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1 저작자표시 - 비영리 - 변경금지 2.0 대한민국 이용자는아래의조건을따르는경우에한하여자유롭게 이저작물을복제, 배포, 전송, 전시, 공연및방송할수있습니다. 다음과같은조건을따라야합니다 : 저작자표시. 귀하는원저작자를표시하여야합니다. 비영리. 귀하는이저작물을영리목적으로이용할수없습니다. 변경금지. 귀하는이저작물을개작, 변형또는가공할수없습니다. 귀하는, 이저작물의재이용이나배포의경우, 이저작물에적용된이용허락조건을명확하게나타내어야합니다. 저작권자로부터별도의허가를받으면이러한조건들은적용되지않습니다. 저작권법에따른이용자의권리는위의내용에의하여영향을받지않습니다. 이것은이용허락규약 (Legal Code) 을이해하기쉽게요약한것입니다. Disclaimer

2 Thesis for the Degree of Master of Science Effect of dietary substitution of sea tangle (ST), Laminaria japonica with rice bran (RB) on growth and body composition of juvenile abalone, Haliotis discus Ye Eun Kim Department of Convergence Study on the Ocean Science and Technology Korea Maritime and Ocean University February 2016

3 Effect of dietary substitution of sea tangle (ST), Laminaria japonica with rice bran (RB) on growth and body composition of juvenile abalone, Haliotis discus Advisor: Prof. Sung Hwoan Cho Ye Eun Kim A dissertation submitted in partial fulfillment of the requirements for the degree of Master of Science Department of Convergence Study on the Ocean Science and Technology Korea Maritime and Ocean University February 2016

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5 CONTENTS Page Contents i List of Tables ii Abstract (in korean) iii Abstract v I. Experiment 1 1. Introduction 1 2. Materials and Methods Preparation of Abalone and Rearing Condition Preparation of the Experimental Diets Analytical Procedures of the Diets and Carcass Statistical Analysis 7 3. Results 9 4. Discussion 13 II. Conclusion 17 III. Acknowledgements 18 IV. References 19 - i -

6 List of Tables Table 1. Ingredients of the experimental diets 5 Table 2. Survival (%), weight gain (g/abalone) and specific growth rate (SGR) of juvenile abalone, Haliotis discus, fed the experimental diets substituting sea tangle (ST) with rice bran (RB) for 16 weeks 9 Table 3. Shell length (mm), shell width (mm), shell height (mm), soft body weight (g/individual) and the ratio of soft body weight to total weight of abalone, Haliotis discus at the end of the 16-week feeding trial 10 Table 4. Chemical composition of the soft body of abalone, Haliotis discus fed the experimental diets substituting sea tangle (ST) with rice bran (RB) for 16 weeks 11 - ii -

7 전복용배합사료내다시마대체원으로서 생미강대체에따른전복의성장 및체조성에미치는영향 Ye Eun Kim Department of Convergence Study on the Ocean Science and Technology Korea Maritime and Ocean University 요약 본연구에서는전복용배합사료내다시마대체원으로서생미강을이용하여전 복의성장및체조성에미치는영향을조사하였다. 실험에이용된전복치패는 실험조건에서 4 주간적응시킨후, 마리당평균 0.43 g의전복 1,260마리를무작 위로선별하여 18개의 70 L 플라스틱수용기에각각 70 마리씩수용하였다. 실 험사료는 1일 1 회만복수준으로사료를공급하였다. 총사육실험기간은 16주 간이었다. 총 6 종류의실험사료를준비하였다. 다시마분말을 20% 첨가하여제 조한대조구사료(RB0), 다시마를생미강으로대체한다시마대체 20% (RB20), 다시마대체 40% (RB40), 다시마대체 60% (RB60), 다시마대체 80% (RB80) 및 다시마대체 100% (RB100) 사료를준비하였다. 전복의생존률은모든실험용배 합사료를공급한실험구간의유의적인차이가나타나지않았으며, 증체량 (weight gain) 은 RB40을공급한실험구가 RB0을공급한실험구를제외한모든 실험구보다유의적으로높게나타났다. 또한 RB0과 RB100을공급한실험구사 - iii -

8 이에는유의적인차이가나타나지않았다. 일일성장률(Specific growth rate, SGR) 은 RB40을공급한실험구가다른모든실험사료를공급한실험구보다 유의적으로높게나타났다. 또한 RB0을공급한실험구의일일성장률은 RB100 을공급한실험구보다유의적으로높게나타났으나, RB20, RB60 및 RB80을공 급한실험구와는유의적인차이가나타나지않았다. 전복가식부의수분, 조단 백질, 회분함량은실험용배합사료에의해영향을받은것으로나타났다. 본 연구결과전복용배합사료내다시마를 20% 첨가시전복의증체량에부정적인 영향없이다시마를생미강으로 100% 까지완전히대체가능하며, 또한다시마 를생미강으로 40% 대체시가장우수한전복의성장률을보였다. Keywords: 전복 (Haliotis discus), 대체원, 다시마, 생미강 - iv -

9 Effect of dietary substitution of sea tangle (ST), Laminaria japonica with rice bran (RB) on growth and body composition of juvenile abalone, Haliotis discus Abstract Dietary substitution effect of sea tangle (ST) with rice bran (RB) on growth and carcass composition of juvenile abalone, Haliotis discus was determined. Juvenile abalone was acclimated to the experimental conditions for 4 weeks. Seventy Juvenile abalone averaging 0.43 g were randomly distributed into each of the 18, 70 L plastic rectangular containers. The experimental diets were fed to abalone once a day at a satiation level with a little leftover. The feeding trial lasted for 16 weeks. Survival of abalone was not affected by dietary substitution of ST with RB. However, weight gain of abalone fed the RB40 diet was higher than that of abalone fed the all other diets except for the RB0 diet. No significant difference in weight gain was found in abalone fed between the RB0 and RB100 diets. SGR of - v -

10 abalone fed the RB40 diet was higher than that of abalone fed all other diets. Also SGR of abalone fed the RB0 diet was higher than that of abalone fed the RB100 diet, but not different from that of abalone fed the RB20, RB60 and RB80 diets. Moisture, crude protein and ash content of the soft body of abalone were affected by dietary substitution of ST with RB. In conclusion, the 100% ST could be substituted with RB without a retardation of weight gain of abalone when the 20% ST was included into the experimental diet. The best growth performance was obtained in abalone fed the RB40 diet substituting 40% ST with RB. Keywords: abalone, Haliotis discus, dietary substitution, sea tangle, rice bran - vi -

11 1. Introduction An annual global production from abalone culture has increasing since 2000 (FAO 2013). Macroalgae such as sea tangle (ST), Laminaria japonica or Undaria are commonly used as feed for abalone culture in eastern Asia, but availability of these macroalgae is exclusively limited during winter season in wild. Therefore, dry or salted macroalgae are commonly supplied to abalone except for winter season. During dry or salting process of these macroalgae, their nutrients like protein (essential amino acid) and lipid (fatty acid) are likely to be destroyed and deficient. To date, many feeding trials have reported that supply of macroalgae produced an inferior growth rate of abalone to the nutrient-balanced feed (Uki et al., 1985; Viana et al., 1993; Lee et al., 1997; Kim et al., 1998; Bautista-Teruel et al., 2003; Cho et al., 2006; Cho et al., 2008; Garcial-Esquivel and Felbeck 2009; Dang et al., 2011). Therefore, development of the nutrient-balanced diet for abalone culture is highly needed. Animal protein source such as casein, fish meal and crustacean meal and plant protein source such as soybean meal were the good protein source for growth of abalone (Viana et al., 1993; Lee et al., 1998; Sales and Britz 2001; Cho et al., 2008; Cho 2010). Garcial-Esquivel and Felbeck (2009) recommended supplementation of kelp meal into the combined fish meal and soybean meal-based diet to improve growth of red abalone, Arthrospira maxima and microalgae, Dunaliella salina into the commercial diet improved growth rate greenlip abalone, H. laevigata

12 An international market price of macroalgae commonly used as feed for abalone has recently increased due to an expansion of the biofuel industry to develop them for ethanol production around the world and high demand for human consumption. Because these macroalgae is no longer to be a cheap feed ingredient in the commercial diet for abalone, development of an alternative feed ingredient that is both cheap and available year-round is highly needed for abalone culture. Donkey's ear abalone, H.asinina well utilized the high levels of carbohydrate rather than high levels of lipid in the diets to support its growth (Thongrod et al., 2003). Mai et al. (1995a) also explained that abalone, H. tuberculata and H. discus hannai seemed to have great potential for utilizing carbohydrate, which is a major component in its natural diet, for energy and perhaps other nutritional purposes. The combined terrestrial leaf meal, Moringa oliefera and freshwater aquatic fern, Azolla pinnata with animal protein (fish meal and shrimp meal) were the promising feed ingredients for the practical diet for the farmed abalone, H. asinina (Reyes and Fermin 2003). Rice bran (RB), one of the agricultural wastes, has a high potential as feed ingredient that is rich in the nutrients such as crude protein and vitamins (Gao et al., 2008), and cheap as a feed ingredient for abalone. In this study, therefore, dietary substitution effect of ST with RB on growth performance and carcass composition of juvenile abalone, Haliotis discus was determined

13 2. Materials and Methods 2.1. Preparation of Abalone and Rearing Conditions Juvenile abalone were purchased from a private hatchery and transferred to an abalone farm (Ocean and Fisheries Research Institute, Jeju Special Self-Governing Province, Jeju, Korea). Before an initiation of the feeding trial, abalone were acclimated to the experimental conditions for 4 weeks and fed with the dry sea tangle once a day at the ratio of 2-3% total biomass. Seventy juvenile abalone averaging 0.43 g (about 1 cm in shell length) were randomly distributed into each of the 18, 70 L plastic rectangular containers (120 cm 36 cm). Nine containers were placed into each of two 9 ton concrete flow-through raceway systems (water volume: 3 ton) as a flow rate of 48.3 L/min/raceway. The sand-filtered seawater at a temperature ranging from 16.7 to 21.8 C (mean ± SD: 18.4 ± 0.99 C) at 15:00 hours was supplied throughout the feeding trial. Aeration was supplied into each raceway and the photoperiod followed natural conditions. The experimental diets were fed to abalone once a day (17:00 hours) at a satiation level with a little leftover (about 2-3.5% biomass). Dead abalone was removed daily and the bottoms of the containers were siphon-cleaned daily. The feeding trial lasted for 16 weeks. At the end of the feeding trial, abalone was harvested and collectively weighed from each container Preparation of the Experimental Diets - 3 -

14 Six experimental diets in triplicate were prepared (Table 1). The 30% fish meal and 7% soybean meal were included into the RB0 diet as the primary protein source. And the 5% wheat flour and 8% dextrin, and 3% squid liver oil and 1% soybean oil were used as the carbohydrate and lipid sources, respectively in the RB0 diet. A 20% ST powder was included into the RB0 diet. The 20, 40, 60, 80 and 100% of ST powder were substituted with RB, referred to as the RB20, RB40, RB60, RB80 and RB100 diets, respectively. The experimental diets were satisfied for dietary protein and lipid requirements for abalone (Mai et al., 1995a; Mai et al., 1995b). Next, a 20% sodium alginate was added to all experimental diets. Thereafter, all the ingredients were mechanically mixed well and water added at a ratio of 1:1. A paste was made from each of the diets by using an electronic mixer and shaped into 0.15 cm thick sheets, which were then cut by hand into 1 cm 2 flakes. The flakes were then dipped into an aqueous solution of 5% CaCl 2 for minute and the excess solution was drained naturally. The flaked were then dried naturally for 2 days and stored at -20 C until use

15 Table 1. Ingredients of the experimental diets Experimental diets RB0 RB20 RB40 RB60 RB80 RB100 Ingredient (%, DM) Fish meal (CP:72.7%, CL:11.3%) Soybean meal (CP:53.5%, CL:2.0%) Wheat flour (CP:14.6%, CL:4.1%) Dextrin Sea tangle (ST) (CP:10.5%, CL:0.1%) Rice bran (RB) (CP:14.9%, CL:16.5%) Squid liver oil Soybean oil Sodium alginate Mineral premix Vitamin premix Nutrients (%, DM) Dry matter Crude protein Crude lipid Ash Mineral premix contained the following ingredients (g/kg mix): NaCl, 10, MgSO 4 7H2O, 150; NaH 2 PO 4 2H2O, 250; KH 2 PO 4, 320; CaH 4 (PO 4 ) 2 H2O, 200; Ferric citrate, 25; ZnSO 4 7H2O, 4; Ca-lactate, 38.5; CuCl, 0.3; AlCl 3 6H2O, 0.15; KIO 3, 0.03; Na 2 Se 2 O 3, 0.01; MnSO 4 H2O, 2; CoCl 2 6H2O,

16 2 Vitamin premix contained the following amount which were diluted in cellulose (g/kg mix): L-ascorbic acid, 200; α-tocopheryl acetate, 20; thiamin hydrochloride, 5; riboflavin, 8; pyridoxine, 2; niacin, 40; Ca-D-pantothenate, 12; myo-inositol, 200; D-biotin, 0.4; folic acid, 1.5; p-amino benzoic acid, 20; K 3, 4; A, 1.5; D 3, 0.003; choline chloride, 200; cyanocobalamin,

17 2.3. Analytical Procedures of the Diets and Carcass Thirty abalone at the start and twenty abalone from each container at the termination of the feeding trial were sampled and frozen for chemical analysis. Prior to examination, all samples were slightly thawed, followed by separation of the shell and soft-body tissue. Shell length and shell width were measured in mm with a digital caliper (Mitutoyo Corporation, Kawasaki, Japan), and the ratio of soft body weight to body weight (the soft body weight + the excised shell's weight) was calculated to determine an index of nutritional status for abalone. Specific growth rate (SGR, % body weight gain/day) was calculated using the formula of Britz (1996): SGR = [(ln(wf) - In(Wi))/days of feeding] 100, where In(Wf) = natural log of the final mean weight of abalone and In(Wi) = natural log of the initial mean weight of abalone. The separated soft body tissue from all abalone from each container was then homogenized and used for proximate analysis. Crude protein content was determined by the Kjeldahl method (Auto Kjeldahl System, Buchi B-324/435/412, Switzerland), crude lipid was determined using an ether-extraction method, moisture was determined by oven drying at 105 C for 24 h, and ash was determined using a muffle furnace at 550 C for 4 h. All methods were according to standard AOAC (1990) practices Statistical Analysis One-way ANOVA and Duncan`s multiple range test (Duncan 1955) were used to determine the significance of the differences among the means of - 7 -

18 treatments by using SAS version 9.3 (SAS Institute, Cary, NC, USA). Percentage data was arcsine transformed prior to statistical analysis

19 3. Results Survival of abalone was all over 80%, but not significantly (P > 0.05) affected by dietary substitution of ST with RB (Table 2). However, weight gain of abalone fed the RB40 diet was significantly (P < 0.05) higher than that of abalone fed the all other diets except for the RB0 diet. However, no significant difference in weight gain was found in abalone fed between the RB0 and RB100 diets substituting 100% ST with RB. SGR of abalone fed the RB40 diet was significantly (P < 0.05) higher than that of abalone fed the all other diets. And SGR of abalone fed the RB0 diet was significantly (P > 0.05) different from that of abalone fed the RB20, RB60 and RB80 diets. However, none of shell length, shell width and the ratio of soft weight to total weight of abalone at the end of 16-week feeding trial was significantly (P > 0.05) affected by dietary substitution of ST with RB (Table 3). Moisture content of the soft body of abalone fed the RB20 and RB100 diets was significantly (P < 0.05) higher than that of abalone fed the RB0, RB40, RB60 and RB80 diets (Table 4). Moisture content of the soft body of abalone fed the RB80 diet was significantly (P > 0.05) higher than that of abalone fed the RB0, RB40 and RB60 diets. Crude protein content of the soft body of abalone fed the RB60 diet was significantly (P < 0.05) higher than that of abalone fed the all other diets. The lowest crude protein content was obtained in abalone fed the RB80 diet. However, crude lipid content of the soft body of abalone fed the RB0 diet was significantly (P < 0.05) higher than that of abalone fed the all other diets. The lowest ash content was obtained in abalone fed the RB100 diet

20 Table 2. Survival (%), weight gain (g/abalone) and specific growth rate (SGR) of juvenile abalone, Haliotis discus fed the experimental diets substituting sea tangle (ST) with rice bran (RB) for 16 weeks Experimental Initial weight Final weight Survival Weight gain SGR 1 diets (g/abalone) (g/abalone) (%) (g/abalone) (%/day) RB ± ± ± ± ab ± b RB ± ± ± ± b ± bc RB ± ± ± ± a ± a RB ± ± ± ± b ± bc RB ± ± ± ± b ± bc RB ± ± ± ± b ± c F-value P-value P > 0.9 P < 0.03 P < Values (means of triplicate ± SE) in the same column sharing a common superscript are not significantly different (P > 0.05). 1 Specific growth rate (SGR) = [(Ln(Wf) - Ln(Wi))/days of feeding] 100, where In(Wf) = natural log of the final mean weight of abalone and In(Wi) = natural log of the initial mean weight of abalone

21 Table 3. shell length (mm), shell width (mm), shell height (mm), soft body weight (g/individual) and the ratio of soft body weight to total weight of abalone, Haliotis discus at the end of the 16-week feeding trial Experimental diets Shell length (mm) Shell width (mm) Shell height (mm) Soft body weight (g) Soft body weight/total weight RB ± ± ± ± ± RB ± ± ± ± ± RB ± ± ± ± ± RB ± ± ± ± ± RB ± ± ± ± ± RB ± ± ± ± ± F-value P-value P > 0.9 P > 0.9 P > 0.9 P > 0.5 P > 0.4 Values (means of triplicate ± SE) in the same column sharing a common superscript are not significantly different (P > 0.05). None of biological parameters of abalone measured was significantly different among treatments

22 Table 4. Chemical composition (%, wet weight basis) of the soft body of abalone, Haliotis discus fed the experimental diets substituting sea tangle (ST) with rice bran (RB) for 16 weeks Experimental diets Moisture Crude protein Crude lipid Ash RB ± 0.09 c 18.9 ± 0.06 bc 2.7 ± 0.02 a 3.4 ± 0.06 a RB ± 0.37 a 18.8 ± 0.05 bc 2.6 ± 0.04 a 3.2 ± 0.06 b RB ± 0.08 c 18.9 ± 0.03 b 2.6 ± 0.05 a 3.2 ± 0.03 b RB ± 0.09 c 19.2 ± 0.04 a 2.5 ± 0.03 a 3.2 ± 0.02 bc RB ± 0.10 b 18.7 ± 0.03 c 2.7 ± 0.01 a 3.2 ± 0.02 bc RB ± 0.15 a 18.9 ± 0.10 bc 2.7 ± 0.05 a 3.0 ± 0.02 c F-value P-value P < P < P > 0.1 P < Values (means of triplicate ± SE) in the same column sharing a common superscript are not significantly different (P > 0.05)

23 4. Discussion A commercial formulated diet might be suitable for juvenile greelip abalone, H. laevigata larger than 4 mm in shell length (Daume and Ryan 2004). Later, Daume et al. (2007) also recommended that moving greenlip abalone into a tank system to feed formulated feed rather than an algal diet consisting of diatoms or macroalgae once they reach 7 mm in shell length. Since an initial size of abalone used in this study was 1 cm in shell length, abalone grew well on the experimental diets. Mai et al. (1995a) reported that improvement in weight gain of abalone, H. discus hannai fed the diets containing % lipid levels was observed when abalone was fed by one of the 25% protein diets with 0.63% to 11.58% lipid levels. Similarly, Green et al. (2011) reported that South African abalone, H. midae was unable to effectively utilize lipid as an energy source at levels in excess of 7% due to inherent physiological constraints. Therefore, abalone fed the RB0 diet containing 28.8% crude protein and 7.4% crude lipid seemed to grow relatively well in this study. No significant difference in weight gain of abalone fed the RB100 diet compared to the RB0 diet in this study indicated that complete substitution of ST with RB could be made without retardation of growth of abalone when the 20% ST was included into the combined fish meal and soybean meal-basal diet. However, no difference and significant difference in SGR of abalone fed the RB80 and RB100 diets compared to that of the RB0 diet, respectively in this study indicated that the 80% ST could be substituted with RB in the diet in terms of SGR of abalone. In addition, slightly and

24 significantly improvement in weight gain and SGR abalone fed the RB40 diet compared to those of abalone fed the RB0 diet, respectively indicated that abalone grew better on the diet substituting 40% ST with RB rather than the RB0 diet containing the 20% ST exclusively. Abalone species, H. discus used in this study seemed to utilize RB as well as ST in the experimental diets. Abalones are known to be herbivorous and feed mostly on macroalgae, which is usually low in lipid but high in carbohydrate, 40-50% (Thongrod et al., 2003). Because RB contained 40.5% carbohydrate calculated by the equation of the difference between 100 and sum of crude protein, crude lipid and ash content, RB seemed to be a good feed ingredient to replace ST of abalone feed. Fleming et al. (1996) also reported that abalone has various enzymes capable of hydrolyzing complex carbohydrates. Similarly the leaf meal, M. oliefera and freshwater aquatic fern, A. pinnata culture in terns of local availability of year round in Philippines (Reyes and Fermin 2003). Because macroalgae used for abalone culture in the past is not a cheap feed ingredient anymore, the development of the alternative feed ingredient (plant meal) that is both cheap and year-round available keeps being needed, In the earlier study (Cho et al., 2008), the possibility of dietary supplementation of byproduct of green tea for abalone (H. discus hannai) was reported. Three different species abalone, H. discus, H. sieboldii and H. discus hannai grew better on the commercial or formulated diet than the macroalgae feed, Undaria reared at C in the 9-week feeding (Kim et al., 1998). The reason for poorer growth of abalone fed the latter

25 compared to the former might be that nutrient content in the latter does not satisfy dietary nutrient requirement for abalone, such as protein and lipid. Similarly, growth of abalone fed the formulated or commercial diet was faster than that of abalone fed the single macroalgae (Lee et al., 1997; Viana et al., 1993; Garcia-Esquivel and Felbeck 2009). However, supplementation of microalgae, Arthrospira maxima and macroalgae, Dunaliella salina into the commercial feed improved weight gain and immunity of greenlip abalone, whereas Ulva lactuca and Spyridia filamentosa could be useful supplements for abalone aquaculture, especially in areas with high risk of herpesvirus infection (Dang et al., 2011). Unlike growth performance of abalone, the distinctive biological parameters of abalone (shell length, shell width, shell height, soft body weight and the ratio of soft body weight to total weight of abalone) was not affected by dietary substitution of ST with RB in this study. Similarly, the biological parameters of abalone would not be different among treatments although weight gain of abalone was significantly affected by dietary lipid level (Green et al., 2011). Unlike this study, However, the biological parameters of abalone measured agreed with weight gain of abalone (Bautista-teruel et al., 2003; Cho 2010). Proximate composition of the soft body of abalone was affected by dietary substitution of ST with RB except for crude lipid content in this. Ash content of the soft body of abalone was relatively well reflected from that of the experimental diets. Proximate composition (Mai et al., 1995a, Thongrod et al., 2003; Cho et al., 2008; Gracia-Esquivel and Felbeck 2009;

26 Cho 2010). Substitution effect of ST with RB in the commercial diet for abalone farm will be reported shortly. In conclusion, the 100% ST could be substituted with RB without a retardation of weight gain of abalone when the 20% ST was included into the experimental diet. However, the best growth performance was obtained in abalone fed the RB40 diet substituting 40% ST with RB

27 Ⅱ. Conclusion The 100% sea tangle (ST) could be substituted with rice bran (RB) without a retardation of weight gain of juvenile abalone, H. discus when the 20% ST was included into the experimental diet. The best growth performance was obtained in abalone fed the RB40 diet substituting 40% ST with RB

28 Ⅲ. Acknowledgments I would like to express my sincere gratitude to Prof. Sung Hwoan Cho, Department of Convergence Study on the Ocean Science and Technology Korea Maritime and Ocean University, Korea for his invaluable advice and continuous encouragement throughout this study. I wish to thanks to my committee members, Prof. Sang Min Lee and Cheol Young Choi for their critical advices for my thesis. For my lab seniors and juniors, Mr. Sang Mok Lee, Byum Hee Park, Sung Choon Ji, Chung Il Kim, Young Jin Cho, In-Cheol Choi, Kyoung Tae Kim, Hee Sung Kim, Gyu Ho Jeon, Won Gwan Jung, Hyeon Jong Kim, Dong Gyu Choi, Bok-Il Jang, Ki Wook Lee, Sang Hyun Lee and, Miss Ah Young Yoon, Min Ju Kim and June Kim at the Feed Nutrition and Engineering Lab in Korea Maritime and Ocean University. I warmly thank for their help at practical things as well as sharing the good and bad moments of research and life. Finally, I would like to express my hearty thanks to my family who have incessantly loved, encouraged and supported me

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