| Objectives:To assess the influence of rh LF expressed by mammary gland bioreactor of transgenic cows on immune function and neurobehavior of offspring rats. According to extended one-generation reproductive toxicity study protocol(OECD443), the experiment was utilized to assess the safety of Recombinant human lactoferrin.Methods:1.1Amino acid detection of rh LF and compositional analysis of rh LF based breed dietsAmino acid detection of rh LF was determined in accordance with Chinese Standard methods.Recombinant human lactoferrin replaced all protein of standard AIN-93 G feed, and it was regarded as feed of rh LF group. standard AIN-93 G feed was as feed of control group.1.2Effects of recombinant human lactoferrin on immune function of offspring ratsAccording to extended one-generation reproductive toxicity study protocol( OECD443), the experiment of immune function was conducted. The focused research was effects of recombinant human lactoferrin on immune function of offspring rats,when recombinant human lactoferrin(rh LF) were fed to parental rats for 14 days prior mating and throughout pregnancy and lactation. Immune function of offspring rats was assessed on PND0, PND21 and PND56.Sixty female and thirty male Wistar rats were 11 weeks of age at the start of the study. All animals were assigned to acclimate to the the experimental housing condition for 7 days. At the beginning of the study, animals were divided into two groups, including control group and rh LF group. Diets containing 20% casein or recombinant human lactoferrin(rh LF) were fed to parental rats for 14 days prior mating and throughout pregnancy and lactation. Offspring rats were randomly selected each group and fed with corresponding diets from weaning to execution. Each group should contain at least 20 pregnant females. Immune function of offspring rats on PND0 was evaluated by percentage of lymphocyte subsets. On PND4, litters were randomly adjusted to eight pups comprising of four males and four females. It should ensure that each group of 20 nests have 10 female rats 10 male rats. Body weight was measured on PND7,PND14 and PND21. Immune function of offspring rats on PND21 was evaluated by percentage of lymphocyte subsets. It should ensure that each group of 20 nests have 10 female rats 10 male rats. Immune function of offspring rats on PND0 was evaluated by percentage of lymphocyte subsets. Immune function of offspring rats on PND56 was evaluated by percentage of lymphocyte subsets and immune function test.This study consulted The ICH S8 immunotoxicity guidance. And the immunological endpoints included: blood routine test, blood biochemistry, flow cytometric analysis of lymphocyte subsets of blood, spleen, and Peyer’s patch, natural killer(NK) cell activity of splenocytes in response to Con A, PFC and pathology. The immunological endpoints covering the humoral immunity, cellular immunity and nonspecific immunity1.3Effects of recombinant human lactoferrin on neurobehavior of offspring rats1.3.1 Early physical development indicators of the offspring ratsEarly physical development indicators of the pups such as the survival rate of offspring rats, sex ratio, Teething time,the separating time of ear,the time of opening eyes and the time of having hair were all observed and evaluated.1.3.2 Early neural development indicators of the offspring ratsEarly neural developmental parameters of the offspring rats such as surface righting, cliff avoidance, Early neural developmental parameters of the F1 pups such as cliff avoidance, surface righting, air righting,forelimb hanging and acoustic startle were all observed and evaluated. The day on which all the pups of a litter attached the criteria for indicators were recognized as the day of reach the standard day. All pups from no less than 20 dames from each group were observed for parameters above mentioned.1.3.3 Sensory-motor function test in the offspring ratsOn PND 28,10 male and 10 female rats randomly selected from each group were conducted sensory-motor function analysis test including hot plate test, rotarod test.1.3.4 Learning-memory behavior test in the offspring ratsOn PND70, 10 male and 10 female rats randomly selected from each group were conducted Morris water maze. Learning-memory behavior effects of rh LF on the offspring rats was evaluated.Results1.1 Amino acid detection of rh LF and compositional analysis of rh LF based breed dietsThe recombinant human lactoferrin and casein(casein, CAS) of 17 kinds of amino acids was determinated. The results of nutrients compositional analysis indicated that primary nutrients of rh LF feed and casein feed were similar.1.2 Effects of recombinant human lactoferrin on immune function of offspring ratsNo significant differences were observed on hematology and percentage of lymphocyte subsets between various groups. PFC, the lymphocyte proliferative response and NK cell activity had no statistical difference(P>0.05).1.3Effects of recombinant human lactoferrin on neurobehavior of the offspring rats1.3.1 Early physical development and neural development indicators of the offspring ratsNeural developmental parameters of the offspring rats such as he survival rate of offspring rats, sex ratio, Teething time,the separating time of ear,the time of opening eyes, the time of having hair, cliff avoidance, surface righting, air righting, forelimb hanging and acoustic startle were all observed and evaluated, but there were no significant differences observed(P>0.05).1.3.2 Sensory-motor function combination analysis in the offspring ratsThe offspring rats were tested for Sensory-motor function by a sensory-motor function combination analysis, including, hotplate test, rotarod test and grip strength test, but there were no significant differences observed(P>0.05).1.3.3 Learning-memory behavior test in the offspring ratsThe learning and memory behavior ability ofthe offspring rats were measured by Learning-memory behavior test. The results suggested that recombinant human lactoferrin exerts no adverse effect on the Learning-memory behavior of the offspring rats.Conclusion1. No significant differences were observed on hematology and percentage of lymphocyte subsets between various groups. PFC, the lymphocyte proliferative response and NK cell activity had no statistical difference(P>0.05). The results suggested that recombinant human lactoferrin exerts no adverse effect on the immune system of the offspring rats.2. The results suggested that recombinant human lactoferrin exerts no adverse effect on the neural development indicators,Sensory-motor function and Learning-memory behavior of the offspring rats. |
PDF Full Text Request