Presentation on

Bovine Genome Database: new

annotation tools for a new
reference genome
PRESENTED BY:
Nabin Neupane
M. Sc. An. Sc
Department of Animal Breeding and
Biotechnology
 INTRODUCTION
BOVINE GENOME DATABASE
 livestock based breeding and research has increased due to availability of
multiple livestock reference genome sequences.
 with complexity and increasing amount in bovine database, BGD has
advanced its practices.
 The characteristics features present in this database are: BGD provides
tools for genome browsing (JBrowse), genome annotation (Apollo), data
mining (BovineMine) and sequence database searching (BLAST).
 To augment the BGD genome annotation capabilities, we have developed
a new Apollo plug-in, called the LocusSpecific Alternate Assembly
(LSAA) tool, which enables users to identify and report potential genome
assembly errors and structural variants.
 bdg was first established to support the bovine
genome sequencing and analysis consortium by
providing web-based tools for annotation of the
bovine genome.
- With time, this software has improved a lot and
added new tools and data and updated existing
datasets.
 LASSA
 Theres is reasonable advancedment in genome assemble and sequencing,
but reference genome assemblies contains errors which will impact genome
annotation.
 Lassa manually corrects local assembly errors or it gives information about
variants.
 genome browsing (JBrowse)
 Fast and full featured genome browser built built with javascript and
html5.
 It helps to assembly genome and annote it.
 The assembly and annotation is in the form of graphical representation.
 Features are finding coding and noncoding genes from refseq and
ensemble (these two are software)
 Finding snp for ensemble.
 QTL from animalqtllab.
 Expression tracks computed using publicly available rna seq data.
 genome annotation (Apollo)
 Annotation tool is apollo.
 Genome annotation is the process of identifying the location and function
of genome encoded features.
 Apollo is the open source software that enables researchers to efficiently
inspect and refine the precise structure and role of genomic features in a
graphical browser based platform.

 Annote means add notes to a text or diagram giving explanation or

comment.
 data mining (BovineMine)
 bovine mine helps in datamining.
 Bovine mine integrates the data at jbrowse with external
source, including protein annotation and domains.
 Sequence Database Searching (BLAST)
 fast spread of genetic abnormalities
 Some males shed virus in semen without clinical signs of disease
(shedders).
 Some bacterial pathogens are resistant to the antibiotics in semen
extenders
 Loss of embryo/ fetus with improper AI.
 Wanting skilled persons
 Requiring special facilities
 The conception rate is not success like Natural Insemination
 Apollo plug-in, called the LocusSpecific
Alternate Assembly (LSAA) tool
a. Directly from vagina after natural insemination
 Oldest and simplest method
 After natural service by bull, semen is collected by long spoon or a syringe
with long nozzle directly from vagina
 Disadvantage: Semen contain more mucus.
b. Rectal massage of ampullar vas defern and seminal vesicles per
rectum
 After rectal palpation, evacuation of feces, seminal vesicle is pressed
towards urethra for few minutes. And the ampullae is massaged against the
floor of pelvis and milked one by one and semen is collected by the
assistant.
newest bovine reference genome assembly,
ARS-UCD1.2
 previous reference genome, UMD3.1.1
 Most common method used for collection of semen.
 The AV is cleaned properly cleaned after each collection.
 Before collection, inner sleeve is placed into outer cylinder and both the ends
are made over the cylinder and watertight space is formed between them.
 Water at 40 –50oC is filled in the created space between the outer cylinder and
inner sleeve.
 The large end of semen receiving cone is attached over one of the ends of this
water jacketed and tightly secured with the help of rubber bands.
 Then the semen collecting vial is attached at the smaller end of receiving cone
 The inner sleeve is then lubricated with sterilized jelly using glass rod.
 Other notable enhancements to BovineMine
 incorporation of genomes and gene annotation datasets for non-bovine
ruminant species (goat and sheep),
 support for multiple assemblies per organism in the Regions Search tool,
 integration of additional ontologies and
 development of many new template queries.
 Advantage and Disadvantage of AV method
Advantages
 The ejaculate is collected in uncontaminated, natural state.
 It is free from the extraneous secretions.
 It is disease free; sterile conditions of the apparatus ensure disease
control.
 The viability of the sperm is better.
 No female is needed if dummy is used successful.
Disadvantages
 It requires equipment and a teaser animal or dummy.
 There is increased danger to the collector.
 It can be very time consuming if the bull is uncooperative or hesitant
 EVALUATION OF SEMEN
Once semen is collected, it is kept at 30-35 degree Celsius until it has been
evaluated and is ready for further processing.

1. Constituent of semen:
 It contains spermatozoa in a watery fluid called seminal plasma. Seminal plasma
consists of combined secretions of seminal vesicles, bulbourethral gland and
prostrate.
 Semen coagulates immediately after ejaculation and then liquefies over a period
of approximately 30 minutes
 Seminal plasma contains constituent for energy (fructose), proteins and ions like
ca, mg, Zn, bicarbonate.
 Criteria for semen evaluation
 Visual evaluation: for volume, colour, consistency/density, smell/odour and
for presence of foreign material is made recorded.
 Microscopic evaluation for motility: phase contrast microscope, Motility
(moving actively forward): > 50% .
 Determination of concentration: sing either Hemocytometer or a calibrated
photometer, Concentration: > 500 million /ml
 Evaluation of live /dead counts: Buffered Nigrosin eosin solution is mixed
with a drop of semen and smeared on a glass, Live sperm: > 50%
 Morphological studies for head and tail abnormalities, and acrosomal
abnormalities.
 DILUTION OF SEMEN
 The main reason : to increase the number of females serviced from one ejaculation.
 A normal ejaculate contains 5 - 10 billion sperm which can be used 300 to 1000 cows
after extension (15 to 20 million spermatozoa per straw for deep frozen semen).
 Dilution rate depends on quality of the ejaculate, number of sperm cells, percent
alive and mobility.
 Normally, the dilution is done at sperm: diluent = 1: 200
Diluents types and extension ratio depends upon preservation type
Deep Frozen Semen (DFS), Chilled Semen (CS) or Room Temperature Semen (RTS).
1. For DFS the recommended diluents are: a) Egg yolk - glycerol extenders b)
Skimmed milk – glycerol extenders c) Tris-buffer based diluents (synthetic)
2. For CS and RTS the recommended diluents are: a) Coconut milk - glycerol extender
b) Egg yolk - citrate - glycerol extenders
Characteristics of semen extender:
1. Provide nutrient and increase the volume.
2. Provide buffer effects to semen (pH 6.7 – 7.0), lactic acid produced
via sperm metabolism lowers pH and effect sperm.
3. Have a proper osmotic pressure and balance of ingredients.
4. Inhibit bacterial growth:- (e.g. Gentamicin, Tylosin, Lincomycin-
spectinomycin, Penicillin, Streptomycin etc
5. Protect against the harmful effects of cooling/ freezing (cold-shock). A
common cryoprotectant is glycerol, egg yolk, milk
 Packing of Semen
 Glass ampules; 1.0ml of extended semen, ampules are sealed in a gas flame
and placed in a bath of 95% ethyl alcohol and controlled amount of CO2. The
semen is kept at -790C for long time by daily addition of ice.
 Straws: Two types (Mini straw- 0.25 ml & Medium straw- 0.50 ml). The
major advantage is that the sperm cells can be frozen more uniformly, less
storage space is required and few sperms are left in the straw upon
insemination.
 Pellets. Composed of lactose (11%), distilled water (18.5%), egg yolk (20%),
glycerol (5%) and antibiotics. ≈ 0.1 - 0.2 ml of extended semen containing 12
to 30 million spermatozoa is put directly in dipressions made in a block of dry
ice and then transferred to storage tubes and put directly into liquid nitrogen.
Most common type of packing used is straw type.
 After equilibrium, semen is filled in previously marked PVC straw and
equilibrated. That straw is then dried using in cold cabinet using absorbent
material and spread over a tray bottom made of mesh wire to allow free
movement of liquid nitrogen vapour.
 The tray with straws is put in liquid nitrogen tank over grill above the level of
liquid nitrogen
 The liquid nitrogen tank is covered with lid. Straw is left in liquid nitrogen
vapour for about 10 minutes.
 Straw are then collected and placed in goblet filled with liquid nitrogen in
nitrogen tank itself.
 Goblet containing frozen semen straw in liquid nitrogen is put in a canister and
canister is immersed in liquid nitrogen.
After packing of semen can be stored in 3 ways depending upon
purpose:
1. Fresh liquid semen can be successfully stored for 1 to 4 days at
40 Celsius (C) or 400 Fahrenheit (F).
2. Dry ice and alcohol (- 700C or -100 degrees F)
3. Liquid nitrogen (-320 degrees F/ -1960C).
 Procedure of Artificial Insemination
 First of all, detection of animal whether it is in estrus or not is estimated. After 12-
18 hours of standing estrus, the cattle is ready for insemination
 Thawing: thawing is the process of increasing the temperature of semen before
insemination. The usual procedure is to immerse the straws in water at 35°C for
20–30 seconds when using 0.25-ml straws or 40–60 seconds for 0.5-ml straws.
 The recto-vaginal technique is the most commonly used for artificial insemination.
 After initial procedure of restraining, cleaning of animal and loading of straw in
AI gun, following procedure is done.
 AI gun is inserted between the lips of vulva into the vagina at 45 º upward angles
to avoid entering the urethral opening and bladder. The rear end of the gun is
elevated upward and gently pushed through the vagina until the surface of cervix
is reached which has a distinct gristly sensation on the gun
 When the gun reaches the anterior part of cervix, external opening of the cervix is located and
thumb is pushed through vagina and cervix forward and away from you to straighten the
vaginal
 With gentle forward pressure on the gun, opening of cervix is located and the gun is slided
slide forward until it contacts the second cervical ring, and by using the flexibility of your wrist,
twist and bend the cervix until you feel the second, and all rings have been passed by the gun
tip (when all rings of the cervix have been cleared, the gun should slide forward freely with
little resistance. Since the uterine wall is very thin, you will once again be able to clearly feel
the insemination gun).
 Gloved hand is retracted until it lies on top of the cervix. With the index finger, far end/anterior
end of the cervix is located.. The gun is pulled back until tip is felt directly underneath your
finger near the internal opening of the cervix.
 Gun is then plunged to place the semen into the uterus. Uterine contractions will then transport
spermatozoa forward to the horns and oviducts with a good distribution of both sides.
SEXED SEMEN/
SEX SORTED SEMEN/
SEMEN SEXING
 HISTORY
 First paper on semen sexing was performed by Larry Johnson.
 sex outcome of a pregnancy could be reliably skewed in either direction.
 Gledhill et al. (1976) commenced the first experiments on flow cytometrical
sperm analysis, but it was Fulwyler (1977) who developed a technical solution
for asymmetric cells to orient them in front of a laser by hydrodynamic
focussing.
 Major breakthrough in Semen sexing: Hoechst 33342 dye easily permeates cell
membranes, selectively binds to the DNA and quantitatively distinguishes the
X- and Y-sperm without any apparent cellular toxicity or impaired sperm
function (Penfold et al., 1998; Garner and Seidel, 2008; Garner, 2009). The dye
selectively binds at AT base pair of minor groove of DNA.
 Methods of semen sexing
 1. Gradient swim down procedure:
• An albumin gradient is used to separate X- and Y sperm
• Y-sperm is small and has greater swimming velocity
• Sperm (22% v) at bottom of column are rich in Y-sperm.

2. Volumetric differences:
• X-sperm is larger than Y-sperm
• Difference in sperm head volume
• Interference microscopy and image analysis
• Flow cytometer is used to separate the sperm.
3. Free-flow electrophoresis
 Presence of electric charge on surface of sperm
 X-sperm differs from Y-sperm based on surface charge of the sperm
 An electric field is used to separate the sperm in two major classes.

4. PCR and FISH

 Specific DNA sequences on X- and Y-sperm have been reported which can be
used to identify the sex of individual sperm and sex ratios of sperm in semen
sample (Wang et al., 2011)
 This method is highly qualitative and quantitative superior (Parrilla et al.,2003).
 Flow cytometer process
 Difference in DNA content between x and y is the discriminating feature to
separate one.
 Difference between x and y chromosome content is 4% in cattle
 In this method, fluorescent dye is used in sperm and laser is used to excite
fluorescent dye.
 The dye used is Hoechast-33342 which is DNA specific fluorescent dye.
 The dye is co-incubated with sperm cells.
 CONTD….
 The stain penetrated sperm membrane and binds with A-T region
of nucleic acid
 The dyed sperm is then passed through flow cytometer into a
discontinuous droplet stream.
 The stream passes through UV laser beam, it illuminates flat
surface of sperm head and excites fluorescent dye.
 The dye shows colour/ shows fluoresces when low wave-length
laser beam is used.
 The fluorescence formed is measured by detector and analyzed by
computer.
Some more insights on: Semen and
Biotechnology
 The sperm genome has to be intact to participate in embryo
development.
 Semen also contains a series of small regulatory non-coding RNA
(ncRNA) that contain 19 to 22 nucleotides (Bartel, 2009).
 These microRNA (miRNA) are found in both the seminal plasma
(Belleannée et al., 2012; ) and spermatoza (Hamatani, 2012; McIver et
al., 2012).
 The miRNA are key post-transcriptional modifiers of gene expression
which acts epigenetically and play an important role in the acquisition
and maintenance of male fertility (Dadoune, 2009).
 They are delivered to the oocyte at fertilization and modulate the first
cleavage division
 Spermatozoa as the vector for transgene
 Sperm-mediated gene transfer (SMGT): DNA from one species is introduced into
another via co-incubation with sperm followed by insemination (Lavitrano et al.,
1989) .
 It is based on principle that the foreign DNA is absorbed by the sperm and then
delivered and incorporated in the genome of future animal during fertilization and
early development.
 it is viewed as a practical method of producing transgenic animals without the need
for surgery, embryo handling, or expensive equipment.
 (SMGT) is highly efficient in integrating transgenes into the genome of the pig
embryo with success rates of 57 to 80%, (Lavitrano et al., 2006).
 However, more applied and basic studies are needed to fully develop this technology
for commercialization.
For
Listening