LEC. GENETICS (1)

8/7/2019 LEC. GENETICS (1)

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Cryopreservation of fish spermatozoa and embryos

Spermatozoa can be cryopreserved more easily for their structural

simplicity and small size.Eggs are more complex than the spermatozoa. They are moresensitive to freezing injury.

Fish eggs are large in size, possess thick chorion and high quantity of yolk which pose problem during cryopreservation.

The cryoprotectants, particularly glycerol and DMSO, can¶t penetrateeasily into the eggs.

Due to above preservation of fish eggs and embryos have not yet beenfeasible.

Spermatozoa of more than 250 species of fishes could be preserved.Successful cryopreservation of spermatozoa depends upon extender composition, cryoprotectant type, extender-semen dilution ratiocooling and thawing rates.

The time gap between spermatozoa collection and freezing also

affects the success rates of cryopreservation.



EXTENDERS

Undiluted milt with or without cryoprotectant is unsuitable for cryopreservation, therefore a suitable extender or diluent isadded to the milt.

Generally the solutions like Ringer¶s or Hank¶s medium mixedwith some adjuvant like egg yolk, skimmed milk or lecithin areused as extender.

The dilution ratio of milt with extender is highly variable andrequires standardization for every species.

The volume of extender varies between 3 to 10 times thevolume of the semen.



C RYOPROTE C TANT

The choice of cryoprotectant and its concentration are of muchimportance.

Equilibration time- time allowed for the penetration of cryoprotectant into the cell before freezing, is not necessary for fish spermatozoa for their small size.

Most commonly used cryoprotectants are- DMSO (7-10%),glycerol (10-20%), methanol (5-15%), ethylene glycol (7%),propen-diol (7-10%).

Glycerol as a cryoprotectant was suitable in Mugil cephalus,

Gadus marhua, Silurus glanis but it was toxic to salmonids andEpinephelus tauvina for which DMSO was used.

Methanol was used successfully to cryopreserve thespermatozoa of T ilapia but had no cryoprotective effect inSillago cillata and Lates calcarifer .



Freezing and thawing protocalFreezing and thawing rates are very critical duringcryopreservation. The cooling rate shoud be moderate.

C ooling rate of about 20-30 0C / min was suitable for salmonidfishes.

Fast freezing by directly immersing the sample in liquidnitrogen causes lethality. However, fast thawing rate generallyprotects the fertilizability.

Slow thawing causes cell death due to ice-crystal formation

around and inside the cell.

After thawing the cryopratectant should be removed bycentrifugation.



In some species, spermatozoa motility has to be induced byactivator solution after thawing. Sodium bicarbonate,caffeine or theophylline in saline water are good activators.

The time gap between thawing and fertilization should be asshort as possible to enhance fertilization rate.

Viability of cryopreserved spermatozoa are assessed by

i) % of motility

ii) duration and intensity of motility

iii) fertilization and hatching rates

Time of collection of milt is of much relevance for success of cryopreservation.

C ryopreservation may prove easier in those species wherespermatozoa swim for a longer time in external medium.



MUTAT ION AND VAR IAT IONS

Mutation involves heritable alteration in the nucleotidesequence of a single gene, blocks of genes or wholechromosomes. C ertain alteration in chromosome number andstructure brings about the changes in the genome size. Pointmutation causes changes in gene structure and function.

Deletion, duplication, insertion or substitution of nucleotidescan cause heritable alteration in the DNA sequence. Nucleotidesubstitutions are of two types: transition and transversion.Transition occurs when a purine is substituted by another purine or a pyrimidine by another pyrimidine. Transversiontakes place when a purine is substituted by a pyrimidine andvice versa.

PO INT MUTAT ION



Effects of point mutation:I. Mutation may have no effect if base substitution in a gene does

not alter the amino acid sequence. This is possible due todegeneracy in the genetic code. This type of mutation is calledsilent mutation.

II- Mutation may result in the change of nucleotide sequenceleading to the alteration of amino acid sequence in thepolypeptide chain. This is called mis-sense mutation.

III - A change in nucleotide sequence may sometimes produce astop codon within gene sequence. As a result, prematuretermination occurs in the synthesis. This type of mutation iscalled non-sense mutation.

IV- If mutation involves the insertion or deletion of nucleotideswhich are not multiple of three, it will disrupt the reading frameand the amino acid sequence will be changed drastically. Thisis called frame-shift mutation.



Table - Types of point mutations



Change in Chromosome number: Polyploidy and Aneuplo

a. Polyploidy:Polyploidy takes place if chromosome replication occurswithout cell division. As a result, number of haploid sets of chromosomes increases. A triploid (3n) would contain threesets of chromosomes, whereas a tetraploid (4n) contains four sets.

b. Aneuploidy:Gain or loss of one or more chromosomes may occur due toimproper division during meiosis. Monosomy (2n-1) occurs, if a

cell contains one chromosome less than the diploid set, whiletrisomy (2n+1) occurs with the addition of one morechromosome.



Change in Chromosome structureThe structural changes in chromosomes are of the following types.

a. Deletion occurs due to loss of a portion in the chromosome. A largedeletion may cause lethality. If deletion occurs in the two arms of achromosome, the two sticky ends join to form a ring chromosome.The deleted portions are lost.

b. Duplication involves addition of a chromosome segment.

c . Translocation refers to the transfer of a segment from onechromosome to another. Reciprocal translocation involves exchangeof fragments between two non-homologous chromosomes.

d. Inversion is a two-break re-arrangement involving a singlechromosome in which a segment is reversed on position. If itinvolves only one arm of a chromosome, without the involvement of centromere, it is known as paracentiric inversion.

e. Fusion / fission occurs when two different chromosomes fuse attheir centromeres or a single chromosome divides into two bytransverse division at the centromere.



Unequal crossing overSometimes, recombination does not occur properly betweenthe homologous chromosomes. For example, pairing mayoccur 'out of register' in the region of tandemly repeatedsequences in homologous chromosomes. As a result, one of the chromosomes will have two copies of a particular sequencethat were present only once in the parental chromosomes. Thisprocess is called as unequal crossing over, which changes thecopy number of a particular sequence.

Transposable elements (TEs) or transposons are DNAsequences capable of inserting copies of them-selves into new

genomic locations. TEs are of two basic types: i) that may be acomplementary sequence produced by reverse transcription of mRNA or ii) a DNA sequence having inverted terminal repeats.Transposable elements may cause mutation with major phenotypic effect.



Random genetic drift

Genetic drift is the random changes in the gene frequency in a basepopulation leading to the loss of allele(s) due to sampling error.

The likelihood of losing an allele is related to its frequency.

A rare allele (whose frequency is low) has greater probability of being lost than a common allele (whose frequency is high).

Two seminal research papers by Allendorf and Phelps (1980) andRyman and Stahl (1980) addressed the problems of hatcherypractices leading to the genetic drift.



The first group identified substantial losses of genetic variationin a hatchery stock of C utthroat trout, O ncorhynchus clarki (Walbaum).

They showed the loss of some alleles in the hatcherypopulation by comparing the allelic frequencies between thehatchery population and its relatives in the wild.

Ryman and Stahl (1980) found significant changes in the allelicfrequencies between different age groups (year-classes) inthree hatchery stocks.The loss of some alleles reduce genetic variation.C onsequently the opportunities for the genetic improvement byselective breeding are drastically reduced.

This has happened in the case of the AU- Ivory coast strain of T ilapia nilotica , where all genetic variance for increased growthrate was reduced. As a result the selective breedingprogramme for growth rate was unsuccessful.

Genetic drift has led to the extinction of a strain of C hannelcatfish (AU-Rio Granade strain) in Auburn University, USA.



Hybridization in fishesHybridization in nature & taxonomic relations

It has been reported in sunfish, Lepomis.

N atural hybridization is commonest in the family cyprinidae andsubfamily, Cy prininae.

Such natural hybridization has also been recorded in salmonids butthere it is limited to interspecific crosses only while in Cy prinidae it isintergeneric also.

It shows that taxonomy within cyprinidae is more complex than other families and makes placing the species in different genera less

justifiable.

Closely related species hybridize more easily than others.

The species lacking specific individual courtship patterns are morelikely to hybridize in nature than those species with specificcourtship patterns.



Intermediacy-

General conclusion is that characteristics in the hybrid areintermediate between those of the putative parents, however thisintermediacy is not always exact.

Hybrid vigor-In natural hybrids vigor as over-dominance has been observed insunfish in which hybrid vigor gave them territorial advantage over non-hybrids.

Hybrid sterility-

Complete sterility reported between bleak × chub hybrids isuncommon. As only F 1 hybrids are found it is taken as hybrid sterilityand lack of introgressive hybridization.



Hybrid Production in India

Interspecific hybrids (1957)

Hybrid

L. rohita L. calbasu

L. bata

L. calbasu

L. calbasu L. rohita

L. rohita

L. gonius

RohuCalbasu

Bata

Calbasu

--

-

-

CalbasuRohu

Rohu

Gonius





1. Catla-rohu grows slightly faster than rohu-catla. Bothhybrids have quick growth of catla and small head of rohu.

2. In reciprocal crosses of hybrids i.e. rohu-calbasu &calbasu ± rohu (94%) or rohu ± mrigal & mrigal ± rohu(90%) had high fertilization rate.

3. Generally hybrids attained maturity in two years.

4. In reciprocal crosses growth rate of hybrids of both thereciprocal crosses were far superior to the parentLabeo calbasu.



C ross of female mrigal ± calbasu with males of

catla, calbasu & mirgal is also successful

Hybrid

C . catla

C . calbasu

C . mrigal

Mrigal ± calbasu

Mrigal ± calbasu

Mrigal ± calbasu

Catla ± mrigal ± Calbasu

Calbasu ± Mrigal ± Calbasu

Mrigal ± Mrigal ± Calbasu



DiseaseDiagnostics and immunomodulationL

ike all other vertebrates fishes also possess, though weak,defense mechanisms against infections.

Following defense mechanisms are known in fishes-

1- Innate defense mechanism- It includes a number of non-specificantimicrobial systems which are constitutional and do notimprove on repeated contact with the same infections agent.These rely primarily on phagocyte cells to deal with lower organisms and natural killer cells for viruses, backed byantimicrobial factors such as by lysozyme.

2- Specific or adaptive immune system- It is based in the largepopulation of lymphocytes and its twin characteristics of specificity and memory of first antigen contact make it theunderlying principle of vaccination.



The specific or adaptive immure system has two basic parts-humoral and cellular.

The agents of humoral immunity are known as lymphocytes or cells. cells produce antibodies or immunoglobulins which areprotein molecules that bind to foreign substances or antigens, onpotentially harmful bacteria and viruses in the bloodstream.

This binding enables other bodily entities to destroy the bacteriaand viruses by various means like phagocytosis by macrophages

-cells originate from the bursa of Fabricus in birds, bone marrowand spleen in mammals and from kidney in teleosts and spleen insharks.

All antibodies on a single -cells are of the same type and bind toa specific antigen. Thesis specific lymphocytes constitute aclone. Each antigen present on the surface of a micro- organismsis recognized by a different clone of -cells. On stimulation by itscorresponding antigen, the -cells reproduces and secretes itantibody.



C ellular immunity is carried out by T-lymphocytes or T-cellswhich originate from the thymus. Thymus is not reported infishes neither differentiated T-cells are found in fishes.

Disease diagnostics-

Diseases affecting fish require rapid, specific and sensitivediagnostic techniques for early diagnosis of the disease and tocarry out appropriate control measures.

The classical diagnotic approach involving isolation andidentification of the pathogen is tedious and time consumingwhich may negate the very purpose of the early diagnosis.



Immunodiagnostics

Antigen-antibody reactions are highly specific and sensitivewhich is basis for the immunodiagnostics.

The tools are used for the qualitative as well as quantitativeestimation of the pathogens and /or the protective antibody.

In this prospect tests namely enzyme L inked ImmunosorbentAssary (E LI SA), L atex Agglutination test, C o-agglutination testetc. can be developed and standardized for the use in

diagnosis of the diseases at the farm level.



C onventional immuno-diagnostic and monoclonal antibodybased techniques are adequately sensitive, specific and rapidbut the specificity of antibodies also limits their usefulness

because major antigens are not conserved between life stagesof certain pathogens. Now it has moved to molecular characterization of pathogen and probe-based diagnostics.

Molecular diagnosis

Every properly classified species of organism has some uniquemolecular sequences that distinguish it from every other species.

Each organisms genetic composition is in essense a fingerprintthat can be used for its identification.

Nucleic acid probes are fragments of DNA labeled in somefashion that can seek out and bind with high specificity tostretches of DNA or RNA that have complimentary sequences(hybridization reaction).





Presently in shrimp diseases diagnosis, hybridization geneprobes are available for many viruses like infectionshypodermal and haematopoitic necrosis virus ( IHHNV),Hepatopancreative parvo-like virus (HPV), Baculovirus penaei(BP), Monodon vaculo virus (MBV), white spot syndrome virus(ESV) etc.

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LEC. GENETICS (1)