Low Cost Alternative Method to Monitor the Electrophoretic Bands Substitution of the Positively Charged Rabbits Seminal Fluids

This paper aimed to evaluate the significance of a non-cost effective double dye technique in visualizing any DNA-seminal proteins interactions. After collecting rabbit’s ejaculate and removing sperm cells, ion exchange chromatography was performed to separate seminal proteins on the basis of their charge. Positively charged seminal proteins were eluted, lyophilized, and involved in this study. After incubating this eluted group with a standard reporter DNA, band shift assay was applied on this group. The results were compared. According to this study, we demonstrated the necessity of utilizing a double staining technique for the same band shift gel in order to ensure weather real or false band substitutions were obtained. Keywords : Rabbits seminal fluids ,DNA , Electrophoresi

Sperm Mediated Gene Transfer in Mammals; a Versatile Platform with Multiple Enhancements Techniques

In the field of animal transgenesis, many attempts have been made recently to simplify facilitate, and reduce the cost and labor required to do such tasks. Although several transgenesis techniques (such as DNA microinjection and somatic cell nuclear transfer) have been applied successfully to produce transgenic animals, these traditional techniques are so tedious and have several disadvantages. Retroviral mediated gene transfer has solved some of these usual problems but has, however, inevitable disadvantages represented most prominently by its biological hazard. Many researchers found that the most simple and non-cost effective way to produce transgenic animals is to focus on the natural ability of the sperm to “carry” the foreign DNA and to “fertilize” the oocyte. The most important breakthrough obtained in this aspect is the accumulated information that demonstrated the ability of foreign DNA to be internalized into the sperm head after simple incubation step. Accordingly, the only manipulation step is restricted into the head of the sperm. Then, nature will be allowed to fulfill its scheduled task of reproduction. This method known as sperm mediated gene transfer or SMGT. However, simple incubation of naked DNA with sperm head is not efficient enough to integrate the foreign DNA into the genome of the sperm. Thus, this review aims to pave the way for every effort to enable the researchers to undergo the transgenesis experiments in the routine laboratories. This is potentially can be done by testing the validity of the most modern enhancement approaches suggested on the original SMGT

The Efficiency of Transgenesis by Restriction Enzyme Mediated Integration - Sperm Mediated Gene Transfer (REMI-SMGT)

The present study tends to  test the validity of REMI-SMGT and to evaluate the efficiency of REMI-SMGT on generating other mammalian species rather than the sheep that made only by one group and to observe the possibility of doing so by using rabbits as a model for this approach and also to reduce the cost of REMI-SMGT by substituting liposomes and highly cost effective media with a high efficient, non-cost effective substitute. Direct protective relationship of liposome with DNA and seminal fluid was identified compared with DMSO.  While different treatments (linearized DNA – restriction enzyme – liposome complex, DNA – restriction enzyme – DMSO complex, DNA –DMSO complex, DNA –liposome complex, and even naked DNA) were all found to be successful to internalize inside the head of the sperm according to PCR results, only three (one by restriction enzyme – liposome treatment and two by restriction enzyme – DMSO treatment) out of fourteen new born babies were found to be transgenic by PCR. Despite the absolute ability of exogenous DNA to be internalized inside rabbit’s sperm head only few percent of transgenic babies were obtained. This may not reflect the weakness of restriction enzyme mediated transgenesis technique itself but it reflects the inability of recombinant sperm to fertilize superovulated oocyte compared with their normal counterparts. Comparable results were found between liposome and DMSO treatment which may reflect direct relationship of DMSO with the cell membrane instead of with the exogenous DNA itself as what is found with liposome. Key words : REMI-SMGT , PCR , DNA – restriction enzyme , DMS

Ion Exchange Fractionation of Rabbits Seminal Fluid: Recognizing a DNA Retardation Activity from the Main DNase Activity

The role of seminal proteins charge on the nature of seminal fluid inhibitory effect that exerted against exogenous DNA. Has been identified and an approach closely with more details to the nature of inhibitory activities available in rabbit seminal fluid proteins that prevent the entry of exogenous DNA into the head of sperm. After collection of rabbit’s ejaculate and removing sperm cells, seminal fluid was incubated with fixed concentration of exogenous DNA. The seminal fluid – exogenous DNA mixture was analyzed by electrophoresis. Ion exchange chromatography was used to separate seminal proteins on the basis of their charge. Positively charged proteins were eluted, lyophilized, and their profile was characterized by SDS-PAGE and native-PAGE. After incubation of this eluted group with the same source of DNA, the same electrophoretic conditions were applied on this group. According to our knowledge, this is the first paper in which ion exchange chromatography was used to separate two DNA counterfeiting activities of the seminal fluid using non-radioactive method in rabbits and even in other mammals. Thus, more than one inhibitory activity were identified and separated. DNA retardation activity (or DNA binding activity) which repressed DNA electrophoretic migration was the only activity that found to be available on the positively charged fractions while the DNase activity was found exclusively on the negatively charged group. Key words : Seminal fluid , transgenesis techniques , DNA electrophoretic migration , DNas

Different Factors Effects In Lactating Mother's Milk Compositions

This study was carried to estimate the effects of different factors on the total protein and lipid concentrations in lactating mother's milk, such as; home, age, body mass index, lactating stage and negative smoking habitat. Total Protein concentration and lipid concentration were estimated using Bradford method and Gravimetric method respectively. Results showed that lipid concentration(L.C) increased with age,  BMI, lactation stage, and affected by  negative smoking and increased in rural,  while protein concentration (P.C)was less effected by age, and decreased in obese and in negative smoking mothers, also it decreased in urban mothers. Protein profile showed slight differences between nonsmoking and negative smoking milk using SDS-PAGE electrophoresis. Key words: negative smoking, BMI, lactating mother’s milk.

Genotyping of Diacylglycerol Acyltransferase 2 Gene in Holstein Cattle Population

This study was conducted to describe the single nucleotide polymorphism (SNPs) within exon 8 and its intron 9 flanking region within bovine Diacylglycerol Acyltransferase 2 (DGAT2) gene and the possible association of these SNPs with the milk productive traits. Blood samples were collected from randomly selected Holstein cattle. Genomic DNA was extracted, and a pair of specific PCR primers was designed to amplify a segment that consists of partial exon 8/partial intron 9 of DGAT2 gene. SSCP experiments were optimized and performed for each amplified PCR fragment. Each set of SSCP resolved bands was sequenced and analyzed. Four SSCP patterns representing four genotypes (BD, BB, CD, and AD) were detected with four alleles. Several novel genetic polymorphisms were discovered. Three SNPs (157 C/A, 158 T/G, and 159 G/A) were found in genotype BD,  while one SNP (94 G/T,  153 C/A, 154 T/A) were found in the genotypes BB, CD, and AD, respectively. The only non-synonymous SNP was found in genotype CD (344 D/Y), and the effect of this missense mutation on the protein three-dimensional structure was visualized. The sequence homology between the bovine DGAT2 gene and other species was also analyzed. The significance of the correlation of each genotype with the productive traits of milk was observed. The Holstein cattle with BD genotype produced significantly more milk for all studied 90 days of lactation, with a significant effect on fat for only the last 30 days of lactation, whereas there was no effect on protein and lactose percentage for the entire lactation period. The results suggest a novel association between bovine DGAT2 genetic variability and the milk yield in Holstein cattle. This opens interesting prospects for future DGAT2 based selection programs and preservation strategies

Seminal fluid; the Natural Guard of Seminal DNA

The ability of foreign DNA to be introduced into the sperm cells faces many problems. These problems have been emerged from the existence of some inhibitory factors available in the seminal fluid. Add to that, other several factors are involved in this internalization. The purpose of this review is to evaluate the protective or interactive roles of these seminal fluid proteins in the process of foreign DNA internalization into the head of the sperm. The ability of foreign DNA to be internalized inside sperm cell is become obvious before more than four decades (Brackett et al., 1971). But, the mechanism by which this foreign DNA has the ability to do so is still under debate. However, several papers described certain factors involved in this process. Thus, in order to describe the mechanism of sperm transfection by exogenous DNA, it is necessary to understand the following natural factors that are playing main roles in this scene and they include; inhibitory factor I (IF1), seminal fluid DNase, DNA binding proteins (DBPs), CD4, major histoincompatibility complex class II (MHCII), topoisomerase type II (TOPO II) and reverse transcriptase (RT). They can be further classified into three main groups. The First group is represented by two factors found in seminal plasma; IF1 and DNase, this group is responsible about inhibiting the internalization process of exogenous DNA.  The fact which refers to the existence of one or more factors in seminal plasma that able to block sperm permeability must be taken into account. This means, extensive washing steps of ejaculate to remove seminal plasma is necessary and should be made before incubating sperm with exogenous DNA. Lauria and Gandolfi reviewed that seminal fluid inhibitors have two ways of inhibition to exogenous DNA, either directly or indirectly (Lauria and Gandolfi, 1993). These seminal plasma inhibitory factors may prevent transfection of intact sperm by foreign DNA (Camaioni et al., 1992).  Gandolfi showed that there is a consensus on the experiments made on seminal fluid of the ejaculated spermatozoa of mammals in the impermeability of sperm cell to the aggression of foreign DNA as long as seminal plasma is not removed (Gandolfi, 2000). Thus, seminal fluid prevents any foreign DNA from binding with its receptor on the sperm cell

Correlation between Leptin Encoding Gene and Some Haematological and Biochemical Parameters in Awassi Sheep

The present study was scanned 104 of different genotypic Iraqi Awassi sheep to determine the relation of genetic polymorphism for leptin gene with several biochemical and haematological features. The results of this study were revealed that most of blood characters were homologous for the genotypes AA, AB, AH, AM, AR, and AS except for RBCs and WBCs count since there were significant differences (P< 0.05) between them, and the following genotypes AH and AB were surpassed on the two genotypes AM and AB concerning the number of RBCs, and the two genotypes AB and AH were surpassed on the genotype AM concerning the number of WBCs. Most of biochemical characters were nonhomologous except for the concentration of urea, since significant differences (P< 0.05) among the genotypes were observed with respect to glucose, protein, cholesterol, and triglycerides. The AH genotype was surpassed the two genotypes AB and AR concerning the glucose ratio. The AB genotype was surpassed the two genotypes AM and AS, the two genotypes AA and AH were surpassed the AB genotype concerning cholesterol ratio, and the AB genotype was surpassed the AH genotype concerning the level of triglyceride. In conclusion, the leptin gene diagnostic tool could be used for selection process to enhance the production levels in Awassi sheep through eliminating the animals that were correlated with the genotypes of some undesired characters in early breeding times. Keywords; leptin, gene, Awassi, parameter, blood, biochemistr

Detection Some Trace Elements in Human Milk and Effect of Some Factors on its Concentrations

Study aim to determination some trace elements in lactating mothers human milk and study effect different factors on its concentration, Age , Body mass index, Home and Smoking habitat, atomic absorption flame less used to measured (Mn, Cd, Co and Zn) in 70 sample of lactating mothers milk which digested using tricolor acetic acid 24%. Result show that concentration of these elements increasing than normal value of these elements,Mn was 1801.40, Cd was 114.64, Co was 288.52 and Zn was 212.10.Also factors were studied effect on minerals concentration,  increasing age was  non effected on minerals concentration, over weight causes increasing it, urban resident causesincreasing in Mn concentration while rural residence causes increasing in others minerals, negative smoking habitat causes increasing in Cd and Co concentrations. Key words: trace element, lactating mother’s human milk, atomic absorptio

Hematological Parameters as Indicators for Litter size and Pregnancy Stage in Awassi ‎Ewes

Physiological status and litter size can indeed have a significant impact on ewes' ‎hematological parameters, which are essential indicators of their health. Therefore, this study ‎examined the hematological profiles of ewes during pregnancy with single and twins in the ‎Awassi ewes. The present study involved 232 ewes in good health and at sexual maturity. ‎Among them, 123 ewes had single pregnancies, while 109 ewes had twin pregnancies. The age ‎range of the ewes included in the study was between 3.5 and 4.5 years. Hematological tests ‎were conducted on the sheep's blood samples promptly following collection. The findings ‎demonstrated variations in hematological parameters among pregnant ewes, with differences ‎based on litter size. Ewes carrying twin pregnancies exhibited significantly higher levels of ‎red blood corpuscular, hemoglobin, packed cell volume, and mean corpuscular hemoglobin ‎concentration during pregnancy. In comparison to single-pregnant ewes, Awassi ewes with ‎twin pregnancies displayed elevated counts of white blood cells, lymphocytes, granulocytes, ‎and granulocyte percentage compared to ewes with single pregnancies. Awassi ewes with twin ‎pregnancies also exhibited a strong positive correlation with the leukocytes and erythrocytes ‎constituents. In conclusion, these findings indicate that litter size significantly influences ‎hematological parameters, highlighting the importance of considering the physiological status ‎and litter size as indicators of ewes' health. The findings have practical implications in sheep ‎breeding and reproduction, as they can be utilized to enhance the diagnosis, prognosis, and ‎treatment of related conditions‎