten snails placed singly sex steroids such as progesterone, in plastic cages for pre-mating stage) oestrogen and testosterone have been and B (ten snails paired into plastic reported in Achatina fulica (Bose et al., cages for mating and post-laying 1997), vulgans and stages). Plastic baskets (0.5m x 0.5m x 0.2m) were used as cages and were Biomphalaria alexandria (Maha et al., prefilled with loamy soils. The 2009). experiment was carried out during the Although it was strongly suggested that raining season of the year (April-June) steroids are involved in control of and was set up in the snailery unit of the mollusc reproduction as well as in Department of Biological Sciences, sexual maturation and during the

58 BAMIDELE ET AL: Steroids concentration in the Giant African Land snail at different reproductive phases College of Natural Sciences, Federal samples were then centrifuged and the University of Agriculture, Abeokuta, supernatant frozen for further analyses. Nigeria. Laboratory Analysis Sample Preparation Prepared samples of the albumen gland, The snails were classified into ovotestis and haemolymph from all the premating (non-mating), mating and four snail samples were analysed for postlaying stages based on their oestrogen and progesterone according reproductive stages. The snails were to the method described by A.O.A.C. dissected according to the method (2000). described by Ademolu et al. (2006) and the albumen gland, ovo-testis and RESULTS haemolymph of each of the Progesterone level was significantly reproductive stages were collected. higher (p < 0.05) in the haemolymph of 0.7g of the albumen gland and the pre-mating, mating and post-laying ovotestis samples were cut and snails than in the albumen gland and the homogenized in a ceramic mortar with ovo-testis (figure 1). Progesterone pestle. The haemolymph was collected concentration recorded an increased by breaking the shell at the apex concentration in the albumen and (Akinloye and Olorode, 2000). The haemolymph from the pre-mating

Figure 1: Progesterone concentrations in the tissues of snails during different reproductive stages

59 Figure 2: Oestrogen concentrations in the tissues of snails during different reproductive stages BAMIDELE ET AL: Steroids concentration in the Giant African Land snail at different reproductive phases (albumen gland – 0.363, haemolymph – 0.697) to the mating stage (albumen gland – 0.469, haemolymph – 0.779). This value increased in the albumen gland but decrease in the haemolymph of the post-laying snails. Concentration of progesterone decreased in the ovotestis from the pre-mating (0.473) to the mating stage (0.279) and up in the post-laying snails (0.298). Oestrogen level was higher in the ovotestis (0.658 – 0.832) than all the reproductive organs of the Giant African land snails tested (0.383 – 0.644) (Figure 2). There was an increased level of oestrogen in the albumen gland, ovotestis and haemolymph from the pre-mating to the mating stage. The values recorded for progesterone increased in the albumen gland and the haemolymph of mating to post-laying snails but dropped in ovotestis of the post-laying snails.

60 BAMIDELE ET AL: Steroids concentration in the Giant African Land snail at different reproductive phases DISCUSSIONS not only the willingness and eagerness This study shows that steroid hormones of an oestrous female to male but also are present in the tissues of giant her ability to mate. Receptivity, African land snails like those of the proceptivity and attractiveness of vertebrates. This could be an indication female mammals were reported to be that, like the other invertebrates and primarily due to estradiol and vertebrates, giant African land snails progesterone (Neil, 2004). The increase also requires the role of steroid in progesterone and oestrogen level hormones for reproduction since during the mating stage of the steroid hormones has been associated experimental snails could therefore be with reproduction (Dixson, 2011). attributed to their sexual activeness as Steroid hormones, especially well as the active role of progesterone progesterone and oestrogen were and oestrogen in the facilitation of present in different levels in the gamete release from the male and albumin gland, ovotestis and female gonads (Wang and Croll, 2003). haemolymph of A. marginata at the As reported by Cosmo et al. (2001), pre-mating, mating and post-laying progesterone levels fluctuate according stages. A low level of both to the reproductive cycle in molluscs, progesterone and oestrogen was being very low during the observed in the albumen gland during nonvitellogenic period, and increasing the pre-mating stage. This is not at the onset of vitellogenesis. The unexpected, since the snails were not ovotestis is the hermaphroditic organ of paired, hence they were not sexually the gastropods, containing both an excited. Maha (2009) also reported that ovary and a testis while the albumen steroid hormones in the snail are gland is the organ opening into the present in very low level during the hermaphroditic duct and secreting a juvenile stage (non-mating) while they viscous substance which surrounds the are abundantly present in adult stage. fertilized ovum and contributes to the There was an increase in progesterone development of the egg. Increase in and oestrogen level during the mating progesterone concentration in the stage. The roles played by these ovotestis and albumin gland of the hormones in the reproductive stages experimental snails from the mating to cannot be underestimated. Increase in the post-laying period could therefore progesterone level has been reported in be linked with their role in the sexually active animals. Neil (2004) vitellogenesis and egg production. described progesterone as a steroid Oestrogen concentration was higher in hormone that maintains the endometrial the ovotestis than in the albumin gland lining of the uterus during the later part and haemolymph of the snails at the of menstrual cycle and during three reproductive stages observed. pregnancy and also promotes Higher oestrogen level in the ovotestis receptivity in female mammals with could be linked to the haemaphroditic oestrous cycle. Wallen (1990) also role it plays in the snails' reproductive pointed out that ovarian hormones system where it acts as both an ovary (progesterone and estradiol) controls and a testis. It could also be connected 61 BAMIDELE ET AL: Steroids concentration in the Giant African Land snail at different reproductive phases with the submission of Awosanya et al. Washington, D.C 1141 pp. (2013) who reported that out of the Ademolu, K.O., Idowu, A.B. and albumen gland, oviduct, ovotestis and Agbelusi, O.M. (2006). Effect spermotheca parts extracted from of stocking density on the Archachatina marginata, expression growth and haemolymph for oestrogen receptor mRNA was only biochemical value of African present in ovotestis. giant land snail (Archachatina Progesterone level was higher in the m a rg i n a t a ) . Tro p i c a l haemolymph than in the albumin Veterinarian 24 (1&2): 6-10 gland and ovotestis of the Akinloye, O.A. and Olorode, O. (2000). experimental snails. A. marginata as Effect of different f e e d i n g c an animal possesses an open o n d i t i o n o n performance, circulatory system (South, 1992; haemolymph biochemical and Ademolu et al., 2006). Akinloye and mineral value of Giant African Olorode (2000) submitted that the Snail (Archachatina haemolymph is the fluid that bathes marginata). Journal of the flesh of snails and any Agriculture and Environment physiological process taking place in 1:143-147. the body of the snail must be reflected Akinnusi, O. (2004). Introduction to by the snail and Snail farming. haemolymph. Thus, it is not Abeokuta: Triolas Ventures. Pp unexpected to have high 1-35 concentration of steroids in the haemolymph since steroids are Awosanya, A. A., Abiona, J. A., chemical messengers and secretions Osinowo, O. A., Ladokun, A. from ductless gland are released into O., Abioja, M. O, Daramola, J. the haemolymph to be taken to all O and Onagbesan, O. M. (2013) target cells (Odiete, 1999). Determination of estrogen receptor gene expression in the CONCLUSION reproductive system of Archachatina marginata. This study has shown that the giant Proceedings of the 2nd African land snail, Archachatina marginata, like its other vertebrate I n t e r n a t i o n a l and counterparts Conference/Workshop on Giant possesses steroid hormones which are African Land Snail (NetGALS) involved with their reproductive 2 - 5 June, 2013 cycle in different concentrations at Bose, R., Majumdar, C. and the different reproductive stages. Bhattacharya, S. (1997). Steroids in Achatina fulica: REFERENCES steroid profile in haemolymph A.O.A.C (2000). Association of and invitro release of steroid Official Analytical Chemsit.Ed. from endogenous precursors by ovotestis and albumen gland. W. Horwitz 13 t h edition 62 BAMIDELE ET AL: Steroids concentration in the Giant African Land snail at different reproductive phases Comparative Biochemistry and Neil, R.C. (2004). Physiology of Physiology 116C: 179-182 behaviour. Pearson Education Carreau, S. And Drosdowsky, M. Inc. U.S.A (1977). In Vitro biosynthesis of Odaibo, W.D. (1997). Snail and snail steroid by gonad of the farming; Nigerian edible land , Sepia officinalis L. snail. Ibadan: Horder Publisher. G e n e r a l C o m p a r a t i v e Pp 20-24 Endocrinology 33: 554-565 Odiete, W.O. (1999). Basic Animal Cosmo, D.A., Cristo, D.C. and Paolucci Physiology. Diversified M. (2001). Sex steroid hormone Resources Ltd., Surulere Lagos. fluctuations and morphological South, A. (1992). Terrestrial : changes of the reproductive Biology, ecology and control. system of the female of Octopus Chapman and Hall, U.S.A Pp vulgaris throughout the annual 66-101. cycle. Journal of Experimental Wallen, K. (1990). Desire and ability: Zoology 289: 33-47 Hormones and the regulation of Dixson, A. (2001). The evolution of female sexual behaviour. neuroendocrine mechanisms N e u r o s c i e n c e a n d regulating sexual behaviour in Biobehavioural Reviews, 14: female primates. Reproductive 233 - 241 and Fertility Development 13: 599-607 Wang, C. And Croll, R.P. (2004). Effects of steroids on gonadal Idowu, A.B. and Akinnusi, O.A. development and gender (2006). The structure of the determination in the sea ovotestits of the common , African land snail found in Abeokuta, South Placopecten magallanics. Western Nigeria, Nigeria Aquaculture 238: 483-498 Journal of Animal. Production. 33(2): 286 -293 Maha, R., Abdel-Hamid, A.Z., Mamlouk, E.T. and Said, N. (2009). Assessment of steroids during maturation and infection of Biomphalaria alexandrina snails, an intermediate host of S c h i s t o s o m a m a n s o n i . Biohealth Science Bulletin 1(1): 7-12

63

Journal of Molluscan Research 1:52-57 (2015)

COMPARATIVE EFFECTS OF PLANT LEAVES (PAWPAW, FLUTED ) AND 20% CRUDE PROTEIN DIET ON GROWTH PERFORMANCE OF Achatina fulica

EJIDIKE, B. N AND AKINNAGBE, O. T

Department of Ecotourism and Wildlife Management, Federal University of Technology, Akure, Ondo State, Nigeria Email:[email protected]

ABSTRACT The dietary effects of plant leaves (pawpaw, fluted pumpkin) and 20% crude protein diet on the growth of Achatina fulica was investigated for 70 days. A total of 99 juveniles of Achatina fulica 5±0.4grams were randomly grouped into three (3) for three specific treatments. The treatments are pawpaw leaves (diet I), fluted pumpkin leaves (diet II), and 20% crude protein diet (diet III). Each diet treatment had three (3) replicates with each replicate having eleven (11) juveniles of Achatina fulica. The snails in each of the three (3) groups were fed with specific diet for the treatment. Pawpaw and fluted pumpkin leaves were obtained within the University and its vicinity. The 20% crude protein diet was formulated using yellow maize (30.6%), groundnut cake-GNC (45.4%), rice bran (5.0%), brewers waste (5.0%), limestone (10.0%), vitamin premix (2.0%) and red oil (2.0%). The ingredients were each milled and sieved into fine powder, and the formulated portions were mixed together using hot water to form clumps which were sun dried for subsequent use in feeding the Achatina fulica juveniles. The Achatina fulica were fed with specific diets at 2% body weight at 1800hour daily. The performance of the snail species- Achatina fulica in each treatment was observed and recorded for a period of ten weeks. It was found that Achatina fulica snails fed on diet I had better growth performance that is significantly different (p<0.05) from the Achatina fulica juveniles fed on diets II and III. This indicates that pawpaw leaves that are available in farms could contribute positively to the growth of Achatina fulica under captive rearing system. This has great potentials for human food source. Achatina fulica fed on fluted pumpkin leaves had better growth performance compared to the Achatina fulica snails fed with compounded diet in terms of numeric body weight though there was no significant difference (p>0.05) in the body weight gain.

Keywords: Achatina fulica, plant leaves, diet, growth INTRODUCTION 64 EJIDIKE AND AKINNAGBE: Effects of plant leaves and 20% crude protein diet on performance of Achatina fulica Ever increasing human population in the et al, 1997). In Nigeria, there are developing countries of the world has different species of snail which vary in consequences of increasing in demand size, colour, adaptability and for necessities of life such as shelter, performance (Amusan and Omidiji, food especially protein in the diets of the 1998). These species are seasonal though people as well as intensified struggle for Archachatina marginata do survive all survival among the populace. In these year round and can be active under moist situations an average citizen in such and proper management (Ejidike, 2002). developing countries needs to have Omole et al., (2000) reported that animal protein sources in the daily diet different snail species found in Nigeria irrespective of the size and species of the are characterized by their best efficiency animal in order to measure up the of nutrient transformation into quality requirements for standard of living. protein. Achatina fulica is a native of Harnessing and utilizing most of the East Africa (Rowson et al, 2010), but it biodiversity a nation is endowed with is has been widely introduced to other parts a good potential of moving a country of the world through trade, as a food forward in achieving economic balance resource, and by accidental as well as attaining sustainability in introduction. This could explain its agriculture. It is wise to encourage existence in some parts of Nigeria. rearing of most animal species a nation is Snail farming on a large scale has not endowed with that can survive and been proportional to snail demand in multiply under captive set up. Most land Nigeria, even though few people have snail species thrive well under captive embarked on rearing snail at different condition as long as food and water are scales. provided. This study evaluated the comparative Snail species which belong to a group of effect of pawpaw leaves, fluted invertebrate animals are one of the most pumpkin and formulated diet on the compact groups of animals which thrive growth performance of Achatina well under a wide food diversity fulica in order to recommend its food (Odunaiya, 1995). Snails are soft bodied under captive management. invertebrates with exoskeleton in the form of a calciferous shell. Land snail MATERIALS AND METHODS species belong to the Phylum Mollusca, Class Gastropoda which is known to be Plant food material the second largest invertebrate group Two different plant leaves (pawpaw found in the Animal kingdom after and fluted pumpkin) were obtained . Snail farming is a good tool for from the Federal University of poverty alleviation (Moyin-Jesu and Technology Akure, Nigeria and its Ajao, 2008) in most countries of the vicinity. The plant leaves were developing world. Snail meat is accepted obtained daily, chopped into pieces and consumed worldwide by the rich and and introduced to the specific group the poor (Murphy, 2001; Paoletti, 2005). of Achatina fulica in the rearing unit. This could be attributed to the fact that Experimental Diet snail meat is tasty, tender and highly Diet of 20% crude protein was nutritional (Akinnusi, 2002; Eruvbetine formulated using the ingredients in 65 EJIDIKE AND AKINNAGBE: Effects of plant leaves and 20% crude protein diet on performance of Achatina fulica Table 1.The feedstuffs were milled Ninety nine juveniles of Achatina fulica into powder using Hammer miller in (5±0.4g) used for the experiment were the Department of Fisheries and handpicked from the garden within the Aquaculture, Federal University of vicinity of Federal University of Table 1: Ingredient Composition of the Experimental Diet Feed formulation Diets % Yellow maize 30.6 Groundnut cake 45.4 Rice bran 5.0 Brewers waste 5.0 Limestone 10.0 Vitamin 2.0 Red Oil 2.0 Total 100.00

Technology, Akure, Nigeria. The Technology, Akure, Nigeria. The snails ingredients composition that include were acclimated in plastic baskets for yellow maize, brewers' dry grain, three days before distributing into groups vitamin premix, rice bran, limestone, for the experiment. Completely groundnut, red oil were thoroughly randomized design (CRD) was adopted mixed. Hot water was added in order to for the feeding trials with a total of nine blend the mixture which was spread in experimental units. At the end of clumps. The diet was sun-dried for three acclimation the juvenile snails were days at 28oC and packed in polyethylene weighed and randomly grouped into bags and stored at ambient temperature three which were assigned each to a for subsequent feeding of the specific specific treatment. The treatments were group of Achatina fulica during the pawpaw leaves (diet I), fluted pumpkin study. leaves (diet II), 20% crude protein diet Experimental layout and Management (diet III) and the snails were randomly of juvenile Achatina fulica distributed in triplicate in nine (9) plastic The experiment was carried out at the baskets of dimensions 90cm x 14cm each wildlife domestication unit of the with lids. The plastic baskets were each Department of Ecotourism and Wildlife filled with loamy soil up to 10cm Management of the Federal University of thickness and randomly stocked with Technology, Akure, Nigeria. Akure in eleven juveniles of Achatina fulica. The Ondo State is a lowland tropical snails were fed with their specific diet rainforest belt with an annual rainfall of treatment at 2% body weight at about 1.613mm per year and an annual 1800hours daily after sprinkling water on mean temperature of about 27°C. The them in their enclosure. Feeding of the study area is located on longitude 15°N snails lasted for ten weeks with daily and latitude 5°SE. In the past dry season observations and weekly sampling of the occurred between October and March snails after which the feed supply to the while the rainy season started from April. snails was adjusted according to the body Since inception of climate change, the weight. The feed supply to the snails and rainy season now exceeds the month of body weight gain were measured using October. electronic balance.

66 EJIDIKE AND AKINNAGBE: Effects of plant leaves and 20% crude protein diet on performance of Achatina fulica RESULTS AND DISCUSSION farming/husbandry plays positive role in In the tropics nutrition among others the welfare and better performance of is one of the major factors that affect animal species. Increase in body weight both growth and reproduction and other morphological parameters performance of most living (Table 2) observed in the A. fulica fed the organisms. Land snail species are no diet treatments at the end of the exception. For optimum performance, experiment proved the conversion of the especially under captive setting, the diet treatments into animal flesh ready requirements of the species in terms for human consumption by the A. fulica. of protein and minerals need to be These supported the report of Omole et adequately supplied. The A. fulica in al., (2000) that different species of snails this study were observed to feed are found in Nigeria and they are actively on the three diet treatments characterized by their best efficiency of throughout the experiment, indicating nutrient transformation into quality acceptance and utilization of the diet protein. Plant food sources are relatively treatments. The acceptance of the diet cheaper and readily available than the treatments by the snails led to animal protein sources in animal hundred percent survival recorded in farming. The diet treatments influenced the study the body weight of A. fulica significantly proving that pawpaw, fluted pumpkin (p<0.05), the A. fulica placed on diet treatment of pawpaw leaves was leaves were suitable food items in land positively affected more than the other two treatments that were placed on fluted snail farming as these plant food materials are also acceptable by pumpkin and 20% crude protein diet. Archachatina marginata (Ejidike, 2002). This significant (p<0.05) influence of Provision of enabling e n v i r o n m e n t pawpaw leaves on A. fulica growth s i n a n i m a l performance had

Table 2: Growth performance of Achatina fulica as affected by different diet compositions

Growth performance index Diet I Diet II Diet III

Initial mean weight (g) 5.0±0.1a 5.1±0 .1a 5.0±0.1a Final mean weight (g) 9.9±0.2b 7.5±0 .2a 8.4±0.2a Initial mean length (cm) 2.0±0.1a 2.0±0 .1a 2.0±0.1a Final mean length (cm) 3.9±0.1a 3.7±0 .1a 3.8±0.1a Initial mean width (cm) 1.6±0.1a 1.6±0.1 a 1.6±0.1 a

2.7±0.1a 2.7±0.1a Final mean width (cm) 2.6±0.1a Survival (% ) 100.0a 100 .0a 100.0 a

67 EJIDIKE AND AKINNAGBE: Effects of plant leaves and 20% crude protein diet on performance of Achatina fulica

Figures…. with the same superscript are not significantly dif ferent(p> 0.05) Fig. 1 showing the shell length of Achatina fulica fed pawpaw, fluted pumpkin leaves and 20% crude protein diet Fig. 2: Effect of different feed treatments composition on the width of Achatina fulica fed 10 weeks been recorded by many authors on the natural plant food materials for easy Archachatina marginata, proving and ready provision of food for the snails pawpaw leaves widely and readily at all times. The diet treatments had acceptable by land snail species similar (p>0.05) effects on (Ejidike, 2002). Similar influence morphological parameters (shell length, (p>0.05) on the body weight of A. and shell width) of the snail fed with the fulica were recorded on the snails fed experimental diets Figures 1 and 2. fluted pumpkin leaves and 20% crude protein at the end of the feeding trial. CONCLUSION The acceptance of 20% crude protein This study demonstrated that Achatina diet by the snail species show that the fulica one of African land snail species snail can be farmed in large scale with accepts and utilizes artificial diet, and it the use of artificial diets to augment 68 EJIDIKE AND AKINNAGBE: Effects of plant leaves and 20% crude protein diet on performance of Achatina fulica grows optimal on pawpaw leaves just Abeokuta, 23-27thNovember, as Archachatina marginata do 1999. 93-97. perform well growth wise when fed Odunaiya, O. (1995).The utilization of pawpaw leaves. snails in Nigeria. M. Sc. Thesis. University of Ibadan, Ibadan, REFERENCES Nigeria, p. 65 Akinnusi, O. (2002). Introduction to Omole A. J, Tewe O. O, Adebowale E. Snails and Snail Farming. A, Oluokun J. A, Ogundola F. J, Triolas Exquisite Ventures. Nworgu F. C (2000). Abeokuta. 25pp. Performance of different breeds Amusan J. A and Omidiji, M. O 1998, of snails under the same m a n a g Edible Land Snail; A e m e n t conditions.Tropical Technical Guide to Snail journal of AnimalScience. 3 Farming in the (1):133-138. Tropics; Ibadan; Variety Moyin-Jesu E. I,and Ajao K (2008). Printers Limited, p-154 Raising of giant snails AOAC, (1990). Association of (Archachatina marginata) in official analytical chemists, urban cities using soil official methods of analysis amendments and feeding 15th edition Washington D.C. materials for food security. Pp71-79. African Journal of Science and Technology (AJST) Science and Ejidike, B. N. (2002) Growth Engineering series. response of African giant land 9(1):118124. snail (Archachatina marginata) fed four single or Murphy B (2001) Breeding and growing combined plant leaves. snails commercially in Australia. Journal of Science, A Report of the Rural Engineering and Technology, Industries Research and Nigeria 9 (4): 4416-4422. Development Corporation Eruvbetine D, Oguntona E. B, Adoye Publication. No 00/188 Project no U. A (1997). Preliminary ARH-IA. studies comparing different Paoletti M G (2005). Ecological diets for intensive rearing of Implication of Mini-livestock – snails. In: Ogunyemi S. and role of insects, rodents, and Ojo L. O. (eds). snails for sustainable d e v e l o p Proceedings of the m e n t . S c i e n c e 1997 Biennial Conference of Publishers, London. the Rowson, B., Warren, B. H, Ngereza C. Ecological Society of Nigeria F (2010) Terrestrial mollusc of (ECOSON) held at the Pemba Island, Zanzibar, University of Agriculture, Tanzania and its status as an 'Oceanic Island Zookeys 70: 1- 69 EJIDIKE AND AKINNAGBE: Effects of plant leaves and 20% crude protein diet on performance of Achatina fulica 39. doc 103897/Zookeys 70.762

70 AJASIN ET AL: Influence of hamecozyme enzyme on performance characteristics of snails Journal of Molluscan Research 1:58-63 (2015)

INFLUENCE OF HAMECOZYME ENZYME ON PERFORMANCE CHARACTERISTICS OF SNAILS (Archachatina marginata) FED BREWER DRY GRAIN BASED DIET

AJASIN1, F.O., OGUNLEKE1, F.O., ADEKUNLE1,O.F., MAKANJUOLA2, B.A., OKPEZE1, C.N., ONONOGBU1, C., OWOSIBO2, O.T. AND OMOLE2, A.J.

1Obafemi Awolowo University, Institute of Agricultural Research and Training Ibadan 2

Federal College of Animal Health and Production Technology, Ibadan, Nigeria. ABSTRACT An experiment was conducted to determine the effects of inclusion of Hamecozyme in the diet of growing snails on performance characteristics and cost benefits. A total of one hundred and twenty growing snails of mean weight 87.32±2.8g were used for the feeding trial. The trial had four treatments, each treatment was replicated thrice with 10 snails per replicate in a completely randomized design. Diet 1, (H1) contained no Hamecozyme, Diet 2 (H2) contained 0.10% Hamecozyme, Diet 3(H3) contained 0.15% Hamecozyme while Diet 4(H4) contained 0.20% Hamecozyme. Parameters measured were feed intake, weight gain, shell length and width. Feed conversion ratio, cost/weight gain and dressing percentage were calculated. The feed intake increased with increased level of enzyme in the diets. Highest weight gain was recorded H4 which was relatively similar to that of Hs. The inclusion of Hamecozyme enzyme had significant influence on the efficiency of feed utilization (P<0.05). Shell length and width were not significantly (P>0.05) affected by inclusion of haemococyme. The results of carcass analysis shows that inclusion of Hamecozyme enzyme (AE) improve the foot weight (edible or fleshy portion) across the treatment the food weight increased from 128.78g in

H1 to 146.35g in H4. The dressing percentage increased from 41.12% in H1 to

42.89% in H4. Hamecozyme improved the weight gain, feed conversion ratio and dressing percentage however the inclusion of Hamecozyme in the diet was not cost effective.

Keywords : Cost/weight gain, feed utilization, fibrous feed, hamecozyme, snails INTRODUCTION playing a good role as an alternative Micro-livestock such as snails, source of animal protein (Ejidike, grasscutters and rabbits have been 2001). There is increase in demand for micro-livestock in Nigeria and Africa 71 in general. The meat of micro-livestock phytate application. It has been used in is palatable with low fat content. broilers and pigs to improve Increasing production of micro- Phosphorus, energy and fibre livestock will reduce dependency on digestibilities with big saving in feed pork, mutton, beef and poultry meat cost. Hamecozyme occurs in both which will consequently reduce price. powder and liquid form and it has not Snails feed on wide varieties of feed, been included in snail's diet hence this mostly in the night and at dust but the study was conducted to determine the growth rate is slow compared to other effect Hamecozyme's inclusion in the livestock (Amusan and Omidiji,1999). diet of snails on growth, carcass and Researchers have been making cost benefits . attempts to improve the growth performance of snails. A lot of works MATERIALS AND METHODS have been done on the use of The experiment was carried out at the formulated diet to improve the Snailery Unit of the Institute of performance with impressive results Agricultural Research and Training and research is still on-going (I.A.R.& T.), Moor Plantation, Ibadan. (Hamzat,2004, kehinde,2009: Omole, A total of one hundred and twenty 2011). Some of the feed ingredients growing snails of mean weight such as maize, brewer dry grains, 87.32±2.8g were used for the feeding offal, palm-kernel cake, rice trial. The trial had four treatments, each bran and corn-bran contain high fibre treatment was replicated thrice with 10 content which cannot be efficiently snails per replicate. Diet 1, (H1) utilized by animal (Esonu,2000; contained no Hamecozyme, Diet 2 (H2) Iyayi,2008). Feed enzymes have contained 0.10% Hamecozyme, Diet important role to play in fibre 3(H3) contained 0.15% Hamecozyme digestion. Enzymes are global protein while Diet 4(H4) contained 0.20% molecule having an active site to which Hamecozyme. The diets were may bind certain specific molecule formulated to contain 24% crude called subtract of the enzyme. protein and energy of 2400 kcal/kgME Inclusion of enzyme in animal feed (Table 1). Feed intake and weight gain have been found to improve the were measured on daily and weekly d i g e s t i b i l i t y o f n o n - s t a r c h basis with the use of sensitive weighing polysaccharides ( NSP) such as balance. Feed intake was calculated by betaglucans and xylans. Also phytase subtracting the left-over feed from the has been found to break down plant feed given while the weight gain was stored phosphorus (Phytate) which in calculated by deducting the initial itself is invariable but form complexes weight from the final weight. Shell with other nutrients and thereby length and width were measured on releasing phosphorus as well as starch, weekly basis with vernier caliper. protein and amino acids (Acamovic, Micrometer screw gauge was used to 2001; Ekenyem and Madubuike,2006; measured the shell thickness on weekly Ani and Omeje, 2008). Hamecozyme is basis. Feed conversion ratio were a multi-enzyme system for NSP and calculated as the ratio of feed intake to 72 AJASIN ET AL: Influence of hamecozyme enzyme on performance characteristics of snails weight gain. Feed cost and cost per selecting eight snails from each weight gain were also calculated. treatment and weighed separately. Carcass analysis was carried out at the Each snail was killed by striking the end of the feeding trial by randomly shell AJASIN ET AL: Influence of hamecozyme enzyme on performance characteristics of snails with a club. The shell, foot and viscerals feed intake increased with increased were separated and weighed separately level of enzyme in the diets. The highest (kehinde, 2009). The chemical feed intake of 1084.33g was recorded in composition of the experimental diets snails fed 0.2% Hamecozyme in the diet and the foot were done according to the which was not significant difference method of A.O.A.C. (1990). All data from those fed 0.15% Hamecozyme. were subjected to statistical analysis The weight gain using analysis of variance and the followed the same trend with the feed means were separated if they are intake (P<0.05). Highest weight gain significantly different using Duncan was recorded H4 which was relatively Multiple Range Test (SAS, 1999).. similar to that of H1 as observed in Table RESULTS AND DISCUSSION 3. The inclusion of Hamecozyme The results of growth performance of the enzyme had significant influence on the snails is shown in Table 2, efficiency of feed utilization (P<0.05). significant differences were observed in The inclusion of Hamecozyme enzyme the mean weekly weight gain and feed improved the feed conversion ratio intake fed different inclusion levels of (FCR) across the treatment (P<0.05). Hamecozyme in the diets (P<0.05). The The feed was best utilized in H4 with FCR of 3.92 which was not significant

Table 1: Gross Composition of Experimenta l Diet. Ingredient (%) H1 H2 H3 H4 Maize 22.00 22.0 22.0 22.0 Brewer dry grain 27.8 27.7 27.65 27.60 Hamecozyme 0.0 0.1 0.15 0.20

*Others 50.2 50.2 50.2 50.2

Total 100.0 100.0 100.0 100.0

24.53 Calculated analysis 24.53 24.53 24.53 Crude protein (%) 2438.1 2438.1 2438.1 2438.1 Metabolizable ener gy

( kcal/KgME ) *Other fixed ingredients , Fish meal-4, Soya bean meal -24, Ground nut cake-10, Bone 73

Initial weight (g) 67.98 67.45 68.32 a 66.95 a 5.10 Final weight (g) 314.18b b 341.95 343.57 273.63 10.13 a 246.2 b 318.5 276.62 5.97 Total weight gain (g) a b 10.57 Total feed intake (g) 1041.5 251.05 0.26 Feed conversion ratio 4.23 a b a 1078.12 1049.4 1084.33 a b b 3.92 b a 3.94 4.18 Shell length increment (mm) 11.78 1 1.87 12.10 12.50 1.02

Shell width increment (mm) 10.18 10.26 10.35 10.98 0.64

Shell thickness increment (mm) 0.13 0.13 0.13 0.14 0.02 Mortality (Number) 0 1 0 0 meal -2.15,Oyster shell-9.8 , Premix-0.25 H1 - 0.0% Ham ecozyme, H2 - 0.1% Hamecozyme, H3 - 0.15% Hamecozyme, H4- 0.2% Hamecozyme Table 2: Growth performance of grow ing snails fed experimental diet.

Means along rows with different s uperscript are significantly different from each other (P<0.05)

H1 - 0.0% Hamecoz yme, H2 - 0.1% Hamecozyme, H3 - 0.15% Hamecozyme, H4- 0.2 % Hamecozyme

Parameters (Means) H1 H2 H3 H 4 ±SEM

different from H4. The shell length and efficiency. Enzyme has been found to width increased numerically but not increase digestibility of fibrous feed significantly different from one ingredients by disrupting the pant cell another (P>0.05). The improvement in wall and thus enhancing nutrients growth rate as the level of enzyme in absorption (Acamovic, 2001; Ekenyem the diet increased could be due to the and Madubuike,2006; Ani and Omeje, effects of hamecozyme in breaking 2008). The mortality recorded in H1 down the fibre content of the feed and H3 could not be traced to the effect ingredients thereby reducing fibre level of Hamecozyme because mortality was and it has been repored that low fibre not recorded in H2 and H4. The snail's diet promotes growth and feed death could not be diagnosed for any

74 AJASIN ET AL: Influence of hamecozyme enzyme on performance characteristics of snails diseases because no sign or symptom . No significant difference was of sickness were observed. It has been observed in H3 and H4 reported by Amusan and Omidiji, (P>0.05). The increase in foot weight (1999) that snails do not show signs or and dressing percentage across the symptoms of a particular sickness and treatments confirmed the reports of Ani there is no specific drug or vaccine for and Omeje,2008 who concluded that disease prevention and control in inclusion of enzyme in the diet snails. The results of carcass analysis improves the carcass quality in term of as observed in table 3 shows that dressing percentage etc. The shell to inclusion of Hamecozyme enzyme weight ratio and offal to live-weight improve the foot weight (edible or ratio were not significantly influenced fleshy portion) across the treatment the by the treatments effect as shown in food weight increased from 128.78g in Table 3.

H1 to 146.35g in H4. The dressing The results of cost analysis in Table 4 percentage increased from 41.12% in shows that the cost per kg feed and total

H1 to 42.89% in H4 as shown in table 3 Table 3: Carcass analysis of growing snails fed experimental diet. Parameters (Means) ±SEM H 1 H2 H3 H 4 Live weight (g) 313.18b 317.22b 340.21 a 341.23 a 3.67 b 73.97 85.27 a b Shell weight (g) 76.51 84.61 a 3.85 Offal weight (g) 69.27b 71.31 b 77.53 a 78.17a 2.95 b b a Foot weight (g) 128.78 143.33 131.23 146.35 a 5.15 Dressing percent (%) 41.12 41.37 42.13 42.89 2.67 Offal/live weight (%) 22.12 22.48 22.79 22.91 2. 56 Shell/live weight (%) 23.62 24.12 24.87 24.99 2.45

Means along rows with different superscript are significantly different from each other (P<0.05) H1 - 0.0% Hamecozyme, H2 - 0.1% Hamecozyme, H3 - 0.15% Hamecozyme, H4- 0.2 % Ha mecozyme Table 4: Cost analysis of growing s nails fed ex perimental diet.

Parameters (Means) H1 H H3 H 4 ± SEM 2 Total feed intake (kg) 1.04 1.05 1.08 1.08 Total weight gain (kg) 0.25 0.25 0.27 0.28 b 51.00 52.94 a a Cost/kg feed (N) 54.1 1 56.17 a 2.26 b.

75

b Total feed cost (N/kg) 53.04 55.59 58.43 60.66 a 3.9 2 a a b Cost/weight gain (N/kg) 212.16c 222.36 a 216.40 b 216.65 b 3.12

H1 - 0.0% Hamecozyme, H2 - 0.1% Hamecozyme, H 3 - 0.15% Hamecozyme, H 4- 0.2% Hamecozyme

76 AJASIN ET : Influence of hamecozyme enzyme on performance characteristics of snails

AL feed cost increased supplementary enzyme. as the level of hamecozyme increased Proceeding of the 13th in diet. The total feed cost (TFC) of Annual Conference of the

N60.66 was recorded in H4 as shown in Animal Science Association of table 5. The cost per weight gain Nigeria (ASAN) September, (CWG) of N212.16 was recorded in H1 1519, 2008. ABN Zaria. while the highest CWG of N222.36 was Association of Official Analytical recorded in H2. Chemist, A.O.A.C. (1990). Official Methods of Analysis, CONCLUSION 13th Edition, Washington, D.C Based on the present results, Ejidike, B. N. (2001): Comparative Hamecozyme improved the weight effect of supplemental and gain, feed conversion ratio and dressing percentage however the inclusion of complete diets on the Hamecozyme in the diet was not cost performance of African giant effective. It is recommended that cost land snail (A. marginata). of enzyme should be reduced by Proceedings of the 26th Annual reducing import duty on it or Conference of the Nigerian subsidizing the products in order to Society for Animal Production, justify its use in the diet of snails. Vol. 26, pp151 – 156. Ekenyem, B.V and F.N. Madubuike REFERENCES (2006). An assessment of Acamovic, T. (2001). Commercial Ipomoea asiri folia leaf meal as application of enzyme feed ingredient in broiler chick technology for poultry production. Pakistan Journal of production. World poultry Nutrition (5) (1) 46-50. science Journal 57 : 225-242. Esonu, B.O (2000). Animal nutrition Amusan, J. A. and Omidiji, M. O. and feeding: A functioning (1999). Edible Land Snail: A Approach, Rukzed and Ruckson Associates. Technical Guide to Snail Hamzat, R.A (2004). Utilization of Farming in the Tropics. Verity cocoa bean shell in the diet of Printer Limited, Ibadan. 96 pg.. growing snail (A.marginata). Ani, A.O and Omeje, O.D (2008). P.hd Thesis, Dept of Animal Carcass and Organ weight of science, University of Ibadan. broiler finishers fed varying Iyayi, E.A. (2008). Prospect and dietary levels of raw bambara challenges of unconventional nut (Vigna subterrranea (L) poultry feed stuffs. Nigerian Verde) waste and Poultry Science Journal 5: 186194.

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Kehinde, A.J. (2009). Utilization of Evaluation Of Cassava Peel – cassava peel products in the life Palm Kernel Cake Mixtures In cycle feeding of snails Snails' Diets. Proceeding of The (Archachatina marginata). 36th Annual Conference of the Ph.D Thesis ,Department of Nigerian Society for Animal Animal Science, University of Production (NSAP) held at the Ibadan.( Unpublished) N a t i o n a l U n i v e r s i t y Omole, A.J. (2011) Nutritional Commission (NUC) and Raw S.A.S. (1999). S.A.S. User's Guide. M a t e r i a l s R e s e a r c h Statistical Analysis System D e v e l o p m e n t C o u n c i l Institute, Inc.Cary, N.C. (RMRDC) Abuja on 13th – 16th 7th edt. 78 pgs. March 2011. Pg 305-307 ( Nigeria )

78 AJASIN ET AL: Influence of hamecozyme enzyme on performance characteristics of snails Journal of Molluscan Research 1:64-77 (2015)

REPRODUCTIVE PERFORMANCE OF Archachatina marginata var. ovum SNAILS IN CALABAR, NIGERIA

IBOM, L. A1., OKON, B1., OKON, F. I2. AND OWOYEMI, J. D1. 1Department of Animal Science, University of Calabar, Calabar, Nigeria. 2Department of Environmental Health, College of Health Technology, Calabar, Nigeria. *Corresponding Author's e-mail address: [email protected], +2348033508663

ABSTRACT A study was conducted to assess the reproductive performance of Archachatina marginata var. ovum (Pfiffer) snails. One hundred and twenty (120) sexually mature Archachatina marginata var. ovum (Pfeiffer) snails, sixty (60) each of the black-skinned (BS) and the white-skinned (WS) ectotypes with weight range of 68.80 to 82.95g sorted out of a base population were used in the study. The snails were grouped into three mating groups on the basis of skin (foot) colour, namely: BS X BS, BS X WS and WS X WS. Each mating group was replicated twenty (20) times in the completely randomized design (CRD) with two snails of similar weights to a cell for purposes of reproduction and managed for twenty-four (24) weeks. The reproductive indices assessed included clutch size (number of eggs laid in a clutch), mean egg weight at lay and weekly, mean egg length and width at lay and weekly, incubation period, number of snailets hatched hatchlings, snailets weight at hatch, percent eggs hatchability and snailets survivability. Results obtained from the study showed that numerical differences existed across the three mating groups for all the measured reproductive indices. The crossbred mating group (BS X WS) performed numerically better than the purebred mating groups (BS X BS) and (WS X WS) in all (mean clutch size, mean incubation period, mean no. of snailets, percent hatchability, mean no. of survived snailets/clutch and mean weight of snailets at hatch), but one (percent survivability) of the parameters assessed. Except for the mean weight of snailets at hatch that expressed statistical difference (P<0.05), all other parameters were not significantly different (P>0.05) across the mating groups. The results of mean egg weight values showed that egg weight decreased with age [from 1.15g at lay to 0.95g before hatch (BS X BS), 1.20g at lay to 0.97g before hatch (BS X WS) and 1.01g at lay to 0. 76g before hatch (WS X WS) mating groups] during incubation. Contrarily, the mean egg length and width values did not change with age [at 79 lay (0 week) and before hatch (4 weeks)]. Based on the results of this study, the crossbreeding of the BS X WS ectotypes could be recommended and/or encouraged. This is because farmers would obtain more eggs per clutch, hatching into bigger snailets with better survival potentials, within shorter incubation periods. Keywords: Archachatina marginata var. ovum, reproductive performance,

80 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails

INTRODUCTION nonconventional sources of animal One of the challenges facing Nigeria protein. According to Ibom (2014), the today in terms of food security is the most known and most often cultivated shortage of animal protein production land snail breeds in Nigeria include: and supply to meet the ever increasing Archachatina marginata (A. marginata demand. This has almost always var. ovum and A. marginata var. culminated in malnutrition, blamed on saturalis), A. degneri (Plates 1 – 3), the low performance and handicap of Achatina (A. achatina and A. fulica) the Nigerian livestock sub-sector, lack (Plates 4 – 5) and Limicolaria species of initiative and drive by livestock (L. aurora, L. martensi and L. flammea) farmers, susceptibility to endemic and (Plates 6 – 8). This study evaluated the infectious diseases and poverty level of reproductive performance of Nigerians. Okon et al. (2009) attributed Archachatina marginata var. ovum, the inadequate supply of animal protein one of snail breeds consumed in in Nigeria to reliance on traditional Nigeria. animal husbandry system which is characterized by low-input, high-cost MATERIALS AND METHODS of feed and low productivity of local Location of experiment animal breeds for the supply of this commodity. Ibom (2009) stated that to The study was conducted at the achieve the much-felt need of Botanical garden, University of improving the animal protein Calabar, Calabar, Nigeria. Calabar is located within latitudes 4º 58'N and 15º production level in Nigeria, there is 39'N and longitudes 8º 17'E and 10º need to look for cheaper and safe 43'E of the equator with a relative animal protein sources to complement humidity of 51 – 99 % and an elevation the supply from conventional sources. above sea level of 99 metres. The Besides, Ibom et al. (2011) stated that annual temperature and rainfall of the snails play very important role in the area are 25o – 30º C and 1260 – 3500 economy of rural families where they mm respectively (Okon and Ibom, serve as the cheapest sources of animal 2011, Google Earth, 2014). The protein, especially during the rainy Botanical garden provided a season (period of abundance). On the microenvironment similar to the other hand, Ebenebe (2000) stated that natural habitat of snails as it is planted there is need to look inward and with trees like mango, oil palm, integrate into Nigeria's farming system gmelina, almond and paw-paw. These some non-conventional meat sources to trees had closed canopies which complement the conventional animals provided shades that protected the as sources of animal protein. hutches from direct sunlight and heavy Ibom (2009) and Okon and Ibom rainfall. (2012) reported that Nigeria is richly endowed with snail breeds, one of the

81 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails

Experimental animals ectotypes sorted out of a base One hundred and twenty (120) sexually population were used for the study. The

P late 1: A rc ha c h a ti n a ma r g in a ta va r . o v u m Pla te 2: A . m a rg in a ta va r . sa tu ra li s

mature (10 months of age) body weights of these two ectotypes Archachatina marginata var. ovum ranged from 68.80 to 82.95g. The (Pfeiffer) snails, sixty each of the description of the selection and blackskinned and the white-skinned management of the snails and breeding Plate 3: Archachatina degneri Plate 4: Achatina achatina

82 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails

Plate 7: Limicolaria martensi Plate 8: Limicolaria flammea

83 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails

84 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails (natural mating) pattern were as package. Significant means were prescribed in Ibom (2009) and Ibom et separated using Least Significant al. (2012). Difference (LSD) of the same software. Data collection and analysis Data collected were clutch size RESULTS AND DISCUSSION (number of eggs laid) as counted, egg The results of parent snails weight (g), egg length (cm) and egg [Archachatina marginata var. ovum width (cm), incubation period (days) (Pfeiffer)] reproductive performance counted as day eggs are laid to are presented in Table1. The results emergence of snailet(s), number of showed that the crossbred [black snailets hatched obtained by difference skinned x white skinned (BS X WS)] between the number of eggs incubated mating group performed numerically and those hatched, and snailets weight better than the purebred [black skinned (g) at hatch. Percent eggs hatchability x black skinned (BS X BS) and white and snailets survivability were skinned x white skinned (WS X WS)] estimated. An electronic scale, KerroTM mating groups in all (mean clutch size, with 0.01g sensitivity and 600g mean incubation period, mean no. of capacity was used to measure weights, snailets/clutch, percent hatchability, while Vernier Caliper was used to mean no. of survived snailets/clutch measure length and width. The data and mean weight of snailets at hatch), were subjected to statistical analysis but one (percent survivability) of the using analysis of variance (ANOVA) parameters assessed. Except for the according to Genstat (2007) software mean weight of snailets at hatch that Table 1: Reproductive performance of parent Archachatina marginata var. ovum snails.

Reproductive traits Mating group BS X BS BS X WS WS X WS Mean clutch size 6.25±0.02 (2–10) 6 .58±0.01 (2–10) 5.08±0.03 (1– 9) Incubation period ( days) 18.00±0.03 (10–25) 17.00±0 .02 (10– 20.00±0.02 (10– 25) 27) Mean no. of snailets/clutch 3.25±0.01 (1–6) 3.33±0 .02 (2–6) 2.90±0.03 (1–6) Percent hatchability (%) 53.75±0.20 (29–100) 58.58±0.30 (38– 50.50±0.10 (0– 100) 83) Mean no. of survived 2.92±0.01 (1–5) 3.00± 0.02 (2–5) 2.40±0.03 (0– snailets/clutch 6)

Percent survivability (%) 92.08±0.50 (69–100) 91.67±0 .40 (67– 83.00±0.30 (0–1 100) 00) Mean weight of snailets at hatch (g) 0.73±0.02b 0.93±0.03 a 0.53±0.01 c

BS = Black Skinned ectotype, WS = White Skinned ectotype. abc w with different superscript are significantly di fferent (P<0.05). Means along the same ro 85

expressed statistical difference The mean weight values of snailets at (P<0.05), all other parameters were not hatch, the only parameter that significantly different (P>0.05) across expressed significant difference the mating groups. The results suggest (P<0.05) across the mating groups fell that the BS and WS parents have good within the range values of 0.66 – 0.93g combining ability, as expressed in the reported by Ewa (2007) and 0.69 – parameters assessed. The clutch size 1.42g reported by Odido (2007) for results of this study fell within the white-skinned and black-skinned range of 2 to 15 and 1 to 16 reported by snailets of Archachatina marginata Akintomide (2004) and Okon and Ibom snails respectively. However, the (2012) respectively for A. marginata snailets mean weight values at hatch of snails. However, the results were this study were lower than the range higher than the range of 1 to 4 and 2 to values of 2.1 – 2.5g and 2 – 7g reported 6 reported for A. marginata var. ovum by Akinnusi (1998, 2002, 2004) and and A. marginata var. saturalis Omole and Kehinde (2005) respectively by Ubua (2004). The respectively for black-skinned incubation period results of this study Archachatina marginata, but higher (Table 1) were lower than the range than the range value of 0.09 – 0.13g value of 30 to 37 days reported in Ubua reported by Hodasi (1979) for snailets (2004) for A. marginata var. ovum, but of A. achatina snail breed. The fell within the range values of 9 to 37 differences in the mating groups' for Achatina achatina snails and 7 to 30 snailets weights at hatch of this study days for A. marginata snails reported in and those in the literature could be Hodasi (1984) and Okon et al. (2011) attributed to differences in the snail respectively. The percent hatchability breeds, the sizes of parent snails used values of this study fell within the range and the sizes of eggs. This is inferred of 0 to 100 % reported by Awesu because snailets size at hatch is (1980), Ogogo (1989) and Odido positively correlated with the size of (2007) for black-skinned snails, while the parent snail and the eggs. This the percent survivability values were corroborated Omole et al. (2007) who very close to the value of 100 % established that the size of baby snails reported by Fapohunda et al. (2007). at hatch correlates positively with the The size of their parents. The mean weight differences between the results of this value at hatch of snailets of the study and those previously reported crossbred (BS X WS) mating group could be attributed to differences in the demonstrated hybrid vigour over the ages and sizes of snails used, genetic mean weight values of the purebred composition of the snail breeds, the (BS X BS and WS X WS) mating management techniques adopted and groups the prevailing environmental (Table 1). conditions. Results of parameters measured on eggs laid by the parent A. marginata

86 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails var. ovum snails are presented in Table numerically higher than that of the WS 2. The results showed that the mean egg X WS mating group. These results were weight values at lay for the BS X BS in line with the views of Ibom (2009), and BS X WS mating groups were that the black skinned snail ectotype

Table 2: Parameters measured on eggs laid by parent Archachatina marginata var. ovum snails.

Trait Mating group BS X BS BS X WS WS X WS Mean egg weight at lay (g) 1.15±0.03 1.20±0.03 1.01±0.02 Mean final egg weight (g) 0.95±0.02 0.97±0.03 0.76±0.01 a a b Mean egg length at lay (cm) 1.50±0.04 1.48±0.03 1.22±0.02 1.50±0.03a 1.48±0.02a 1.22±0.01b Mean final egg length (cm) Mean egg width at lay (c m) 1.23±0.03a 1.18±0.03a 0.97±0.01b Mean final egg width (cm ) 1.23±0.04a 1.18±0.03a 0.97±0.02b

BS = Black Skinned ect otype, WS = White Skinn ed ectotype. ab Means along the same row with different superscript are signific antly different (P<0.05).

IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails naturally lays larger (bigger) eggs than weights at lay for parities 1 and 2 the white skinned ectotype of the same respectively of albino A. marginata snail breed. The numerical differences snails. However, the mean egg weight between the mean egg weight values at values at lay of the BS X BS and BS X lay and before hatch (mean final egg WS mating groups were higher than weight) were not significantly different the mean value at lay (1.05g) reported (P>0.05). The mean egg weight values in Ibom et al. (2008) for albino A. at lay obtained in this study for the WS marginata snails, but lower than the X WS mating group was similar to the mean values of 1.80g and 3.0g reported mean values of 1.01 g and 1.04 g in Ibom et al. (2008) and Amubode reported in Okon et al. (2010) as (1994) respectively as weights at lay

87

for black skinned ectotype of A. mean egg width values at lay of this marginata snails. The mean egg length study were close to the mean values of and width values at lay and before 10.78 mm and 10.94 mm reported in hatch expressed significant differences Okon et al. (2010) for parities 1 and 2 (P<0.05) across the mating groups respectively of albino A. marginata (Table 2). The mean egg length and snails. The mean egg width values at width values at lay and before hatch lay of this study were higher than the remained the same in this study. The mean values at lay (1.29 mm) and (1.05 mean egg length values at lay obtained mm) reported in Ibom et al. (2008) for in this study were close to the mean black skinned and albino A. marginata values of 14.32 mm and 15.35 mm snails respectively. The differences in reported in Okon et al. (2010) for mean egg length and width values parities 1 and 2 respectively of albino recorded in this study with earlier A. marginata snails. However, the reports could be attributed to the age mean egg length values at lay of these and sizes of the snails used, differences mating groups were higher than the in egg sizes among the mating groups, mean values at lay (1.61 mm) and (1.43 incubation conditions like uptake of mm) reported in Ibom et al. (2008) for water and other prevailing black skinned and albino A. marginata environmental conditions like snails respectively. The mean egg temperature, relative humidity and soil length values at lay were lower than the conditions. mean values of 19.70 mm reported in Table 3 shows the results of parameters Amubode (1994) for black skinned measured on eggs laid by parent A. ectotype of A. marginata snails. The Table 3: Eg.g parameters of parent Archachatina marginata var. ovum snails during incubation

Trait Mating group Age (Week s) 0 1 2 3 4 Egg weight (g) BS X BS 1.15±0.03 1.10±0 1.05±0.02 0.99±0.02 0.95±0.02 .02 BS X WS 1.20±0.02 1.10±0 1.03±0.01 0.98±0.01 0.97±0.01 .01 WS X WS 1.01±0.02 1.00±0.01 0.98±0.01 0.80±0.01 0.76±0.01 Egg length (cm) 1.50±0.04 1.50±0.04 BS X BS 1.50±0.04 1.50±0.04 1.50±0.04 BS X WS 1.40±0.03 1.40±0 1.40±0.03 1.40±0.03 1.40±0.03 .03 WS X WS 1.22±0.02 1.22±0 1.22±0.02 1.22±0.02 1.22±0.02 .02

88 IBOM ET AL: Reproductive performance of Archachatina marginata var. ovum Snails Egg width (cm) BS X BS 1.23±0.02 1.23±0 1.23±0.02 1.23±0.02 1.23±0.02 .02 BS X WS 1.18±0.01 1.18±0.01 1.18±0.01 1.18±0.01 1.18±0.01

WS X WS 0.97±0.01 0.97±0.01 0.97±0.01 0.97±0.01 0.97±0.01

BS = Black Skinned ectotype, WS = Whi te Skinned ectotype. marginata var. ovum snails during developing embryo and exposure of incubation. The results showed that eggs to fluctuating environmental mean egg weight values decreased with conditions which differed from their age [from 1.15g at lay to 0.95g before near constant uterine environment hatch (BS X BS), 1.20g at lay to 0.97g (Tracey et al., 1978, Ibom, 2009). before hatch (BS X WS) and 1.01g at Unlike the mean egg weight values, the lay to 0. 76g before hatch (WS X WS) mean egg length and width values did mating groups] during incubation. not change with age, at lay (0 week) to Ibom (2009) opined that the decrease in before hatch (4 weeks). The trend in egg weight with age during incubation egg parameters (decreasing egg weight might be as a result of the values with age, near constant egg transformation of the embryonic liquid length and egg width values with age) mass into a solid shelled snailet, as well obtained in this study was in line with as the snailet feeding on the egg the findings of Ewa (2007) and Odido contents. It could also be as a result of (2007). uptake and loss of moisture, increased transpirational water loss resulting CONCLUSION from increased heat produced by the

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The results of this study showed that Reproductive Performance of the crossbred (BS X WS) mating group Two Species of African Giant performed numerically better in all the Snail (Achatina achatina and evaluated parameters. These results Archachatina marginata). could suggest that crossbreeding of the Journal of Tropical Forestry ectotypes will yield better offspring Resources. 9 and 10:68 – 74. which can be of benefit to farmers as Awesu, M. O. (1980). The biology and they yearn to get snails that will Management of the African produce more eggs per clutch, hatching Giant Land Snail (A. into bigger snailets with better survival marginata). M. Phil. Thesis. potentials, within shorter incubation University of Ibadan. periods. Further studies are therefore recommended. ( Unpublished ). Ebenebe, C. I. (2000). Mini livestock REFERENCES production in Nigeria. The present and future. Proceedings Akinnusi, O. (1998). Life history of the 5th Annual Conference of studies of Archachatina Animal Science Association of (Calachatina) marginata (Swainson). Proceedings of the Silver Jubilee Anniversary Conference of Nigerian Society for Animal Production. (NSAP). March 21 – 26, 1998. Abeokuta, Nigeria. Pp. 403 – 404. Akinnusi, O. (2002). Introduction to Snails and Snail Farming. Abeokuta, Nigeria. Triolas Exquisite Ventures. Pp. 89. Akinnusi, O. (2004). Introduction to Snails and Snail Farming. 2nd ed. Abeokuta, Nigeria. Triolas Exquisite Ventures. Pp. 90. Akintomide, T. O. (2004). Tropical snail farming. 1 s t . Ed., Abeokuta, Nigeria. Oak Ventures. P. 56. Amubode, F. O. (1994). Growth and

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