V.

Discussion   Reproductivebiotechnologies as IVF and ET may increase genetic improvement and used as toolsto increase offspring of superior animals so, there is a positive relationshipbetween the use of this technology and production. Buffalo embryos can becultured under different conditions and there are indications that the culturesystem as well as the composition of the medium, can affect embryo quality.Various studies have been shown that while the innate quality of the oocyte isthe major factor that determines the blastocyst yield, the IVC environmentwhich the embryos are exposed after fertilization is the key determinant of theblastocyst quality.      Theassessment of embryo quality is one of the most important factors, whichdetermine a successful embryo transfer. Its significance is related to theessential correlation between embryo quality and the stage of its development.  Many embryo quality assessment systemshave been proposed in order to increase the pregnancy rate.

The predominantnon-invasive technique for selecting viable embryos is based on morphology,where parameters such as rates of cleavage and blastocyst formation as well asdevelopmental competence are evaluated. Methods of assessment also include staining of embryos by differentstains as trypan blue (to assess cell viability) and Hoechst stain (cellcount) and assessment of mitochondrial function and cell growth by3-(4.5-Dimethylthiazol-2-yl)-2.

5-diphenyltetrazolium bromide (MTT). V.1.

Evaluation the quality of in vitroproduced buffalo embryos in relation to the type of monolayers: Thisstudy revealed that addition of oviductal cell monolayer to culture mediaresulted in a significant increase in morula and blastocyst developmentcompared with cell free media and media with granulosa cell monolayer. Thesefindings agreed with who indicated that co-culture with BOCM exerted apronounced beneficial effect on development of in vitro fertilized bovineoocytes. Embryo co-culture systems offer advantages attempting to mimic the invivo conditions. reported that the beneficial effect of monolayers ismediated by their contribution of some specific component as growth factors andenergy substrates to the culture medium (these factors improve the developmentof embryos) or the removal of inhibitory substances as reactive oxygen species(ROS), ammonia and phosphate from the culture medium. Theseresults were nearly in accordance with, who found that development ofearly embryos (8-cell stage) improved in TCM199 with BOCM co culture thanTCM-199 alone so he concluded that addition of bovine oviductal cell monolayer(BOCM) to TCM-199 improved the developmental capacity of early embryo. He alsosaid that addition of follicular granulose cells and cumulus cells result in ahigher maturation rate of buffalo follicular oocytes than addition of BOCM.

These beneficial effects resulted from providing nutritional molecules that arenecessary for growth and development of embryos and by transmitting signalsthat regulate oocytes maturation. BOCM have a role in intimate contact withgametes and embryos during fertilization and early embryo development, and areconsidered the most suitable in vitro model to study early embryonic maternalinteractions. BOCM modify their transcription in the presence of developingembryos, demonstrating that co-culture systems allow a dynamic exchange ofnutrients and cell secretions.  BOCM alsohelp overcome the developmental block occurring at the 8- to 16-cell stage inin vitro produced cattle embryos. The last author also reported superiordevelopment of bovine zygotes and early embryos in simple medium with BOCM,compared with a complex medium.evaluated the developmental potential ofpronuclear-stage ovine embryos co-cultured on both oviductal epithelial anduterine fibroblast monolayers.

Both of these monolayer systems were capable ofsupporting embryo development to the early blastocyst stage. After 6 days ofincubation, 42% of the ovine embryos developed into expanded blastocyst usingoviductal cell co-culture compared with only 5% co-cultured on uterinefibroblast monolayers. The transfer of embryos co-cultured on oviductal cellmonolayers resulted in higher pregnancy rates in recipient sheep compared withthose co-cultured on fibroblast monolayers. A higher percentage of morula andblastocyst stage embryos resulted when IVF derived one cell bovine embryos wereco cultured on oviductal cell monolayers compared with that of culture inmedium alone (22% and 3%, respectively).

It was suggested that the majordifference between uterine fibroblast and oviduct epithelial cell co-culturesystems is that fibroblast feeder-layers are capable of removing embryotoxiccomponents, reducing oxygen tension, thereby, improving embryo development overthat of culture medium alone but are unable to secrete embiyotropic factors.Results thus far suggest that oviductal cell monolayers are the most effectiveco-culture system for early-stage farm animal embryos while fibroblastmonolayers are adequate for development of later-stage embryos (morulae toblastocyst) but less effective for developing earlier stage embryos through thehatched blastocyst stage. reported that bovine oviductal monolayers were easilyobtained and could be subcultured for 14 to 18 passages before the appearanceof an in vitro crisis phase. Similarly, rabbit oviductal monolayers could bemaintained for 10 passages before the appearance of altered growth andmorphology. However, limited numbers and slower growth rates of human oviductalcells were noted compared with other species. also evaluated protein secretionsby subpassaged oviductal monolayers of different species using radiolabelledmethionine incorporation and noted that monolayers maintained high proteinsecretion rates following 96 h in culture.

Evaluation of radiolabelled proteinsecretion by monolayers during in vitro culture will likely provide vitalinformation on embryotropic factors produced by monolayers during embryoco-culture.A variety of culture media have been used toestablish oviductal cells prior to embryo co-culture experiments. Excellentgrowth of bovine epithelial cells has been obtained with Menezo’s B2 medium,TCM-199. In contrast to our study, noted significantly higher rates ofdevelopment of IVF-derived bovine embryos to morulae and blastocyst (32%) usinggranulosa cell co-culture compared with oviductal epithelial cell co-culture(17%).Although most results indicate that granulosa cell co-culture systems aresimilar to other co-culture systems, the major advantage of the granulosa cellculture system is that the cells are available for harvesting from the follicleat the time of oocyte collection. This provides a simple and cost efficientco-culture system without lowering success rates of in vitro culture systems.   The present study  also revealed that addition of oviductal cellmono- layer on culture media resulted in a significant increase in the numberof good quality embryos (grade 1) and decrease in the number of  poor quality embryo grade 4 compared withcell free media grade 1 embryos  and poorquality embryos (grade 4). This effect resulted from the action of BOCM in a protective manner through the reductionor removal of potentially harmful substances or modifying the concentration ofthe medium constituents to levels more appropriate for embryo development  and they also probably secreted specificembryo trophic factors  which werebeneficial for the development of embryos in vitro.

stated that the presence of BOCM at earlystages of embryo development, up to four days, improved embryo development andembryo quality in terms of specific gene transcripts, concluding that thisperiod reflects the in vivo conditions where the embryo is still in theoviduct.BOCMco-culture may reduce O2 tension and glucose concentrations in the culturemedium reducing glucose toxicity in early embryos. BOCM also secretedantioxidant enzymes which are able to detoxify O2, H2O2, and organic peroxides.

  Thisstudy also revealedthat there are no significant differences in viability of embryos using trypanblue stain  among different treatments(cell free, cumulus and oviductal cells). This finding came in agreement with who stated that trypan blue staining method was veryunsatisfying concerning the objectivity of marking viable embryos. Althoughthis method is often used routinely to assess the viability of cell lines inculture, is not suitable for evaluating embryo viability because its entry intothe cell is much held back by the zona pellucida of the embryo.    This study also recorded that additionof oviductal cell and granulosa cell mono- layers to culture media resulted ina significant increase in cell number. This result came in agreement with whonoted that embryos co cultured with bovine oviduct epithelial cell monolayersand granulosa cell monolayers averaged more cells than did embryos inconditioned medium. Addition of oviductal cell and granulosa cell mono- layersto culture media also resulted in a significant increase in mitochondrialfunction compared with cell free media. These findings may resulted from secretionof some growth factors such as platelet-derived growth factor, activin,insulin-like growth factor and basic fibroblast growth factor  and energy substrates by cell monolayers(these secretions increase metabolic activity and subsequently increase cellnumber and mitochondrial function) . These monolayers also secreted moreprotein than other cell lines.

This secretory activity may explain stimulatoryeffect of their co-culture on blastocyst rate and this increased proteinproduction by these cells reduced serum requirement during co-culture and/orinhibit the detrimental effect of high FCS concentration. BOCM also suppressedapoptosis in addition to stimulating proliferation of blastomere.IV.2. Evaluation the quality of in vitroproduced buffalo embryos in relation to cumulus cells (denuded and compact):      The present study  revealed that using of compact oocytesresulted in a significant increase in morula and blastocyst development (improvedevelopmental competence of in vitro produced buffalo embryos)  compared with denuded oocytes. This findingcame in agree with who stated that cumulus cells supported IVM ofoocytes to the MII stage and were involved in the cytoplasmic maturation neededfor optimal developmental competence, such as male pronucleus formation anddevelopment to blastocyst stage.

Cumulus cells might be a good indicator  for an oocytes ability to undergo meiosis I invitro and that the developmental problems of denuded oocytes were due to deficientcytoplasmic maturation.The oocyte cumulus cell gap junction isrequired for the coordination of nuclear and cytoplasmic meiotic competence andare vital for oocyte maturation and subsequent embryo development. Moreover,Physical contact between oocytes and cumulus cells has been considerednecessary for the transfer of nutrients and factors essential for oocytesdevelopment. Cumulus cells removal before in vitro maturation decreases bovineoocyte developmental competence without affecting nuclear meiotic maturation.Cumulus cells are physically and metabolicallycoupled with an oocyte during its growth and maturation and also participate inovulation and fertilization and in tight relation with the oocyte through gapjunctions that allows a bidirectional paracrine signaling, thus regulatingdifferent processes, including chromatin remodeling and RNA synthesis in theoocyte. This study also clarified that using of compact oocytes resulted in asignificant increase in number of grade 1 embryos and decrease in poor qualityembryos compared  with denuded oocytes.

This effect of cumulus cells may result from its secretion of soluble factors,which induced developmental competence, or from removing an embryo developmentsuppressive component (affects the quality of embryos) from the medium. Theoocyte also contributes to cumulus cells functioning through the secretion ofdifferent oocyte-specific factors and so regulates its own microenvironment toacquire a better capacity to develop into an embryo.  In this study, there was no significant difference inviability of embryos between compact and denuded oocytes using trypan bluestain.This study also recorded that usingof compact oocytes resulted in a significant increase in cell number andmitochondrial function compared with denuded oocytes. This may result fromcumulus cells secretion of some factors which improve the development ofembryos and subsequently increase cell number and mitochondrial function of invitro produced embryos. Cumulus cells are particularly involved in antioxidativeand metabolic processes, such as reducing cystin to cystein and metabolizingglucose to pyruvate, respectively.

These components are provided to the oocytesby cumulus cells and known to improve oocyte quality and subsequently improveembryo development. Glucose is the main energy source for the oocyte, andtherefore glucose metabolism is very important for oocyte maturation and earlyembryo development. However, because of the low capacity of the oocytes toutilize glucose, this substrate is mainly metabolized by cumulus cells viadifferent pathways, including glycolysis and the pentose phosphate pathway toprovide an oocyte with pyruvate for energy production. Cumulus cells areinvolved in providing energy substrates as fatty acids, carbohydrates, andamino acids from surrounding fluids to the oocyte.IV.3. Evaluation the quality of in vitroproduced buffalo embryos in relation to the concentration of Epidermal GrowthFactor (EGF):    The present study revealed that addition ofEGF by concentration 50ng/ml to culture media resulted in a significantincrease in morula and blastocyst development compared with control. Moreover,there were no significant differences in the morula and blastocyst stagesdevelopment among other concentrations and control.

This finding came inagreement with who stated that the in vitro buffalo embryo developmentmatured in the presence of EGF showed significantly higher rate of morula andblastocyst (transferable embryos) (40.9-42.7 %) than that without EGF(20-33.5%) and showed higher significance in the cell number of the blastocyst and with who reported that theaddition of EGF to the culture medium resulted in higher developmentalcapacities than other growth factors used. Moreover, stated that addition of EGF even at a low concentration(5 ng/ml) enhanced blastocyst development of cat embryos cultured singly. EGFexerts its effect on preimplantation embryo development by binding to itsreceptor located within an embryo’s plasma membrane, which, in turn, stimulatesdownstream signaling pathways regulating cell proliferation and differentiation.EGF has been shown to enhance blastocyst formation via stimulating proteinsynthesis especially in trophectoderm cells and decreasing the rate ofapoptosis.found that EGF treatments had no effect ondevelopment of pre-implantation  zygotesto the 4-cell stage compared to the control.

A concentration of 5 ng/ml of EGFsupplementation during embryo culture significantly improved blastocystformation from the 4-cell stage compared to the control. EGF seems to play a moreimportant role in embryonic cell differentiation than in cell proliferation.  In the present study, addition of  EGF by concentration 50 ng/ml  to culture media improved the development ofin vitro produced buffalo pre-implantation embryos said that addition ofsome growth factors, including EGF produced no positive effects on blastocystdevelopment when TCM-199 was used as defined culture medium.

The present studyrevealed that addition of  EGF improveddevelopment of   blastocyst  when CR1aa was used as culture medium. stated thatabsence of growth factors or disruption of paracrine/autocrine signaling oftenresults in growth retardation, chromosomal abnormalities, alteration of geneexpression, metabolic disruption and increased apoptosis levels. Growth factorsupplementation can compensate for the adverse effects of culture in largevolumes and thus enhance embryo development in vitro.      Thepresent study also revealed that addition of EGF by concentration 20 and 50ng/ml to culturemedia resulted in a significant increase in number of good quality embryos(grade 1) and decrease in number of poor quality embryos compared with control.This finding may result from the effect of EGF on proliferation of cells formingthe intracellular mass and trophectoderm, compaction and formation of theblastocyst and decreasing rate of apoptosis and protection against oxidativestress so EGF causes increase of grade 1 embryos and decrease of poor qualityembryos. EGF also activates transport systems responsible for the uptake ofglucose, enhance endocytosis and influence the processes of replication,translation, and degradation of proteins.

This study also revealed that there were no significantdifferences in viability of embryos among different treatments.Thepresent study also revealed that addition of EGF by concentration 20 and 50ng/ml to culture media resulted in a significantincrease in cell number and mitochondrial function compared with control. Thesefinding came in agreement  with  who found higher blastocyst cell numbersafter EGF supplementation irrespective to concentration  and  whosaid that EGF has been shown to increase total nuclei of bovine blastocystsby increasing numbers of trophoblast cells. While found that numbers ofICM nuclei and trophoblast cell nuclei were unaffected by adding exogenous EGF. The effect ofEGF on cell number and mitochondrial function  may result from stimulation of some signaling pathways regulatingcell proliferation, differentiation and metabolic activity of  in vitro produced embryos.  EGFexerts its action by binding with high affinity to epidermal growth factorreceptors on the cell surface and stimulating the intrinsic protein tyrosine kinaseactivity of the receptor. The tyrosine kinase activity, in turn initiates asignal transduction pathway  that resultsin a variety of biochemical changes within the cell,  an increase in intracellular calcium levels,increased glycolysis and protein synthesis and increases in the expression ofcertain genes including the gene for epidermal growth factor receptors  that lead to DNA synthesis and cellproliferation.

It was suggested that at higher concentrations,blastocyst development had been reduced due to a phenomenon called growthfactor induced receptor down regulation. So the presence of high concentrationof EGF caused a significant down regulation or acceleration of EGF receptorsdegradation.IV.4. Evaluation the quality of in vitroproduced buffalo embryos in relation to the concentration of lactoferrin:The present study demonstrated that addition oflactoferrin by concentration 50µg/ml to culture media resulted in a significantincrease in cleavage rate, morula and blastocyst development compared with control. While there were no significant differencesin cleavage rate, morula and blastocyst development among other concentrationsand control group. Moreover, a significant increase in number of  good quality embryos (grade 1) and decreasein number of  poor quality embryoscompared with control. Although the addition of lactoferrin to culture mediumimproved the developmental competence and quality of in vitro produced buffaloembryos,  the  present study was the first study that recorded this effect.

 This effect of Lf may result from playing akey role in maintaining cellular iron levels. LF also has a greater resistanceto proteolysis and has antioxidant activity. Its protective effects are directantimicrobial activities against a large number of microorganisms, includingbacteria, viruses and fungi. Theantimicrobial activity of LF is due to two mechanisms.

The first issequestration of iron in sites of infection, which deprives the microorganismof this nutrient, thus creating a bacteriostatic effect. The other mechanism isthe direct interaction of the LF molecule with the infectious agent. Thepositive amino acids in LF can interact with anionic molecules on somebacterial, viral, fungal and parasite surfaces, causing cell lysis.

LF has the ability to function as an enzyme insome reactions (DNAse, RNAse and ATPase activities). The basis for LF’s various enzymaticactivities is unknown. The variety of activities can be attributed tovariations in the nature of the protein: multiple isoforms, degrees ofglycosylation and tertiary structure (holo- or apo LF). The discovery of LF’senzymatic activities has helped to explain several of its physiologicalmechanisms. These effects of lactoferrin may be the cause of improvement ofbuffalo embryos development when LF added to culture medium.

Thepresent study   revealed that there were no significantdifferences in viability of embryos among different treatments of LF and alsorevealed for the first time that addition of lactoferrin by concentration 20and 50µg/ml to culture media resulted in a significantincrease in cell number compared with control and concentration 10 and 100 µg/ml and significant increase in  mitochondrial function  compared with control. This increase in cellnumber and mitochondrial function may be attributed to antioxidant (decreaseoxidative stress and apoptosis of the cells), antimicrobial and enzymaticactivities of LF. IV.5.

Evaluation the quality of in vitroproduced buffalo embryos in relation to the concentration of vitamin B12:     Thepresent study showed that addition of vitamin B12by concentration 20 µg/ml to culture media resulted in a significantincrease in the cleavage rate and  morulaand blastocyst  development compared withcontrol. While there were no significance differences in the cleavage rate andmorula and blastocyst development among other vitamin B12concentrations and control group. This study also revealed that addition of vitaminB12 by concentration 20 µg/ml to culture media resulted in a significantincrease in number of good quality embryos (grade 1) and decrease in poorquality embryos compared with control while there was no significance in numberof grade 1 and poor quality embryos among other concentrations and controlgroup. The present study was the first study that recorded the positive effectof vitamin B12 on developmental competence and quality of in vitroproduced buffalo embryosThiseffect of vitamin B12 in improvement of development and quality ofbuffalo embryos may be due to its involvement in the metabolism of every cell  especially affecting DNA synthesis, fatty acid and amino acid metabolism and energyproduction. Vitamin B12 functions as a coenzyme, meaning that its presence isrequired for enzyme-catalyzed reactions as isomerases and methyltransferases.VitaminB12 is a co-substrate of various cell reactions involved in methylationsynthesis of nucleic acid and neurotransmitters.

It is required for themethylation of homocysteine in the production of methionine, which is involvedin a number of biochemical processes including the monoamine neurotransmittersmetabolism.The best known function of B12 that which is involved with DNAsynthesis and cell-division is actually an important function which is mediatedby B12-conservation of an active form of folate which is needed for efficientDNA production so reduced availability of folate results in ineffectiveproduction of cells.This study revealed that there was no significantdifference in viability of embryos among different treatments and also revealedfor the first time that addition of vitamin B12 by concentration 10and 20 µg/ml to culture media resulted in a significantincrease in cell number and mitochondrial function compared with control. Whilethere was no significant difference in cell number and mitochondrial functionbetween concentration 50 µg/ml as compared with control group. Thisincrease in cell number and mitochondrial function may be attributed to theimportant role of vitamin B12 in DNAsynthesis and cell-division and in the metabolism of every cell  especially affecting  fatty acid and amino acid metabolism and energyproduction.

     

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