| Vitamin B12,also called Cobalamin,is the only vitamin containing metal elements.The Vitamin B12 in nature is synthesized by microorganisms.Higher animals and plants can't produce vitamin B12,and there is little vitamin B12 in plants.Brachionus calyciflorus is a good natural diet for freshwater fish and an important member of the food chain in natural water body.As an open biological diet,it owns the advantages of comprehensive nutritional value,rapid reproduction,and easy for mass cultivation.In addition,free from contamination,it can meet the nutritional requirements of different fish,and greatly improve the seedling survival rate.However,in the actual production process,the peak period of rotifer population is difficult to control,and the supply in the seedling breeding stage is seriously insufficient,the seedling production efficiency will be affected greatly.Therefore,it is very important to explore how to increase the density of rotifer and prolong the duration of rotifer peak period in seedling production.In combination with the reproductive biological characteristics of rotifers,this thesis conducts the research on the effects of the Vitamin B12 on the rotifer population dynamics with the aim to provide a theoretical reference for the actual rotifer production.The main research findings are as follows:1.The effects of Vitamin B12 at different temperatures and concentrations on population parameters of Brachionus calyciflorusThe experimental condition is as follows:the light intensity is about 4 OOOlx;the day length ratio is L:D=16:8;the density of the fed Chlorella pyrenoidosa is 3.0×106 cells/mL.When the temperature was 20 ? after 24 hours' inoculation,the population growth rates(r)of the treatment group with the VitaminB12 of 0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1.0?g/ml were 0.26,0.36,0.33,and 0.35 respectively,which were significantly higher than that of the control group(0.057)(p<0.05).After 48 days,the population began to show a downward trend.The population growth rates(r)of the treatment group with the VitaminB12 of 0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1.0 ?g/ml were 0.21,0.34,0.28,and 0.40 respectively,which were still higher than that of the control group(0.12).The growth rate of the treatment group with the concentration of 1.0 ?g/ml Vitamin B12 after one day to five days was obviously better than that of the control group and other groups.When the temperature was 25 ?,each group showed a rapid increase trend in the first three days.On the 3rd day,the control group began to decrease.The population growth rate of the other treatment groups increased slowly from the third day to the fifth day.On the fifth day,the r values of the control group and the treatment groups with 0?g/ml,0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1.0?g/ml were 0.76,0.89.0.91,0.92,and 0.93 respectively.The growth rates of each treatment group were significantly higher than that of the control group.From the 9th day,the growth rate of each group gradually decreased(lower than that of the first day).The population growth rate of the 10th day was lower than that of the first day,and the growth rate of the experimental group with the concentration of 0.2 ?g/ml was the lowest.The 0.8?g/ml treatment group was significantly higher than that of the control group(P<0.05).On the tenth day,the population density of each concentration group was as follows:10.69±0.02,11.82±0.10,5.79±0.02,5.79±0.04,and 3.39±0.04ind./ml.The difference of population density was significant(p<0.05).At 30?,every experimental group increased rapidly on the first day,and showed a tendency of slow increase on the second day.On the third day,the population growth rate reached the peak.The growth rate(r value)of the control group and each concentration group were 1.18,1.26,1.37,1.32 and 1.40 respectively.After the third day,every experimental group showed a decrease tendency.At the fifth day,the population began to decline and the growth rate was lower than that of the first day.The growth rates(r value)of each concentration group were 0.97.0.90,1.09,and 1.03 respectively,but all the vitamin B12 treatment groups were higher than that of the control group(0.88%).The growth rate of the control group has been in the lowest state since the third day.2.The Effects of different concentrations of Vitamin B12 on the average lifespan expectancy of Brachionus calyciflorusThe experimental condition is as follows:the light intensity is about 40001x;the day length ratio is L:D=16:8;the temperature is 25?;the density of the fed Chlorella pyrenoidosa is 3.0 ×106 cells/mL.The experimental findings of the effects of different vitamin B12 concentrations on the lifespan of Brachionus calyciflorus are as follows:The average lifespan of the Chlorella pyrenoidosa in the experimental group with concentration of 1 ?g/ml was the longest(137.3 ±6.6 h).In compared with the control group,the Rotifera's average lifespan of each vitamin treatment group was significantly prolonged.The average life spans of the Chlorella pyrenoidosa with the experimental concentration of o?g/ml,0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1 ?g/ml were 97.33±2.56 hours,101.00±6.039 hours,97.33±2.906 hours,108.66±5.129 hours,and 99.000±4.698 hours respectively.The total lifespan of rotifers were prolonged with the increase of the Vitamin B12 concentration.3.The Effects of different concentrations of Vitamin B12 on Mortality Table Parameters of Brachionus calyciflorusThe experimental condition is as follows:the light intensity is about 4000lx;the day length ratio is L:D=16:8;the temperature is 25?;the density of the fed Chlorella pyrenoidosa is 3,0 ×106 cells/mL.The experiment conducted the research on the Mortality Table parameters,including net reproductive rate(R0),generation time(T),intrinsic growth rate(rm).From the results of the experiment,we drew the conclusions as follows:The net reproductive rate(R0)showed an upward trend with the increase of vitaminB12 concentration.The net reproduction rates of 0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1.0?g/ml in each experimental group were 6.66 ±0.41,0.66 ±0.55,6.88 ±0.51,and 6.39 ±0.57 respectively,which were higher than those of the control group(6.18±0.74).In the group with the concentration of 0.8?g/ml,RO showed the maximum value:6.88 ±0.51.The generation time(T)and the intrinsic rate of increase(rm)were all higher than those of the control group.The generation time(T)was higher than that of the control group(71.75 ±1.13),except for 0.40 ?g/ml(68.93 ±0.26).The intrinsic rates of increase(rm)of each experimental group were 0.03 ±0.001,0.03 ±0.007,0.03 ±0.001,and 0.022 ±0.001 respectively.The general trend of the intrinsic rate of increase was an increase with the increase of concentration,while the growth rate of high concentration group had a fall.However,it was still higher than that of the control group(0.02 ±0.001).In general,the parameters of the three Mortality Tables increased with the increase of concentration.The experimental results showed that at 108 hours,the lifespan of each concentration group was significantly different.Before 120 hours,all the life spans of the concentration were higher than the control group.After 156 hours,the experimental groups with 0.2 ?g/ml concentration were all higher than the rest of groups.However,the trend was basically the same.4.The Effects of VitaminB12 on the average offspring numbers and the prophase reproduction duration of the Brachionus calyciflorusThe experimental condition is as follows:the light intensity is about 4000lx;the day length ratio L:D=16:8;the temperature is 25?;the density of the fed Chlorella pyrenoidosa is 3.0 ×106 cells/mL.With the increase of the concentration of vitamin B12,the average offspring numbers of the Brachious calyciflorus increased.In addition.the duration from hatching to laying the first egg prolonged with the increase of concentration.The average offspring numbers of the experimental groups of O?g/ml,0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1?g/ml were 14.50 ± 1.40,15.33 ±0.60,16.00 ±0.23C,16.67 ±0.36,and 19.83 ±0.19ind respectively.In comparison with the control group,the offspring numbers of 0.2?g/ml,0.4?g/ml,0.8?g/ml,and 1?g/ml increased by 5.52%,10.34%,14.94%,and 36.78%respectively.The difference was significant(P<0.05).The prophase reproduction duration of the Brachionus calyciflorus with the concentration of 1?g/ml was the longest(21.000±0.34h).The prophase reproduction durations of the rest experimental group were 16.33±0.18.16.67±0.11,17.00±0.06h respectively,which was higher than the control group(15.67±0.30h).Therefore,it can be concluded that vitamin B12 in the high concentration group can affect the average offspring numbers and the prophase reproduction duration of the Rotifers.In combination with other experiments,it can be seen as follows:although the high concentration group takes a long time in the early stage,the vitamin B12 can effectively increase the offspring number of rotifers and keep it at a relatively stable height.5.The Effects of Vitamin B12 on the expressive quantity of the CAT Gene,MnSOD Gene,and HSP60 Gene of the Brachionus calyciflorusIn this research,the purpose of the real-time quantitative PCR was to explore whether the nutritional enhancement of vitamin B12 could increase the expression quantity of the Genes of CAT,MnSOD,and HSP60 so as to observe the effects of vitamin B12 on the antioxidant activity of the Rotifers.The experimental condition is as follows:the light intensity is about 4000lx;the day length ratio L:D=16:8;the temperature is 25?;the density of the fed Chlorella pyrenoidosa is 3.0 ×106 cells/mL.From the analysis of the experimental results,we can see that vitamin B12 could promote the expression of the genes of CAT,MnSOD,and HSP60 of the Rotifers.In addition,they showed an increasing trend roughly with the increase of the experimental concentration.The expressive quantities of the Rotifers,CAT Gene in the experimental group with 0.2? g/ml,0.4?g/ml,0.81 ?g/ml,and 1 ?g/ml were 0.07,2.22,6.11,and 7.56 respectively.The expressive quantities of CATmRNA in rotifers'bodies were significantly up-regulated(P<0.05);the expressive quantities of MnSOD Gene were 5.98,6.82.6.58,and 10.20 respectively.The expressive quantities of mRNA in rotifers' bodies were significantly up-regulated(P<0.05);the expressive quantities of HSP60 Gene of Rotifers were 5.54,9.25,11.8,and 8.34;the expressive quantity of HSP60mRNA in rotifers' bodies showed a significant up-regulated tendency(P<0.05).Therefore,we hold that vitamin B12 can stimulate the Genes of CAT.MnSOD.and HSP60 in the Rotifers' bodies to some extent so that the antioxidant activity is enhanced during the course of rotifers' metabolism.In addition,the higher the concentration of vitamin B12 is,the more obvious the rising trend is.This indicates that the addition of vitamin B12 for the large-scale cultivation of the rotifer has a certain effect on the population and lifespan of the rotifers. |
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