Department: Biotechnology Supervisor: Prof Xu Pao EFFECT OF DIETARY ALOE (ALOE VERA) ON SEX RATIO, GROWTH AND PHYSIOLOGICAL RESPONSES IN GIFT-TILAPIA AFTER STREPTOCOCCUS INIAE CHALLENGE (MSc-Thesis Defense) By Ndakalimwe N Gabriel Student No. 2013113050 Major: Aquaculture Department: Biotechnology Supervisor: Prof Xu Pao Sept. 2013-Dec. 2015 This template can be used as a starter file for presenting training materials in a group setting. Sections Sections can help to organize your slides or facilitate collaboration between multiple authors. On the Home tab under Slides, click Section, and then click Add Section. Notes Use the Notes pane for delivery notes or to provide additional details for the audience. You can see these notes in Presenter View during your presentation. Keep in mind the font size (important for accessibility, visibility, videotaping, and online production) Coordinated colors Pay particular attention to the graphs, charts, and text boxes. Consider that attendees will print in black and white or grayscale. Run a test print to make sure your colors work when printed in pure black and white and grayscale. Graphics, tables, and graphs Keep it simple: If possible, use consistent, non-distracting styles and colors. Label all graphs and tables.

Outline Research Objectives Introduction Experiment 1 Experiment 2 Sex ratio Growth performance & feed utilization Digestive enzymes Haematological parameters Biochemical parameters Introduction Experimental Designs Data analysis Results Give a brief overview of the presentation. Describe the major focus of the presentation and why it is important. Introduce each of the major topics. To provide a road map for the audience, you can repeat this Overview slide throughout the presentation, highlighting the particular topic you will discuss next. Conclusion of the study Publications Acknowledgements

Research Objectives To determine the effect of dietary Aloe vera extracts on sex ratio of sexually undifferentiated GIFT-O. niloticus fry. To investigate the potential effects of dietary A. vera on growth performance, digestive enzymes, & proximate chemical composition in GIFT-O. niloticus Juveniles. To investigate the effect of dietary A. vera on the physiological parameters of GIFT-O. niloticus juveniles when exposed to bacterial pathogen, Streptococcus inae.

Experimental design Experiment 1 The experiment was a one factorial design, whereby 150 first feeding fry (9 days, post hatch) were randomly allocated to 15 tanks after acclimatization, with 3 tanks assigned as replicates per group (0, 0.5, 1.0, 2.0, 4.0% A. vera powder/kg basal diet). The experimental diet started after stocking, 4 times a day until satiation for 30 days. G1 No Aloe Control G2 0.5% Aloe G3 1.0% Aloe G4 2.0% Aloe G5 4.0% Aloe This is another option for an overview slide. After 30 days, experimental diet was withdrawn & was replaced with a commercial diet & fed for 6 weeks. Then, 70 fish were randomly sampled from each tank. Establish sex ratio (♂ :♀)

Growth & feed utilization assessment Muscle prox. Chemical analysis Experiment 2 25 GIFT tilapia juveniles were randomly allocated to 15 tanks after acclimatization, with 3 tanks assigned as replicates per treatment. The experimental diet started after stocking, 3 times a day until satiation for 60 days. G1 No Aloe Control G2 0.5% Aloe G3 1% Aloe G4 2% Aloe G5 4% Aloe After 60 days, 15 fish from each tank were inoculated with S. inae pathogen & were sampled after every 48 hrs. for 96hrs After 60 days fish were randomly sampled from each tank. Growth & feed utilization assessment Muscle prox. Chemical analysis Haemato-biochemical analysis Digestive enzymes

Data Analysis All results for each parameter measured were expressed as mean  standard error (MSE). Data were analyzed using one-way ANOVA) with Aloe vera inclusion levels as a factor, using (SPSS). Duncan’s multiple range tests at a significant level of 5% was used to determine significant differences between treatments. The second order polynomial regression model (Zeitoun et al., 1976) was used to estimate the optimum dietary aloe requirement in GIFT-tilapia juveniles. Meanwhile, a Chi-squared analysis was used to test for significant deviation from the expected 1♂ :1♀sex ratio at a 5% probability level for each treatment.

Results Sex ratio

Growth performance & feed utilization

2nd polynomial analysis: Optimum inclusion level

Digestive enzymes

Haematological parameters: immune response Fig. 2: RBCs (A), Hb (B), Ht (C), MCV (D), MCH (E), and MCHC (F) of Gift-O. niloticus fed four Aloe vera supplemented diets (0.5, 1, 2, 4% /kg feed) and unsupplemented diet at initial sampling (pre-infection), 48 hours and 96 hours after S. iniae challenge

Haematological parameters cont..:immune response Fig. 3. WBCs (G), neutrophils (H), lymphocytes (I), monocytes (J), and eosinophils (K); and neutrophil to lymphocytes count ratio (NLCR) (L) of GIFT O. niloticus fed Aloe vera powder supplemented diet at different levels before and after S. iniae challenge.

Biochemical parameters: immune response Fig 4. Serum total protein content (M), lysozyme activity (N), glucose (O) and cortisol concentration (P) in GIFT-O. niloticus fed Aloe vera powder supplemented diet at different levels, before and after S. iniae challenge.

Biochemical parameters cont….: antioxidation

Biochemical parameters cont….: antioxidation Fig. 5: Liver CAT (S), GSH-Px (T), SOD (U), MDA (V) activity in GIFT-O. niloticus fed Aloe vera powder supplemented diet at different levels, before and after S. iniae challenge.

Biochemical parameters cont….: hepatoprotection Fig. 6: Plasma AST (W) and ALT (X) activity in GIFT-O. niloticus juveniles fed A. vera diet and control diet, pre and post S. iniae challenge.

Conclusion of the study Aloe extracts have the potential to be used as a sex reversal agent in tilapia farming. However, more research: purification, types of extracts, mode of administration, and proper treatment regimes is deemed necessary to yield 100% male tilapia and reduce mortality. Aloe extracts especially around 2%/kg diet could be used as growth promoters, appetizers, immunostimulants, antioxidants, & hepatoprotective agents in tilapia culture. But, inclusion level greater than 2% may affect fish survival rate & growth parameters and may lead to vulnerable fish which hardly cope with stress.

Publications 1. Full length research article: Dietary Aloe vera supplementation on growth performance, some haemato-biochemical parameters and disease resistance against Streptococcus iniae in tilapia (GIFT). Fish & Shellfish Immunology, 2015, 44: 504-514, (First Author) 2. Full length research article: Dietary Aloe vera improves plasma lipid profile, antioxidant, and hepatoprotective enzyme activities in GIFT-tilapia (Oreochromis niloticus) after Streptococcus iniae challenge. Fish Physiology & Biochemistry, 2015 DOI 10.1007/s10695-015-0088-z, (First Author). 3. Review article: Use of herbal extracts in controlling reproduction in tilapia culture: trends and prospects - A review. The Israeli Journal of Aquaculture-Balmidgeh, IJA_67.2015.1178, 29 pages, (First Author). 4 Full length research article: Dietary supplementation of Aloe vera crude extracts on sex ratio, digestive enzyme activities and muscle proximate composition in GIFT-tilapia (Oreochromis niloticus). Fish Physiology & Biochemistry, revised, (First Author). 5. Full length research article: Changes in the physiological parameters, fatty acid metabolism, and SCD activity and expression in juvenile tilapia (Oreochromis niloticus) reared at different temperature. Fish Physiology & Biochemistry, 2015, DOI 10.1007/s10695-015-0059-4, (Co-author). 6. Full length research article: Effect of Feeding-Intensity Stress on Biochemical and Hematological Indices of GIFT Tilapia (Oreochromis niloticus). Turkish Journal of fisheries & Aquatic Sci, ( Co-author).

Acknowledgements The people and institutions: Nanjing Municipal Government & Nanjing Agricultural University for their sincere financial support towards tuition fees. Namibian Government Scholarship & Training Program (NGSTP) for their valuable financial support towards accommodation fees and living costs during my study in China. Wuxi Fishery College of Nanjing Agricultural University International Training office staffs in Wuxi, China, particularly Prof. Yuan Xinhuan, Mr Jing Xiaojun, and Ms. Zhang Lin for their unwavering support from the very beginning to the end of this project. My supervisor Prof. Xu Pao for his fatherly love and sincere academic support; your leadership skills and commitment to your busy schedule is an inspiration to me, God bless you abundantly. Our research team (Dr. Jun Qiang, Ms. Ma Xin Yu, Mr. He Jie, Dr. Mathew D Kpundeh & Ms. M. Omyia), particularly Dr. Jun Qiang for his patience, valuable guidance in experimental set up, and procuring of all the required research materials; Ms. Ma Xin Yu for her loyal and continuous support especially when I needed a helping hand during sampling and guidance in the lab.

The end Comments & questions are most welcome Thank you!