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NATURAL RESOURCES DEVELOPMENT COLLEGE
ANIMAL PRODUCTION 111 MODULE
ANIMAL REPRODUCTIVE PHYSIOLOGHY,
BREEDING, GENETICS, FEEDING AND BY-
PRODUCTS
ELLISON MSIMUKO (MPVS, B.Agric. Dip.Agric)
7/7/2015
Application of animal breeding and genetics through appropriate understanding of animal
physiology and reproduction in a key to achieving maximum response to selection that brings value
to quality and quantity of livestock products and by products. in a sustainable environment
i
Contents
Introduction..............................................................................................................1
Structure And Function Of The Male Reproductive System.......................................2
Male Macrostructure And Function ...........................................................................2
Penis And Related Structures: ................................................................................2
Testes And Related Structures:...............................................................................3
Epididymis And Related Structures:.......................................................................3
Accessory Glands: .................................................................................................3
Semen.......................................................................................................................4
Male Microstructure And Function .........................................................................4
Hormonal Regulation Of Testicular Function ........................................................6
Structure And Function Of The Female Reproductive System ..............................6
Placenta....................................................................................................................8
Cervix .......................................................................................................................9
Vagina.......................................................................................................................9
Female Microstructure And Function ..................................................................10
Oogenesis ..............................................................................................................10
Prenatatal Maturation........................................................................................ 11
Postnatal Maturation.......................................................................................... 11
The Mature Ovum ..................................................................................................12
Comparison Of Spermatocytogenesis And Ovigoogenesis ...................................13
Ovulation................................................................................................................ 14
Transport Of Gametes, Fertilisation And Implantation ........................................14
References..............................................................................................................16
Chapter Two..........................................................................................................16
Artificial Insemination.......................................................................................... 16
Principles Of Artificial Insemination ....................................................................16
1.0 Introduction .....................................................................................................16
History Of Artificial Insemination .........................................................................16
Aim Of Ai ................................................................................................................ 17
Role In Animal Breeding........................................................................................ 17
Utilization Of Bulls.............................................................................................. 17
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Role With Regard To Health And Sterility ......................................................... 18
Scientific Role .....................................................................................................19
Critics Of Artificial Insemination ...........................................................................19
Advantages Of Artificial Insemination:.............................................................. 19
Disadvantages Of A.I:.......................................................................................... 20
Semen Collection Methods And Evaluation:......................................................... 20
Artificial Vagina Method..................................................................................... 20
Recovery Method................................................................................................ 22
Massage ..............................................................................................................22
Electro-Ejaculator............................................................................................... 22
Semen Analysis ......................................................................................................23
Semen Storage....................................................................................................24
Processing Semen............................................................................................... 26
Packaging............................................................................................................26
Calculation Of Sperm Concentration .....................................................................26
Insemination Methods........................................................................................... 27
Vaginal Method:..................................................................................................32
Symptoms Of Heat .................................................................................................32
Detecting Estrus As Aid To Artificial Insemination..............................................33
The Timing Of Insemination ..............................................................................34
Chapter Three .......................................................................................................34
Embryo Transfer....................................................................................................34
Super-Ovulation .................................................................................................34
Methods Of Embryo Recovery And Transfer......................................................... 35
Methods Of Collection Or Recovery ...................................................................35
Surgical Methods................................................................................................ 35
Site Of Transfer ......................................................................................................36
Transfer Into The Receipient................................................................................. 36
Surgical Method..................................................................................................37
Synchronization.....................................................................................................37
Advantages .........................................................................................................37
Disadvantage ......................................................................................................37
Chapter Four .........................................................................................................38
iii
Principles Of Animal Genetics................................................................................ 38
Mendel’s Laws........................................................................................................38
Genetic Material.....................................................................................................38
Gene Coding........................................................................................................39
Considering Multiple Traits-Dihybrid Cross ......................................................... 41
Other Concepts In Genetics.................................................................................... 43
Mutations And Other Chromosomal Abnormalities .............................................43
Sex-Linked Traits...................................................................................................44
Quantitative Vs Qualitative Traits .........................................................................44
Measuring Heritable Variation ..............................................................................44
Heritability.............................................................................................................45
Estimating Heritability From Regression .......................................................... 46
Comparison Slope............................................................................................... 46
Role Of Reproduction In Genetic Improvement.................................................... 46
Methods Of Selection ............................................................................................. 47
Factors Affecting Genetic Change And Progress ................................................... 48
Modern Genetics ....................................................................................................49
Cloning................................................................................................................ 49
Chapter Five..........................................................................................................49
Animal Breeding Systems...................................................................................... 49
Objectives Of The Topic ......................................................................................... 49
Breeding Systems............................................................................................... 49
Linebreeding ..........................................................................................................50
Linecrossing .......................................................................................................50
Outcrossing.........................................................................................................50
Grading Up..........................................................................................................51
Crossbreeding (X) ..................................................................................................51
Beef Crossbreeding Systems ..............................................................................51
Two-Breed Rotation........................................................................................... 52
4 And 5 Breed Rotations .................................................................................... 52
Static Terminal Sire System ...............................................................................52
Rotational –Terminal Sire System .....................................................................53
Composite Breeds............................................................................................... 53
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Breed And Crossbred Means (Reciprocal Crosses) For Various Traits Of Cattle In
Zambia ................................................................................................................ 53
Extra Beef Production........................................................................................... 54
Intensive Beef Production Systems.......................................................................54
Production Systems............................................................................................ 54
Principles Of Ration Formulation For Livestock................................................... 56
Ration Functions ................................................................................................ 57
Balancing Rations............................................................................................... 58
Summary.............................................................................................................60
Relationship Between 100 Percent Dry Matter Basis And As-Fed Basis ..........60
Rules Of Thumb For Balancing Beef Rations ..................................................... 61
Energy System....................................................................................................61
Protein System.......................................................................................................62
Least Cost Formulation ...................................................................................... 62
Beef Cattle: High-Grain Finishing Rations............................................................ 65
All-Concentrate Rations......................................................................................... 65
Disease Problems Of Animals On All-Concentrate Rations ............................... 66
Application In Zambia............................................................................................ 66
Use Of Other Grains With Or Without Roughage Supplementation.................. 66
Sources Of Roughage In High-Grain Rations ..................................................... 68
Ration Characteristics............................................................................................ 75
The Metabolisable Energy (Me) System................................................................ 75
Introduction:.......................................................................................................75
Evidence Of Energy Balance Regulation............................................................ 75
Responses To Changes In The Environmental Heat Loas :................................ 75
Eefects To Exrcise :............................................................................................. 75
Effect Of Lactation : ............................................................................................ 76
Effect Of Metabolic Rate And Growth :............................................................... 76
Short Term Regulation Of Feed Intake..................................................................76
Chemostattic Theories ....................................................................................... 76
Thermostatic Theory.......................................................................................... 76
Effect Of Gut Digestion :...................................................................................... 77
Role Of Gut Hormones........................................................................................ 77
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By-Pass Protein :.................................................................................................77
Regulation Of Energy Balance Through Feed Intake.........................................77
Internal (Or Animal) Factors :...........................................................................77
External (Or Env Iromnental) Factors...................................................................77
The Prediction Of Voluntary Feed Intake.............................................................. 78
Equations Used To Predict The Voluntary Intake Of Feed By Growing Animals .78
The Metabolisable Energy (Me) System................................................................ 79
Feed Energy:.......................................................................................................79
Chapter Six.............................................................................................................80
Flaying .................................................................................................................... 81
Hand Flaying .......................................................................................................81
Machine Flaying ..................................................................................................81
Damage After Flaying ............................................................................................. 81
Preserving ...............................................................................................................81
Methods Of Preservation...................................................................................... 82
Salting ................................................................................................................. 83
Brine Curing ........................................................................................................83
Storing Of Preserved Hides...................................................................................... 83
The Grades ..............................................................................................................85
1st Grade..............................................................................................................85
2nd Grade ............................................................................................................85
3rd Grade.............................................................................................................85
4th Grade.............................................................................................................85
Rejects................................................................................................................. 86
Packing.................................................................................................................... 86
Ellison Msimuko (MPVS, B.Agric DipAgric)
.Mr. E. Msimuko studied at Natural Resources Development College, University of
Zambia and University of Adelalide Australian. He works has been dedicated to
improving Livestock in Zambia through handson experience obtain from Zambezi
Ranching and Copping Limited for nine years, the government of Republic of Zambia
(ZCA-MPIKA and NRDC) as Assistant Farm manager/ Farmer Manager. He was also
involved in lecturing animal science and genetic courses at NRDC and Lusangu
University for years. Currently, he has dedicated his work in molecular and qunatitative
genetics in Zambian Livestock to assist in developing and consrving well and adapted
genetic material in Zambia Livestock. He has made several publicastions, attended
several internation conferences as invited speaker. Inaddition he is member of ISAG,
AAABG, ASGN.
Acknowledgements
This task was huge and needed help from other colleagues elsewhere. Therefore, I am
indebted to numerous people and organisations that contributed significant time and
resources to enable me to complete this research.
I would like to acknowledge and thank the providers of the open and distance learning, for
the considerable financial support I received to undertake this work.
In recognition of my principal supervisor Associate Professor Wayne Pitchford, thank you
very for your guidance that is exceptional and ever valuable in imparting research knowledge
and skills. Your dedication to work has indeed motivated me to take any future challenges
without fear or favour. Out of your busy schedule, you always found time for me without that
I would not have completed my work as scheduled. You are truly my living example across
numerous domains of my life ranging from professional development, teaching to general
outlook.
A significant appreciation goes to my co supervisor Associate Professor Cynthia DK Bottema
(Animal Genetic Technologies, South Australia), for her swift and tremendous feedback on
written and oral work that was provided during my work In particular many thanks for your
assistance in addressing Mendelian inheritance and genomics notes. Your input really was
tremendous special to me. Thank you ‘Cindy’
Finally, I acknowledge several individuals whom I may not include in the lists including from
other universities I had contacted for assistance in data analysis and allowing me to use their
writtern notes and author of various books
2
Introduction
Reproduction is the process by which animals produce offspring for the purpose of
continuing the species. The process of reproduction begins with copulation, which is the
mating of a male and female of the species. Sperm cells from the male are deposited in the
female reproductive tract and try to unite with an egg cell. When fertilization (a sperm cell
and egg cell unite) occurs, an embryo begins to develop. The embryo attaches to the wall of
the uterus where it is protected, receives nourishment, and develops. When the new offspring
reaches the end of the gestation period, it is delivered from the female reproductive tract in a
process called parturition. To completely understand the process of reproduction, a basic
knowledge of the reproductive tract structures and functions is required.
Structure and Function of the Male Reproductive System
The male reproductive system is made up of several organs, glands, and muscles; The major
functions of the male reproductive system include: Production, storage, and deposition of
sperm cells, Production of male sex hormones, Serve as passageway for expelling urine from
the urinary bladder.
Figure 1. Male reproductive system of a bull
Male Macrostructure and Function
Penis and Related Structures:
Penis an organ that allows for the deposition of semen into the female reproductive tract. The
penis of the stallion is vascular, which means that it depends on the engorgement of blood
within certain tissues for erections to occur and it forms no sigmoid flexure when relaxed.
The penises of the bull, ram, and boar are fibroelastic, which means that they are primarily
composed of connective tissue and depend little on blood for erections. The rear portion of
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the fibroelastic type penis forms an S-shaped curve or sigmoid flexure when relaxed The
penis of a bull, muscle contracts to retract the penis and form the sigmoid flexure and relaxes
to extend the penis upon sexual excitement
Testes and Related Structures:
Testes a paired, ovoid shaped organs that produce sperm cells and testosterone the male sex
hormone that is responsible for the development of secondary male characteristics and sex
behaviour (libido). It has a spermatic cord a protective fibrous sheath consisting of smooth
muscle, blood vessels, and nerves; cremaster muscle, primary muscle supporting testes and
coursing the length of the spermatic cord and scrotum, a sac outside the body cavity that
protects and supports the testes. The spermatic cord extends from the body through the
inguinal ring to suspend the testes within the scrotum. The cremaster muscle, spermatic cord,
and tunica dartos muscle raise and lower the testes to maintain a constant temperature (4 – 6
degrees below body temperature) for sperm to develop. Sheath is an external portion of the
male reproductive tract that serves to protect the penis from injury and infection
Epididymis and Related Structures:
Figure 2. Epididymis and related structures of a bull
Epididymis is a coiled tube connected to each testis that is responsible for the maturation,
storage, and transport of sperm cells. Deferent Duct (Vas Deferens) is part of the spermatic
cord that is the passageway for sperm from the epididymis to the urethra. Ampulla is an
enlargement of the deferent duct that opens into the urethra and may serve as a temporary
storage depot for sperm. Urethra that is a passageway for both semen and urine that extends
from the ampullae and bladder to the end of the penis.
Accessory Glands:
The accessory glands are responsible for the production of secretions that contribute to the
the liquid non-cellular portion of semen known as seminal plasma. Semen and ejaculate are
terms given to the sperm plus the added accessory fluids. Vesicular glands (seminal vesicles)
paired accessory glands that secrete seminal fluid that nourishes the sperm and provides
protection and transportation medium for sperm upon ejaculation. It adds fructose and citric
acid to nourish the sperm. Seminal vesicles empties into ejaculatory duct produces about 60%
of semen and contains fibrinogen with high pH
4
Prostate Gland located at the neck of the bladder, secretes thick, milky fluid that mixes with
seminal fluid to provide nutrition and substance to the semen. It cleans the urethra prior to
and during ejaculation, provides minerals for sperm, acts as medium for sperm transport and
provides the characteristic odor of semen. Produces about 30% of semen thin, milky secretion
with high pH and contain clotting factors, and fibrinolysin
Bulbourethral glands (Cowper’s glands) secretes fluid that cleanses and neutralizes the urine
residue that can kill sperm cells in the urethra. it contribute about 5% to semen, mucous
secretion just before ejaculation and helps neutralize pH of female vagina.
Semen
2-5 mL of fluid expelled during orgasm 60% seminal vesicle fluid, 30% prostatic, 10% sperm
normal sperm count 50-120 million/mL Other components of semen fructose energy for
sperm motility fibrinogen causes clotting enzymes fibrinolysin liquefies semen within 30
minutes prostaglandins stimulate female peristaltic contractions spermine is a base stabilizing
sperm pH at 7.2 to 7.6. Secretions of all three accessory glands plus sperm cells referred to as
semen.
Emission is the discharge of semen into prostatic urethra while Ejaculation is referred to as a
forceful expulsion of semen from urethra caused by peristalsis. Temporary coagulation as
fibrinogen becomes fibrin then fibrinolysin breaks up the coagulation. Sperm swim up
vagina. Emission: accumulation of sperm cells and secretions of the prostate gland and
seminal vesicles in the urethra controlled by sympathetic centers in spinal cord peristaltic
contractions of reproductive ducts. Seminal vesicles and prostate release secretions and
accumulation in prostatic urethra sends sensory information through pudendal nerve to spinal
cord
Male Microstructure and Function
Microscopic cellular parts within the testes produce sperm cells and testosterone.
Spermatogenesis refers to the development of sperm cells (spermatozoa) through a process of
cell division and maturation. Firstly, spermtogenic cells form rounded cells called spermatids
(Spermatocytogenesis) and secondly, spermatids differentiate into specialized cells known as
sperms-Spermiogenesis. It is a process by which spermatogonia are transformed into
spermatozoa. It begins at puberty. At birth: germ cells in the male present in the sex cords of
the testis are surrounded by supporting cells (Sertoli cells). At this time the primordial germ
cell divides to give spermatogonia. At puberty the germ cells awaken and start the actual
process of spermatogenesis. These cells increase in number by simple mitosis to form cells
known as spermatogonia ; type- A and type- B. Type-B spermatogonia, replicate DNA to
have 46 double structured chromosomes to begin meiosis-1 and are called primary
spermatocytes. Meiosis 2 follows immediately without DNA replication. Only 23 double
structured chromosomes are involved 2 secondary spermatocytes quickly undergo meiosis-2
(Centromeres split in metaphase 2) and end with the formation of 4 sperrmatids each with 23
single structured chromosomes and1N DNA. Two spermatids bear X chromosome
complement and other two bear Y chromosome complement.
5
Figure 3. Process of sperm formation
As steps of spermatogenesis continue, the spermatocytes progressively move from basement
membrane to the luminal side of seminiferous tubule. The cells of Sertoli provide nutrition
and pockets of support to developing spermatocytes, support production of spermatozoa,
androgen-binding protein intratesticularly (tebg), form blood-testes barrier, produce inhibin
and seminiferous tubule fluid (bathing medium, nutrients, capacitation, motility).
Spermatids are rounded cells. They modify to assume specific shape of the sperm. The
changes include;
 Golgi apparatus forms acrosomal cap-proteolitic enzymes
 Nucleus is condensed
 Centriols: make collar around neck
 Microtubules, form flagellum,
 Mitochondria arrange as spiral around neck
 Excess cytoplasm cast off as residual body
 Cytoplasmic bridges break and sperms release from Sertoli cells to lie free in lumen
of seminiferous tubules.
About 64 days are required to go from a spermatogonium to a sperm. A mature sperm has
head, neck and tail. From lumen of seminiferous tubules sperms enter duct of epididymis and
they take 20 days to travel this 4-6 meter long tortuous duct. If ejaculation does not occur
they die and degenerate.
6
Hormonal Regulation of Testicular Function
Testicular regulation involves three sets of hormones:
GnRH - gonadotropin-releasing hormone secreted by hypothalamus and stimulates secretion
of anterior pituitary secretion hormones (FSH/LH). LH act as Interstitial Cell Stimulating
Hormone secreted by anterior pituitary and directly stimulate the testes (interstitial cells) to
secrete testosterone while FSH stimulates formation of ABP (androgen binding protein).
Testosterone secreted by cells of Leydig (Interstitial cells of seminiferous tubules) exert
negative feedback on hypothalamic and ant. pituitary hormones and stimulates
spermatogenesis by binding to ABP and development of secondary sex characteristics.
QUIZ???
1. Describe the structure of the testis and the process of spermatogenesis.
2. What is spermatogenesis.
3. Describe the normal and abnormal morphology of the sperm.
4. Appreciate functions of different hormones produced from Male spp
5. Describe cell cycle
Structure and Function of the Female Reproductive System
The functions of the female reproductive system include: Produce egg cells (ova), serve as
receptacle for the penis during copulation, and house and nourish the fetus until parturition as
well as production of hormones.
7
Figure 3. Female reproductive system of a cow
Ovaries are paired structures that produce eggs (ova) and the female hormones, estrogen and
progesterone. Broad ligaments that support the female reproductive tract and arteries, veins,
and nerves of the ovaries in the abdominal cavity. The ovary is comparable to the male
testicle and is the site of gamete production. A bovine animal has 20,000 potential eggs per
ovary. Ova are fully developed at puberty and are not continuously produced as in the male.
All species contain two functional ovaries except for the hen that has only a left functioning
ovary. The ovaries have three major functions:
 Gamete production (oovigenesis)
 Secrete estrogen (hormone)
 development of mammary glands
 development of reproductive systems and external genitalia
 fat deposition on hindquarters and belly (source of energy)
 triggering of heat
 Form the corpus luteum that secrete Progesterone
Oviducts (Fallopian tubes), paired tubes that transport the eggs from the ovaries to the uterus
and serve as the site where sperm and ova meet and fertilization occurs.
Infundibulums two funnel-like openings of the oviducts that pick up the eggs at ovulation and
direct them to the body of the oviducts.
8
Uterus a major reproductive organ that consists of the uterine body and two uterine horns.
The embryo attaches to uterine body or uterine horn, depending on the species. The uterus
varies in shape between livestock species from long uterine horns of the sow to relatively
short uterine horns in the mare. This varying degree of sizes reflect the species differences in
litter sizes, monotocous species having short horns and polytocous animals having much
longer uterine horns.
Figure 4. Types of uterus found in various species
Functions of the uterus include:
 Passageway for sperm during copulation,
 Incubation and nourishment of the embryo during pregnancy, and
 Expulsion of the fetus during parturition by contractions.
 Hormonal production P2G
PLACENTA
Classification is based on the thppgross shape, the distribution of contact sites between fetal
membranes and endometrium, the number of layers of tissue between maternal and fetal
vascular systems and type of tissue (figure below)
9
Figure 5. Types of Placentas and common examples
CERVIX
Cervix is a thick-walled mass of connective tissue with a small tube-like opening that joins
the uterus to the vagina; it serves as a passageway for semen during copulation. It also
contains glands that secrete a waxy-like substance that seals off the uterus during pregnancy
and between heat periods to protect against infection, disease, or foreign matter.
Figure 6. Cervix of Cow
Vagina
Vagina is a reproductive structure that serves as the receptacle for the penis during copulation
and the birth canal at parturition; it also serves as a passageway for expelling liquid wastes, as
the urethra joins the bladder to the vagina prior to the opening at the vulva. It receives
semen (except in sow) while the vulva an external portion of the female reproductive tract
that serves to protect the internal system from infection, to initially receive the penis at
copulation, and to act as a passageway for urine.
10
Female Microstructure and Function
Oogenesis is the process of producing ova (eggs) in the follicles of the ovaries. Oogonia cells
develop in the ovaries of the fetus and mature into oocytes by birth. Only a small proportion
of oocytes develop into ova or reach ovulation. Follicle which is a blister-like mass on the
surface of the ovary that contains a developing ovum and produces and stores estrogen. The
follicle secretes estrogen as a signal to the rest of the reproductive tract to prepare for
ovulation (release of the ovum from a mature follicle). Corpus hemorrhagicum that develop
after ovualation is a small hemorrhage or blood-clotted area that develops at the site of a
ruptured follicle and lasts 2 – 3 days. Then later Corpus luteum (CL), a yellow body of cells
that develops in place of the corpus hemorrhagicum and produces progesterone.
Figure 7. An ovum with different stages of follicle development
Progesterone prepares the female reproductive system for pregnancy; it is produced by the
corpus luteum and lasts about twelve days, unless the ovum is fertilized. During luteal phase
corpus albicans, a white body of connective tissue that is the result of the degeneration and
re-absorption of luteal tissue.
Oogenesis
The number of ova produced per cycle varies with each species. For example a cow or mare
normally produces one ovum per cycle, ewe produces two ova per cycle whiles a sow
produces eight to fifteen ova per cycle and a hen approx. 28 eggs/month. Oogenesis means
differentiation of the primordial germ cells (oogonia) that are present in the cortex of the
ovary into mature ova. Oogenesis passes into two stages of maturation: prenatal and postnatal
maturation:
11
Prenatatal maturation
The primordial germ cells (oogonia) undergo mitotic divisions then they are arranged in
clusters surrounded by a layer of flat epithelial cells. The flat cells are called follicular cells
and are derived from the epithelium covering the ovary. The development of ova mature,
unfertilized eggs cells happens in the ovary. Some oogonia differentiate into large primary
oocytes where DNA replication occurs and they enter the prophase of the first meiotic
division. The surviving primary oocytes become surrounded by flat epithelial cells. Together
they are called primordial follicles. Oogonia (stem cells that give rise to ova) multiply and
begin meiosis STOPS at prophase 1. At this phase, the cells are called primary oocytes and
remain in this phase until the onset of puberty, when they are activated by hormones LH and
FSH. Near birth, all the primary oocytes have started prophase of the first meiotic division
then they are arrested till puberty. The number of primary oocytes at birth ranges from
700,000 to 2 million. At puberty only 400,000 primary oocytes remain and about 400 only
will be ovulated during the female life time.
Postnatal maturation
With the onset of puberty, 5-15 primordial follicles begin to maturate with each ovarian
cycle. The primary oocyte begins to increase in size and the surrounding flat cells change
12
from flat to cuboid and proliferate to produce a stratified epithelium of granulosa cells. The
follicle is now called the primary follicle. The granulosa cells rest on a basement membrane
separating them from the stromal cells of the ovary that form the theca folliculi. The
granulosa cells and the oocyte secrete a layer of glycoprotein on the surface of the oocyte
called the zona pellucida. Fluid-filled spaces appear between the granulosa cells then they
coalesce together forming the follicular antrum and the follicle is now called the secondary
follicle. The follicle enlarges and is called the Graafian follicle
In a 46 diploid individual, the primary oocyte continues the first meiotic division leading to
two unequal daughter cells with 23 chromosomes each (22 autosomes + X). The large cell is
called the secondary oocyte and the small one (having little cytoplasm) is the first polar body.
The secondary oocyte and the first polar body enter the second meiotic division without DNA
replication. The second meiotic division is completed only if fertilization occurs to give
fertilized oocyte (mature ovum) and a second polar body that soon degenerates. FSH
periodically stimulates a follicle to grow and induces its primary oocyte to complete meiosis
1 and start meiosis 2 meiosis then STOPS again and LH stimulates the completion of meiosis.
The mature ovum
It is a large oval cell that varies from 117 – 142 µ in diameter. It has two membranes; the
inner thin one is the vitelline membrane and the outer one is the zona pellucida. The corona
radiata is two or three layers of cells surrounding the zona pellucida externally when the
ovum is shed from the follicle. .
13
Comparison of spermatocytogenesis and Ovigoogenesis
14
Ovulation
Mechanisms of ovulation involves hormonal surge that leads to increase in enzyme activity;
collagenase and increased intrafollicular pressure. Ovulation a rupture of ovarian wall and
follicle releases gamete. Gamete is transported and fertilised in uterine tube. Granulosa cells
and theca interna become luteinised and form corpus luteum(CL), a transitory endocrine
organ. In the Luteal phase progesterone is secreted from CL and maintains pregnancy. Life
span of CL dependent on luterophic LH and luteolytic PG2a. Formation, maintenance and
regression of CL always under hormonal control.
Transport of gametes, fertilisation and implantation
Ova ovulated from ovary, transported through uterine tube. Sperm deposited in vagina where
it is transported and also capacitation occurs. .Fertilisation at ampulla-isthmic junction of the
reproductive tract. After fertilisation, the Zygote is transported and get implanted in
endometrium.
Fertilisation
Fertilisation consists of binding of sperm to zona pellucida and acrosome reaction.
Thereafter, the sperm head penetrate of Zona Pellucida through cortical reaction that help
fusion of plasma membranes of sperm and egg. Sperm nucleus enters oocyte, forms
pronucleus, to complete meiosis II.
15
Fertilization that is the union of the haploid sperm and the haploid ovum to form a diploid
individual; the actual beginning of pregnancy. Insemination can be natural or artificial. When
bred naturally, the male deposits the semen into the vagina of the female. The sperm make
their way through the cervix, into the uterine body, both uterine horns, and finally into the
oviducts. At first, the embryo floats freely in the uterus obtaining its nourishment from fluids
secreted by the uterine wall. The embryo will then attach to the wall of the uterus (20 days
in cattle; varies with species) and begin taking a recognizable form, at which time it
becomes a fetus. The embryo is encompassed by a fluid filled membrane called the amnion,
which protects the embryo from mechanical disturbances. The amnion is surrounded by the
chorion, which functions as a protective coat and point of nutrient exchange.
In ruminants (cow and ewe), the chorion develops cotyledons (raised button-like nodules)
that attach at certain places on the uterus called caruncles. In the mare and sow the placental
attachment is made over most of the surface area of the chorion. The fetus receives
nourishment from its mother through these attachment sites. The placenta is the term given to
the membranes (chorion and uterine mucosa) that surround the embryo and attach to the
uterus.
Parturition
16
The fetus is expelled from the uterus, passing through the cervix and vagina, out of the
reproductive tract. The membranes that had formed around the fetus are also expelled and,
collectively, are called afterbirth. After parturition, the corpus luteum on the ovary begins to
degenerate, a developing follicle on the ovary begins to release estrogen, estrus occurs, and
the estrous cycle begins again.
References
Alberts, B; Johnson, A; Lewis, J; Martin, R; Roberts, K; & Walter, P(2002). The Molecular
Biology of the Cell. 4th.Edition. Garland Science Chapter 20.
Dyce, K; M.,Sack, W;O, & Wensing, C;J;G.(2006). 3rd. Edition. Textbook of Veterianry
Anatomy.Pages183 – 202, 435 -453
Neill,J;D.(editor-in-Chief). Physiology of Reproduction. 3rd. Edition. Elsevier.
London.Volume 1. Page3-113.
Chapter two
ARTIFICIAL INSEMINATION
PRINCIPLES OF ARTIFICIAL INSEMINATION
1.0 introduction
This chapter will be devoted solely to technical aspects of artificial insemination (AI). This is
a tool for improving livestock production. A productive tool used to bring out the potential of
animals by using superior sires. Artificial insemination is the technique in which semen with
living sperms is collected from the male and introduced into female reproductive tract at
proper time with the help of instruments. It is a breeding tool that involves methods of
collecting semen from the bull and its deposition in the female reproductive tract by any
insemination technique rather than natural. It is a very modest expedient aimed solely at
collection of semen and its insertion into the female genital passages.
History of Artificial Insemination
Documents from around 1322 A.D. state that an Arab chief wanted to mate his mare to a
stallion owned by his rival. Then in 1780, Spallanzani successfully bred two dogs with the
use of A.I. Leading to him being named the inventor of artificial insemination. The mass
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breeding of cattle however didn’t come until later, in 1931. His experiments proved that the
fertilizing power reside in the spermatozoa and not in the liquid portion of semen. Few
further studies under research station conditions helped this technique to be used
commercially all over the world including India.
Artificial insemination is not merely a novel method of bringing about impregnation in
females. Instead, it is a powerful tool mostly employed for livestock improvement. In
artificial insemination the germplasm of the bulls of superior quality can be effectively
utilized with the least regard for their location in far away places. By adoption of artificial
insemination, there would be considerable reduction in both genital and non-genital diseases
in the farm stock.
Student Learning Objectives
1. Describe the importance of animal reproduction.
2. List the parts and explain the functions of female and male reproductive systems.
3. List and describe the phases of the estrous cycle.
4. Explain how artificial insemination is performed.
5. Explain the advantages and limitations of artificial insemination.
6. Explain new technologies that are being used in reproductive management of animals.
AIM OF AI
It is a breeding to tool used to enhance improvement in breeding stock and it can never be
sufficiently emphasis that artificial insemination is essentially a method essentially a method
whose aim is better livestock breeding
ROLE IN ANIMAL BREEDING
Utilization of Bulls
a. Use of outstanding males by the majority of breeders. Most breeders are able to use
best bulls which otherwise would have been impossible for many of them owing to
distance, high cost of purchasing such a bull or the prohibitive cost of service.
b. Testing of young bulls. It helps produce, test, discover and use new genetic material
c. Reduction in the number of bulls. Reducing the number of males reduces direct and
indirect costs. With natural insemination a bull giving 100 services per year, or say
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80 calves, will total 250 descendants in three years: with AI this number has to be
multiplied by a coefficient which may be as high as 40:250 = 10,000.
d. Withdrawal of bulls from small or average farms. Many farms carry one bull to ten
cows. This makes service very expensive for poor semen. Removal of bull will
allow a productive cow to be added to the stock
e. Use of superior males that are old or already dead. Semen collection methods enable
old bulls to be used that for various reasons, such as excess weight, tired legs etc are
incapable of service. Use of refrigerated semen enables bulls that have long since
dead to be used, facilitates testing.
f. Use of bulls in various regions. Bulls of the best breeds are generally delicate and
exacting in their demands and it has been hazardous and onerous task to transport
them from region to another: sending semen eliminates such journeys. in addition as
a results of the rigours of acclimation these animals often provide a poor
reproductive service, whereas semen rapidly conveyed to where it is needed and
inseminated with all its own qualities and those of the donor into indigenous females
well accustomed to the environment will give cross-breed of good quality.
g. Use of bulls restricted by international sanitary regulations. AI overcomes this
obstacle by sending semen instead of the bull which makes easier to comply with the
requirement.
h. Use of bulls in cross-breeding. AI solves the problems of the sexual repulsion that
exist between breeds and thus facilitates cross-breeding.
Role with regard to health and sterility
a. Prophylaxis of non- venereal diseases. By avoiding any contact between animals
b. Prophylaxis of venereal diseases. Control sexually transmitted diseases. The bulls
are tested before use.
c. Therapy and sterility. Direct introduction of semen into the uterus may vaginal and
cervical spasms to be overcome and the injurious influence of the vaginal
environment to be avoided. It overcomes the effect of granular vaginitis
d. Recognition of sterility. Renders worth service gynaecologic ally. Tool for
investigation of reproductive issues of females
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Scientific role
a. Synchronization of estrus. If the animals are synchronized it is difficult to mate them
but AI makes it easier.
b. Makes possible the histo-physiological study of semen, study of its fertilizing
capacity and examination of factors affecting if especially nutrition, health and sex
life of the bull.
c. It enables optimum time of coitus to be established.
d. The effects of medicaments and the possibilities of disinfection and antiseptics of the
semen can be studied
Critics of artificial insemination
a. It is difficult in developing countries for most farmers to properly detect heat.
b. In developing countries, the delivery system is poor such that semen may be
received later than the day you request it.
c. Very accurate breeding records are needed to know when the animal comes on heat.
d. Safety on the farm. In some dairy bulls are very dangerous and aggressive.
ADVANTAGES OF ARTIFICIAL INSEMINATION:
There are several advantages by artificial insemination over natural mating or servicing.
These include:
1. There is no need of maintenance of breeding bull for a herd; hence the cost of
maintenance of breeding bull is saved.
2. It prevents the spread of certain diseases and sterility due to genital diseases e.g:
contagious abortion, vibriosis.
3. By regular examination of semen after collection and frequent checking on fertility
make early detection of interior males and better breeding efficiency is ensured.
4. The progeny testing can be done at an early age.
5. The semen of a desired size can be used even after the death of that particular sire.
6. The semen collected can be taken to the urban areas or rural areas for insemination.
7. It makes possible the mating of animals with great differences in size without injury
to either of the animal.
8. It is helpful to inseminate the animals that are refuse to stands or accept the male at
the time of oestrum.
9. It helps in maintaining the accurate breeding and cawing records.
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10. It increases the rate of conception.
11. It helps in better record keeping.
12. Old, heavy and injured sires can be used.
Disadvantages of A.I:
1. Requires well-trained operations and special equipment.
2. Requires more time than natural services.
3. Necessitates the knowledge of the structure and function of reproduction on the part
of operator.
4. Improper cleaning of instruments and in sanitary conditions may lead to lower
fertility.
5. If the bull is not properly tested, the spreading of genital diseases will be increased.
6. Market for bulls will be reduced, while that for superior bull is increased.
SEMEN COLLECTION METHODS AND EVALUATION:
Various methods of collection of semen have been devised from time to time. The older
unsatisfactory methods have gradually replaced by the new modern techniques. There are
four common methods.
 Use of artificial vagina
 Electro-stimulation method.
 Massaging the ampulae of the duct us differences through rectal wall.
 Recovery method
The ideal method of semen collection is use of artificial vagina which is safe for sire and the
collector also.
ARTIFICIAL VAGINA METHOD
The artificial vagina has the following parts: A heavy hard rubber 2" lose, open at both ends
with a nostle for air and water in and outlet, inner sleeve of rubber or rubber liner and the
semen receiving cone or rubber cone; the semen collection tube made of glass or plastic
graduate as well as insulating bag. Before using for semen collection all the parts must be
washed thoroughly and sterilized properly, and assembled. As in artificial vagina, the rubber
liner is inserted into the hose; inverting both ends back by folding back from either side
opening, and fastening with rubber bands. Now the space between the hard rubber hose and
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inner rubber liner forms a water tight compartment. The nostle at one end of the hose can be
fixed .
Turning through the threaded nut up or down. The water jacket of the Artificial -vagina is
filled with hot water at a temperature of 45°C (113°F) by opening the nostle. The graduated
semen collection tube is fixed to the narrow end of the artificial vagina hose, and fastened by
a rubber band. The inner side of the rubber liner on the anterior side of the artificial vagina is
lubricated with sterile jelly to a length of 3 to 4 inches. Air is blown through the nostle into
the water jacket, to create pressure in if, and the same is exerted the rubber linear, to simulate
natural vagina.
The temperature of the artificial vagina is to be checked, at each collection, and it should
simulate natural vagina at mounting time.
If the artificial vagina is to mount later. If it is too cold ejaculate may not be there after a
thrust, or even if ejaculate is there; it may be contaminated with urine, and becomes unfit for
use. The artificial vagina assembled is held at 45° angle from the direction of penis, and the
thrust is that angle. The artificial vagina is held with the left hand by a right handed person;
and when the bull mounts the cow, the sheath of the bull will be graphed by the operator,
directing the gland penis into the artificial vagina, and then the bull gives a thrust to ejaculate
(Figure). The operator should evince care so as not to touch the exposed past of the penis.
After the bull dismounts, the artificial vagina is taken off from penis and the air vent is
opened to release the pressure from the jacket.
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The water from the jacket is also drained by opening the nostle. This allows the ejaculate to
flow from the cone to the semen collection tube. The semen collection tube is detached from
the cone, plugged with cotton wool, and taken to the laboratory for examination. The rubber
cone and the semen collection tube can be protected from external contamination or heat or
higher, by covering with an insulation bag with zip.
Recovery Method
This method allows the male to mate with the female and you use a spoon to take the sperms
that were ejaculated. Some us a spongy in the female reproductive system and just take the
spongy out which will have some semen. The main disadvantages is that spermatozoa is of
poor quality because they may mix with secretion from the female. it also increases chances
of sexually transmitted diseases.
Massage
The hand is passed in the rectum of the bull (gloves or plastic should be used). If there is a
lot of fecal matter it should be removed. The hand is passed over the vesicles glands and
ampullae of the vas deferens to induce the female of semen. The cause the penis to protrude
the sigmoid flexure can also be massaged so that the semen collected is less contaminated.
Disadvantage is semen collected may be contaminated with urine and debris.
Electro-ejaculator
An electro-ejaculator probe powered ether by battery or battery cum-electric transistorized
circuits is used by inserting it into the rectum and stimulating nerves of the reproductive
system by gradually increasing voltage(10-15 volts) in a rhythmic fashion with a rheostat for
short period. The electricity is passed on in pulses. The change of voltage causes stimulation
which causes erection and ejaculation.
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This type of collection is recommended because; Semen is clean because the area around the
penis is cleaned and it is of high concentration. Incapacitated animals (lame) that cannot
mount can also be used provided they are genetically fine. Advantage is that it does not
require female or a dummy is required, however, the disadvantage of this method require an
expensive machine and may affect sciatic nerves.
Semen Analysis
Sperm concentration- Total sperm is determined by multiplying concentration (sperm per ml)
by ejaculate volume (ml). Semen traits include Volume-sperm/g and Density classified as:
 Very good(VG)-750-1 billion sperms (Creamy)
 Good (G)-400-750 spermatozoa (Milk-like)
 Fair (F)-250-400 spermatozoa (Skim milk-Like)
 Poor –less than 250 (translucent)
Sperm motility is the percentage of sperm that are progressively motile. A progressively
motile sperm swims briskly forward in a relatively straight line.
 Very good(VG)-rapid dark swirls
 Good (G)-slower swirls
 Fair (F)- No swirls, but permanent individual cell motion
 Poor – little or no individual cell motion
Sperm morphology - the percentage of sperm with normal shape and size is determined.
Double heads,
not tails, double
tails, no heads,
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SEMEN STORAGE
The discovery that bull semen could be successfully frozen and stored for indefinite periods
has revolutionized AI in cattle. In 1949, British scientists discovered that addition of glycerol
to the semen extender improved resistance of sperm to freezing. Glycerol acts to remove
water from the sperm cell prior to freezing and prevents the formation of cellular ice crystals
which would damage the sperm. There are two methods of freezing and storing semen: dry
ice and alcohol (-100 degrees F) and liquid nitrogen (-320 degrees F). Liquid nitrogen is
preferred because there is no evidence of fertility deterioration with age. Fertility gradually
declines in semen stored in dry ice-alcohol.
Frozen semen can be stored indefinitely if proper temperature is maintained. A recent report
told of a calf born from frozen semen stored for 16 years. Fresh, liquid semen can be
successfully stored for 1 to 4 days at 40 degrees F. Semen is usually stored in glass ampoules.
Other methods appear promising, particularly the French-straw. Several AI organizations
have gone to this method exclusively. Artificial colouring is frequently added to semen
extenders in order to distinguish one breed from another. Complete identification of the bull
is required on each individual semen container.
Freezing of semen for successful preservation of spermatozoa, for long periods, is of great
importance in livestock breeding and farm management. It has made it possible” to make
available the use of outstanding proven sizes for larger number of cows, covering larger area,
frozen semen shipment has become possible to different continents in the globe to any place
connected with any service. Now a day if farmer wants to use of an outstanding size for
inheritance of high milk yield, he can go in for frozen semen service provided his area is,
covered by Artificial insemination, with supply of frozen semen.
At present frozen semen is used in most of the states in India. The technique of semen
preservation in straws was developed in France . Freezing of semen is done with a special
diluents, which has the following composition.
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Sodium citrate dihydrate (angular) 2.4 y. 2.0 gm 8.0 ml 25.0%byvolume 50,000 units per 100
ml of semen Fructose Glycerol Egg Yolk Penicillin dilulent. Dihydro-streptomycin 50.0 mg
per .100m1 of semen dilulent. Distilled water double glass distilled 100.Om1. The addition of
glycerol to the dilulent makes the cells more resistant to the rigours of freezing and icy
crystals, which form are smaller and smoother thus creating less damage to the spermatozoa.
The addition of fructose to the diluent luprores sperm resistance to glycerol; and also
provides nutrition.
Frozen semen is packed in single dose glass vials or plastic straws at +5°C. The final level of
glycerol should be 7.0 to 7.6% during the freezing process. The antibiotics are added to
inhibit bacteria and to kill pathetic organisms. The semen to be diluted in such a way that one
ml. of extended semen will contain 20 million motile spermatozoa. The semen must be
cooled carefully for spermatozoa to remain with life. The final temperature is lowered to -
79°C or still lower. Quick freezing is done for a period of 3 to 5 minutes to -75°C with the
help of atmosphere created by liquid nitrogen. In the slow freezing technique cooling is done
at the rate of 1 °C per minute from +5°C to -15°C. From -15°C to -31 °C at the rate of 2°C
per minute. From -31°C to 75°C at the rate of 4 to 5°C per minute. Thus taking 40 minutes in
total, further cooling to -96°C can be done quickly as it is not critical after freezing. Before
freezing the diluted semen in equilibreated for 3 to 5 hours or for the best 16 to 20, hours
period in refrigerator at 5°C. Frozen semen facilitates the percent use of the semen diluted
and frozen, and thus the delivery price is reduced, and it can be supplied with the gaps of
months to the A.I technicians as against the supply of fluid semen every days or alternate
days. Liquid nitrogen plays a vital role for storing the frozen semen straws, at a temperature
of -196°C for longer periods.
The sperms after being ejaculated cannot survive foe a long time outside the female
reproductive tract unless various agents are added. The agents added have the following
characteristics;
 Isotonic with the semen (ions) contained should not be different from those of semen.
 Buffering Capacity. The agents should have the buffering capacity to avoid shifts in
pH as the semen is being processed.
 Protect the semen from cold shock. Lipo-proteins are added to the extender which
prevent cold shock as the sperms are being frozen.
 Nutrients. These agents should provide nutrients for metabolism of sperm. Milk, egg
yolk and simple sugars are added.
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 Microbial Contamination should be controlled by use of antibiotics such as
penicillin.
 Should be able to protect cells during freezing -Glycerol is added to prevent water
crystals forming during freezing.
Examples of Diluters (Extenders)
Egg Yolk Extender. Add 4 parts egg yolk to 6 parts of Nacitrate (2.9g Nacitrate in 100ml
distilled water) add 500 IU penicillin and 1mg streptomycin per ml of the mixture.
Skim Milk Extender. Heat skim milk at 95o
C for 10 minutes then cool and add glucose at
1g/100ml milk. Then penicillin and streptomycin are added to protect sperm against bacteria
PROCESSING SEMEN
Extending (or diluting) the semen increases the number of females that can be inseminated
from one ejaculation. There are several good semen extenders. Those made from egg yolk or
pasteurized, homogenized milk are two of the most widely used. A good extender not only
adds volume to the ejaculate, but favors sperm survival and longevity. Dilution rate depends
on quality of sperm. Antibiotics such as penicillin and streptomycin are added to semen
extenders to inhibit bacterial growth and reduce the spread of diseases.
PACKAGING
Extended semen frozen as pellets, in straws or in ampules is held at 50o
C prior to freezing.
Straws are frozen in nitrogen vapour and stored at –196o
C. Ampules are frozen at 3o
C/minute
to –15o
C and then the rate of freezing increased to –15o
C and then the semen is transferred to
liquid N at –196o
C in the ampules on canes. Pellets freeze in few seconds to –79o
C on a
block of ice.
Calculation of sperm concentration
If 8ml ejaculate containing semen evaluation showed the following
Volume of semen 8ml
Concentration 900 x 106
Motility 80%
Normal cells percentage 95%
1. Estimate the total number of cells in the ejaculate
= volume x concentration
= 8ml x 900 x 106
= 72 x 108
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2. Live cells
= total number of cells x motility
= 72 x 108
x 80/100
= 576 x 107
live cells
3. Live normal cells
= live cells x normal cell %
= 576 x 107
x 95/100
= 5472 x 106
Therefore live normal cells = volume x concentration x normal cell x motility
For bull semen recommended;
10 x 106
live normal cells per insemination (not frozen)
15 x 106
`live normal cells per insemination pre-freezing.
INSEMINATION METHODS
The recto-vaginal technique is the most commonly used method of artificially inseminating
(AI) cattle. The basic skills required to perform this technique can be obtained with about 3
days practice under professional instruction and supervision. Additional proficiency and
confidence come with time and practice. Regardless of whether the inseminator is left or right
handed, it is recommended that the left hand be used in the rectum to manipulate the
reproductive tract and the right hand be used to manipulate the insemination gun. This is
because the rumen or stomach of the cow lays on the left side of the abdominal cavity,
displacing the reproductive tract slightly to the right. Thus it may be easier to locate and
manipulate the tract with the left hand.
Step #1: Restrain the animal to be inseminated. There are several things that should be kept in
mind when choosing a location for inseminating cattle. Some of these include safety of both
the animal and the inseminator, ease of use, and shelter from adverse weather. A gentle pat
on the animal’s rump or a soft spoken word as the inseminator approaches will help to avoid
startling or surprising the cow.
Step #2: Raise the tail with the right hand and gently massage the rectum with the lubricated
glove on the left hand. Place the tail on the back side of the left forearm so it will not interfere
with the insemination process. Cup the fingers together in a pointed fashion and insert the left
hand in the rectum, up to the wrist.
Step #3: Gently wipe the vulva with a paper towel to remove excess manure and debris. Be
careful not to apply excessive pressure which may smear or push manure into the vulva and
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vagina. With the left hand, make a fist and press down directly on top of the vulva. This will
spread the vulva lips allowing clear access to insert the gun tip several inches into the vagina
before contacting the vaginal walls.
Step #4: Insert the gun at a 30° upward angle to avoid entering the urethral opening and
bladder located on the floor of the vagina. With the gun about 6 to 8 inches inside the vagina,
raise the rear of the gun to a somewhat level position and slide it forward.
To become a successful inseminator, it is very important to always know where the tip of the
insemination gun is located. The walls of the vagina consist of thin layered muscle and loose
connective tissue. The insemination gun can be easily felt with the left hand in the rectum. As
the breeding gun is inserted into the vagina, keep the gloved hand even with the gun tip
(Figure #1).
Figure #1: Keeping the gloved hand even with the tip of the inseminator gun.
Manure in the rectum can often interfere with the inseminator’s ability to palpate the cervix
and gun tip. However, it is seldom necessary to remove all the manure from the bowel.
Instead, keep the open hand flat against the floor of the rectum, allowing the manure to pass
over the top of the hand and arm (Figure #2).
Figure #2: Allowing manure to pass over the top of the hand and arm.
With the hand in the rectum, the inseminator may notice colon constrictions or "rings"
attempting to force the left arm from the cow. To relax these rings, place two fingers through
the center of a ring and massage back and forth. The constriction ring will eventually relax,
pass over the hand and arm, and the inseminator can continue the palpation process (Figure
#3)
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Figure #3: Dealing with colon constrictions.
Because the reproductive tract is freely movable, cows that have strong rectal and abdominal
contractions in response to being palpated may actually push their reproductive tract back
into the pelvic cavity. This will cause many folds to form in the vagina. In such cases, the
insemination gun can get caught in these folds and little or no progress will be made until
they are removed. If the cervix can be located, grasp it and gently push it forward. This will
straighten the vagina and the gun should pass freely up to the cervix (Figure #4). The
inseminator will note a distinct gristly sensation on the gun when it contacts the cervix.
Figure #4: Grasping the cervix and gently moving it forward.
In most cows, the cervix will be located on the floor of the pelvic cavity near the anterior
(front) end of the pelvis. In older cows, the cervix may rest slightly over the pelvic bone and
down into the abdominal cavity. Once the gun is in contact with the external surface of the
cervix, the inseminator is ready to begin threading the cervix over the end of the gun. Place
the cervix on or over the insemination gun; the gun is not passed through the cervix.
Excessive movement or probing with the insemination gun during this step is seldom
productive. The key to mastering this step of the insemination process is knowing how to
hold and manipulate the cervix and concentrating on doing the work with the hand inside the
cow, not the one holding the gun. When the gun first contacts the cervix, the inseminator will
usually find that the tip is in the fornix area directly over the top of the opening of the cervix.
If this happens, grasp the external opening to the cervix with the thumb on top and
forefingers underneath (Figure #5).
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Figure #5: Finding the opening of the cervix.
This closes the fornix at top and bottom. It is also still important to know the location of the
gun tip. This is accomplished by contacting the gun tip with the palm and 3rd and 4th fingers
of the hand in the rectum. Use the palm and these two fingers to guide the gun tip to the
cervical opening located between the thumb and forefingers. With gentle probing, the
opening of the cervix should be located. The inseminator will feel the gun slide forward until
it contacts the second cervical ring.
Maintain gentle but steady forward pressure on the gun and slide the thumb and forefingers
just in front of the gun tip and re-grasp the cervix. Because the cervix is composed of dense
connective tissue and muscle, it is difficult to clearly distinguish the gun tip when it is located
within this structure. However, the inseminator can determine the approximate location by
bending the cervix. Using the flexibility of the wrist, gently twist and bend the cervix until
the second ring of the cervix slides over the gun tip (Figure #5). Repeat the process until all
the rings have been passed over the gun tip. Remember, the cervix is being placed over the
gun, not the gun through the cervix. For the most part, gentle forward pressure is all that is
necessary and gun movement should be minimal. 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, the inseminator will once again be able to feel the tip of the gun.
It is now time to check the gun placement and deposit the semen. Rotate the gloved hand
until it lies on top of the cervix. With the index finger of that hand, locate the far end of the
cervix (Figure #7). Pull back on the gun until the tip of it is directly underneath the index
finger near the internal opening of the cervix. Raise the finger and slowly deposit the semen.
Push the plunger slowly so that drops of semen fall directly into the uterine body.
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Figure #7: Locating the end of the insemination gun and depositing the semen in the body of the
uterus.
With proper AI technique and gun placement, semen will be deposited in the uterine body.
Uterine contractions will then transport spermatozoa forward to the horns and oviducts with a
good distribution of both sides (Figure #10).
Figure #8: Good distribution of the semen to both uterine horns while Improper distribution of
the semen into one horn because the insemination gun is pushed too far forward.
When the insemination gun is more than 1 inch through the cervix, all the semen will be
deposited in only one horn (Figure #8). Be sure to raise the index finger after checking gun
placement. Not doing so may obstruct one horn, creating a situation of uneven semen
distribution. When checking gun tip placement, be careful not to apply excessive pressure.
The delicate uterine lining is easily damaged, potentially causing infections and reduced
fertility.
Make sure to push in with the plunger and do not pull back on the gun. Pulling back may
result in much of the semen dose being deposited in the cervix and vagina instead of the
uterine body. Although the recommended site of semen deposition is in the uterine body,
research suggests that when exact gun tip placement is in doubt, depositing semen slightly
into one uterine horn is less likely to compromise fertility than depositing it in the cervix.
However, if the cervical mucous of a cow that has been previously inseminated feels thick
and sticky on the gun, the cow may be pregnant. In this case, deposit the semen about
halfway through the cervix.
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After properly depositing semen, slowly pull the gun from the reproductive tract. Remove the
gloved hand from the rectum. Check the gun tip for signs of blood, infection or semen
leakage inside the sheath. Make the necessary notes for future reference and for the local
veterinarian. Remove the sheath from the gun and hold it in the gloved hand. Check again to
see which bull was used. Remove the glove starting at the top of the arm by turning it inside
out trapping manure, the sheath, and dirt inside. Dispose of the used glove in a proper
receptacle. Wipe the gun clean and dry and return it to the proper storage location.
Correct insemination procedures will result in better breeding efficiencies. More selection
pressure can then be placed on economic traits such as milk and beef production, enabling a
higher return on semen investment dollars.
VAGINAL METHOD:
Hand is passed through the vagina and the inseminating tube is guided by hand to the site of
insemination and semen is deposited. Here there is a risk of contamination and injury of
female genitalia.
Advantage- The semen collected is of high quality and is highly concentrated.
Type of breeding method (AI - artificial insemination vs. live coverage):
SYMPTOMS of HEAT
The estrous cycle is a series of steps that occur from estrus to estrus. These are the phases of
reproductive readiness in the reproductive system of a mature female. The cycle does not
occur during pregnancy nor when a female is in anestrus. Anestrus is the absence of cycling.
Anestrus may occur due to disease, not being of reproductive age, or other conditions. The
estrous cycle is comprised of four phases. The phases occur in a definite sequence unless the
female is pregnant.
1. Estrus is the phase when a female is in heat. The animal is receptive to mating
and will stand for copulation with a male. Females exhibit signs of heat. An
enlarged vulva, restlessness, and a mucus discharge are signs. Some females
exhibit behaviours indicating readiness for mating such as when a cow mounts
another cow in the mating position. Ewes, sows, mares, and some small animals
ovulate at this time. Some animals usually release only one egg while others may
release twenty eggs at one time.
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2. Metestrus is the phase following heat. Ovulation occurs during metestrus in does
and cows as do other processes that help maintain a pregnancy should conception
occur. During metestrus luteinizing hormone (LH) causes the corpus lutea (CL) to
develop.
3. Diestrus is the phase in the estrous cycle when the reproductive system assumes
that conception has occurred, even if it has not. Diestrus is several days long
depending on the species of animal. High levels of progesterone cause the uterus
to begin preparing for pregnancy.
4. Proestrus is the period following diestrus in which preparation is being made by
the reproductive system for the next heat period and ovulation. If conception has
occurred, the estrous cycle ceases until it is renewed after gestation and
parturition.
The following table outlines the length of estrous, length of estrus, and when ovulation occurs
for a variety of species.
Detecting estrus as aid to artificial insemination.
1. Standing heat is one of best indicators of estrus in cows. Cows will stand to be
mounted and will try to mount other cows. They will be restless and will oftentimes
have an enlarged vulva and discharge.
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2. Mares will “wink” to expose their clitoris and will urinate frequently.
3. Redness in the vulva is an indicator for sows.
4. Ewes will not show signs of estrus until a ram is present. Producers will use a
castrated ram to detect heat.
5. Goats will act aggressively, be noisy and active, stand to be mounted, and have a
mucous discharge.
The timing of insemination
The timing of insemination varies greatly by species and can vary between individuals to a
degree.
1. Cows are usually inseminating in the evening after showing signs of estrus in the
morning or in the morning after showing signs the evening before.
2. Mares are usually inseminated on the third, fifth, and seventh days of estrus.
3. Turkeys are inseminated three times over the course of several weeks. About one
week should pass between the first two inseminations and then a little over a week
between the second and third inseminations. A female turkey has a storage gland for
semen making this possible.
4. Twenty-four hours after the initiation of estrus sows can be inseminated. Some
producers will inseminate the sow after another 24 hour period.
CHAPTER THREE
EMBRYO TRANSFER
The mammalian ovary contains thousands of ova but the number of off-springs produced is
small. The number of times an animal can become pregnant is affected by the length of
gestation. Under normal conditions the number of ova release per estrus is one or two in non
litter bearing animals. The number of offspring that a female can bear can be greatly
increased by allowing it to temporally become pregnant recovering the embryo in early
pregnancy and transferring them to the reproductive tracts of other females to complete
gestation. The process can be amplified if the donor super-ovulates
Super-ovulation
This is the procedure in which the female is treated with hormones to cause her to produce a
large number of ova instead of the one or two that she normally produces at each estrus.
35
This procedure is done so that these ova can be transferred to another female. About 60-70 %
of the super-ovulated ova form normal embryos. Reliable super-ovulation has not yet been
effected in the mare but can be accomplished in the sow. Superior germplasm could be
identified earlier and utilized more. To date response has been variable and fertilization rates
have been very low.
Methods of Embryo Recovery and Transfer.
Embryo transfer refers to the technique by which fertilized ova are collected from a female
called the donor and transferred for development to term to another female known as the
recipient. The chain of events in the process of embryo transfer includes
1. management of the donor for production of a suitable number of viable ova,
2. mating or A.I,
3. collection,
4. evaluation,
5. short term storage of the embryo from that donor,
6. transfer of these embryos to suitable recipients.
Also associated with embryo transfer a number of procedures including maintenance of
vigorous health program of donors and recipients and the keeping of a detailed series of
records for every aspect of the program. The main objective of embryo transfer is the
improvement of animal populations through better utilization of superior females.
Methods of Collection or Recovery
Early collection techniques involved either slaughtering the females and excising (cutting out
part of the body) the oviduct or surgically removing the oviduct from the live females at 72
hours post ovulation so that the embryos could be recovered by flushing. This however
defeated the primary purpose of superovulation so other methods were developed.
Surgical Methods
Laparotomy. This is surgically opening of the abdomen (is performed to expose the
reproductive tract). A clamp or thumb and forefinger can be used to block the distal of
1/3 of the uterine horn so that fluid injected into that segment can be forced through the
oviduct with a gentle milking acting and collected at the infindibulum.
An alternative method is to occlude (block) the uterine horn at the body of the uterus
and introducing culture medium through a puncture at the uteral junction or through the
36
oviduct until the uterus is turgid. The uterus is then punctured with a blunt needle
attached to a flexible catheter. The pressure causes the medium to gush through the
catheter with enough turbulence to carry the embryos into a collection tube. Although
these procedures allow for the recovery of a high percentage of the embryos, they can
be repeated only a few times because of the surgical tromer and the resulting adhesions.
The adhesions make it difficult if not impossible to expose the reproductive tract
repeatedly.
Now surgical techniques that give results equal to surgical methods have been developed for
cows. This involves the use of a Foley catheter which is a two ways or three way flow
catheter. It allows flushing fluids to pass into the uterus at the same time allows fluids to be
returned from the uterus to a collecting receptacle. A feature in this catheter which can be
inflated just inside the uterine horn to prevent the flushing fluid escaping through the cervix.
Non surgical techniques for collection of ova are desirable because all surgical technics may
lead to the formation of adhesions and because there is less risk to life and health of the donor
with non surgical methods. The non surgical methods have been used for repeated recovery
from the same donor practically. Ova are being collected from cows without superovulatioin.
Each uterine is filled with 30-50ml of medium which is then allowed to flow into the
collection vessel while the uterus is gently massaged through the rectum. This is repeated
until 300-800ml of medium has been used. The Foley catheter is then inserted into the other
uterine horn and the process repeated.
After ova are collected (recovered) they are analyzed under the microscope. Some ova are
not fertilized and others are deformed. Those that can develop are then frozen for storage.
SITE OF TRANSFER
If the embryo has less than 8 cells in it then it is transferred to the oviduct because the
conditions in the uterus are not conducive for their development. The uterus produce a toxic
environment for theses cells. If the embryo has more than 8 cells then the uterine transfer is
recommended.
TRANSFER INTO THE RECEIPIENT
As in embryo collection the method of transfer are similar to those in embryo collection.
There are surgical and non-surgical methods.
37
Surgical Method
In this method the cow is excised to expose parts of the reproductive system. When embryo is
being transferred into the oviduct a capillary pipette is used to drop ova with medium at the
infundibulum and ampula of the oviduct. When the transfer is made into the uterus puncture
the uterine wall with blunt needle and then expel the embryo from the tip of the capillary
pipette inserted into the uterine lumen. Now non surgical methods are used similar to A.I
ones to deposit ova in the uterus.
Synchronization
The recipient should be in the similar period of estrus with the donor either naturally or by
synchronization. The recipient should be in estrus within 12 hours as the donor. Pregnancy
rates reduce if the difference is more than 24 hours in cows. The recipient for frozen embryos
should be in physiological synchrony with the embryo. If the embryo was frozen 3 days then
the recipient should have been in estrus 3 days.
Advantages
Embryo transfer is an experimental technique used to evaluate reproductive problems and
improve the genetic performance of the cattle population.
Disadvantage
There is high variability in the results due to unpredicted response to super ovulation and
many ova are unfertilized and abnormal
Assignment. Discuss factors that limit the use of AI and ET in Zambia.
38
Chapter four
PRINCIPLES OF ANIMAL GENETICS
Genetics is the science of heredity and variation. It is the scientific discipline that deals with
the differences and similarities among related individuals. All animals have a predetermined
genotype that they inherit from their parents. However, an animal’s genotype can be
manipulated by breeding and more advanced scientific techniques (genetic engineering and
cloning). For many years, managers of agricultural systems have manipulated the genetic
makeup of animals to; mprove productivity, increase efficiency and adaptability. Successful
manipulation of the genetic composition of animals requires a depth understanding of
fundamental principles of genetics
Mendel’s Laws
Gregory Mendel is recognized as the father of genetics and in the 1850’s and 1860’s, he
developed his theories without any knowledge of cell biology or the science of inheritance-
he failed his teachers exams. In later years, genes, chromosomes, and DNA were discovered
and people began to understand how and why Mendel’s theories worked. Mendel proposed
three principles to describe the transfer of genetic material from one generation to the next.
To understand Mendel’s principles and the relationships between phenotype and genotype, it
is necessary to understand; what makes up the genetic material of animals and how this is
transferred from one generation to the next.
Genetic Material
The body is made up of millions of cells which have a very complicated structure. These cells
are made up of many parts that have specialized roles. These include:
1. Nucleolus 5. Rough Endoplasmic Reticulum 9. Mitochondria
2. Nucleus 6. Golgi Apparatus 10. Vacuole
3. Ribosome 7. Cytoskeleton 11. Cytoplasm
39
4. Vesicle 8. Smooth Endoplasmic Reticulum 12.Lysosome13.Centriole
The nucleus contains chromosomes that are visible under the microscope as dark-staining,
rod-like or rounded bodies. Chromosomes occur in pairs in the body cells. The number of
chromosomes in each cell is constant for individual species, but it differs among species.
Suis-38, Caprine-60, Carnis 78, Galus-78, Bovine. Chromosomes are made up of tightly-
coiled strands of DNA and is a complex molecule composed of deoxyribose, phosphoric acid,
and four bases. Individual genes are located in a fixed position (known as the loci) on the
strands of DNA. A chromosome is made up of two chromatids and a centromere. The
chromatids are formed from tightly coiled strands of DNA. If these strands of DNA are
stretched out, individual genes can be identified.eg DT
GENE CODING
A gene is made up of a specific functional sequence of nucleotides, which code for specific
proteins. A specific protein is produced when the appropriate apparatus of the cell (the
ribosome) reads the code. In animals, chromosomes are paired and, therefore, genes are
paired. These paired genes code for the same trait, but they are not identical and can have
different forms, known as alleles. For example, sheep and cattle can be polled or horned. One
gene codes for this trait and two possible forms (alleles) of the gene are polled or horned
Identified by Mendel as three fundamental principles of genetics.
The Principle of Dominance A capital letter is used to denote the dominant form of the gene
(P) and a small letter is used to denote the recessive form of the gene (p). In the example, the
polled allele is dominant and is, therefore, denoted by P, while the horned allele is recessive
40
and denoted by p. Because genes are paired, an animal can have three different combinations
of the two alleles: PP, Pp, or pp. When both genes in a pair take the same form (PP or pp), the
animal is referred to as being homozygous for that trait. An animal with a PP genotype is
referred to as homozygous dominant. An animal with the pp genotype is referred to as
homozygous recessive. If one gene in the pair is the dominant allele (P) and the other gene is
the recessive allele (p), the animal is referred to as being heterozygous for that trait and its
genotype is denoted as Pp. If an animal has the allele combination PP, it will be polled. If the
combination is pp, the animal will be horned. If it is a heterozygote, the animal will have
both traits (Pp), but the animal will be polled because the polled allele (P) is the dominant
form of the gene.
Mendel’s principle of dominance states that in a heterozygote, one allele may conceal the
presence of another.
The Principle of Segregation When animals reproduce, they only pass on half of their
genetic material to their offspring. The offspring will only receive one allele from each
parent. The Principle of Segregation explains some of the differences that are observed in
successive generations of animals and can be used to predict the probability of different
combinations of alleles occurring in offspring. In a heterozygote, two different alleles
segregate from each other during the formation of gametes. Aa individual will produce two
gametes- A-alleles and a-alleles. Considering these three types of individuals, six
combinations of the various genotypes are possible:
 PP x PP (both parents are homozygous polled),
 PP x Pp (one homozygous polled parent and one heterozygous polled parent),
 PP x pp (one homozygous polled parent and one homozygous horned parent),
 Pp x Pp (both parents are heterozygous polled),
 Pp x pp (one heterozygous polled parent and one homozygous horned parent), and
 pp x pp (both parents are homozygous horned)
The genotypes of the parents can be used to predict the phenotypes of the offspring.
41
Predicting the Genotypes and Phenotypes of Offspring by A punnett square - grid-like
method that is used to display and predict the genotypes and phenotypes of offspring from
parents with specific alleles. The male genotype is normally indicated at the top and the
female genotype is indicated in the vertical margin (Figure)
When crossing homozygous
dominant parents (PP x PP) all
heterozygous parent with a
homozygous dominant parent
(Pp x PP), 1:1
two heterozygous parents
are crossed (Pp x Pp)
hen crossing homozygous
recessive parents (pp x pp)
This happens because Alleles separate during meiosi. In others words the punnet chart maybe
presented in this
Considering Multiple Traits-Dihybrid Cross
Commonly, there are multiple traits that need to be considered when mating animals. For
example, consider that cattle can be horned or polled and white-faced or red-faced.The horns
and red-faced coloring are recessive traits. If two individuals with two pairs of heterozygous
genes (each affecting a different trait) are mated, the expected genotypic and phenotypic
ratios would be:
 Genotypes: 1 PPWW, 2 PPWw, 2 PpWW, 4 PpWw, 1 PPww, 2 Ppww, 1 ppWW, 2
ppWw, and 1 ppww;
 Phenotypes: 9 polled, white-faced; 3 polled, red-faced; 3 horned, white-faced; and 1
horned, red-faced offspring
42
The Principle of Independent Assortment the alleles of different genes segregate, or assort,
independently of each other. When considering multiple traits, Mendel hypothesized that
genes for different traits are separated and distributed to gametes independently of one
another. Therefore, when considering polled and white-faced traits, Mendel assumed that
there was no relationship between how they were distributed to the next generation. In most
cases, genes do assort independently. However, advances in genetics have shown that an
abnormal situation, called crossing-over, can occur between genes for different traits.
Crossing-over is an exchange of genes by homologous chromosomes during the synapses of
meiosis prior to the formation of the sex cells or gametes. PpBb x PpBb gives 9:3:3:1
43
Other Concepts in Genetics
Non-traditional inheritance involves alleles that are not dominant or recessive. Incomplete, or
partial dominance, & co-dominance are two examples of non-traditional inheritance. Recent
studies in sheep has indicated another form of inheritance called POLAR DOMINANCE
Partial, or incomplete, dominance occurs when the heterozygous organism exhibits a trait in-
between the dominant trait and the recessive trait. eg Homozygous mice are black (BB) or
white (bb) and the heterozygous mice will be grey (Bb). When a pure, brown-eyed sheep is
crossed with a pure, green-eyed sheep, blue-eyed offspring are produced
Codominance occurs when a heterozygote offspring exhibits traits found in both associated
homozygous individuals. An example of codominance is the feather color of chickens. If a
homozygous black rooster is mated to a homozygous white hen, the heterozygous offspring
would have both black feathers and white feathers. Roan is a coat color in horses (sometimes
dogs and cattle) that is a mixture of base coat colored hairs (ex. black, chestnut) and white
hairs. Neither the base coat color or the white hairs are dominant nor do they blend to create
an intermediate color. Under these circumstances, neither allele is dominant and neither is
recessive. Therefore, each allele is denoted by a capital letter.
Epistasis It is possible for more than one gene to control a single trait. This type of
interaction between two nonallelic genes is referred to as epistasis. When two or more genes
influence a trait, an allele of one of them may have an epistatic, or overriding, effect on the
phenotype. Comb shape in chickens is an example of an epistatic relationship.
Mutations and Other Chromosomal Abnormalities
Genes have the capability of duplicating themselves, but sometimes a mistake is made in the
duplication process resulting in a mutation. The new gene created by this mutation will cause
a change in the code sent by the gene to the protein formation process. Some mutations cause
defects in animals, while others may be beneficial. Mutations are responsible for variations in
coat color, size, shape, behavior, and other traits in several species of animals. The beneficial
mutations are helpful to breeders trying to improve domestic animals.
Changes in chromosomes are reflected in the phenotypes of animals. Some chromosomal
changes will result in abnormalities, while others are lethal and result in the death of an
animal shortly after fertilization, during prenatal development, or even after birth. Changes
that can occur in chromosomes during meiosis include:
 Changes in the chromosome number,
44
 Translocation or deletion – chromosome breakage, and
 Inversion and insertion – the rearrangement of genes on a chromosome.
Sex-Linked Traits
Sex-linked traits involve genes that are carried only on the X or Y chromosomes, which are
involved in determining the sex of animals. The female genotype is XX, while the male
genotype is XY. The X chromosome is larger and longer than the Y chromosome, which
means a portion of the X chromosome does not pair with genes on the Y chromosome.
Additionally, a certain portion of the Y chromosome does not link with the X chromosome.
The traits on this portion of the Y chromosome are transmitted only from fathers to sons.
Sex-linked traits are often recessive and are covered up in the female mammal by dominant
genes. The expression of certain genes, which are carried on the regular body chromosomes
of animals, is also affected by the sex of the animal. The sex of an animal may determine
whether a gene is dominant or recessive (Ex. Scurs in polled European cattle).
In poultry, the male has the genotype XX, while the female has the genotype Xw. An
example of a sex-linked trait in poultry is the barring of Barred Plymouth Rock chickens. If
barred hens are mated to non-barred males, all of the barred chicks from this cross are males,
and the non-barred chicks are females
Quantitative vs qualitative traits
Quantitative traits are controlled large number of genes. They exhibit normal distribution and
phenotypes and show continuous express with additive gene effect. They traits affect more
genes with large effect. In selection, quantitative traits are economical traits and expensive to
select for (Carcass quality, Milk Yield). Single dominant or recessive genes control
qualitative traits. They tend to exhibit Non additive gene and non- continuous e.g (Colour and
Polled)
Measuring Heritable Variation
The value of quantitative traits such a mohair length or size or milk yield is determined by
their genes operating within their environment. The size of how a spp grows is affected not
only by the genes inherited from their parents, but the conditions under which they grow up.
For a given individual the value of its phenotype (P) (e.g. the weight of a broiler in grams)
can be considered to consist of two parts genotype (G) and environment (E).
P = G + E.
45
The quantitative genetics approach depends on taking a population view and tracking
variation in phenotype and whether this variation has a genetic basis. We measure variation
in a sample using a statistical measure called the variance. The variance measures how
different individuals are from the mean and the spread of the data. Mean and variance are two
quantities that describe a normal distribution. For Your Information:
 Variance is the average squared deviation from the mean.
 Standard deviation is the square root of the variance.
 Covariance is correlations among characters or relatives
Heritability
Heritability is the proportion of the total phenotypic variation controlled by genetic rather
than environmental factors. The total phenotypic variance may be decomposed:
VP = total phenotypic variance
VG = total genetic variance
VE = environmental variance
VP = VG + VE
Estimation heritability (H2
) = VG/VP (broad-sense)
The total genetic variance (VG) may be decomposed:
VA = additive genetic variance
VD = dominance genetic variance
VI = epistatic (interactive) genetic variance
Variance
Mean
46
Estimation heritability = h2
= VA/VP (narrow sense)
h2
= VA / VP = VA / (VG + VE)
Heritability ranges from 0 to 1 (Traits with no genetic variation have a heritability of 0).
Since heritability is a function of the environment (VE), it is a context dependent measure.
It is influenced by both, The environment that organisms are raised in, and the environment
that they are measured in
ESTIMATING HERITABILITY FROM REGRESSION
one common approach is to compare phenotypic scores of parents and their offspring:
junco tarsus length (cm)
Cross Midparent value Offspring value
F1 x M1 4.34 4.73
F2 x M2 5.56 5.31
F3 x M3 3.88 4.02
Slope of a plot of two variables (x,y) = Cov (x,y) / Var (x)
Comparison Slope
Midparent-offspring h2
Parent-offspring 0.5 h2
Half-sibs 0.25 h2
First cousins 1/8h2
As the groups become less related, the precision of the h2
estimate is reduced
Role of reproduction in genetic improvement
Heritability important because it allows us to predict a trait’s response to selection
Let S = selection differential
Let h2
= heritability
Let R = response to selection
R = h2
S
Slope (h2
)
47
Figure 8. Response to selection. The selection differential (S) =
mean of selected individuals - mean of the base population.
The response to selection,: for a given intensity of selection, the response to selection is
determined by the heritability which can either be high or low. The higher the slope, the
better offspring resemble their parents. In other words, the higher the heritability, the better
offspring trait values are predicted by parental trait values. The response to selection can be
predicated for example in the large ground finch, Geospiza magnirostris
Mean beak depth of survivors = 10.11 mm
Mean beak depth of initial pop = 8.82 mm
S = 10.11 – 8.82 = 1.29
h2
= 0.72
R = h2
S = (1.29)(0.72) = 0.93
Beak depth next generation = 10.11 + 0.93 = 11.04 mm.
Therefore, the Response to Selection and is defined as the difference between the mean trait
value for the offspring generation and the mean trait value for the parental generation i.e. the
change in trait value from one generation to the next.
Gy = SD * h2
GI
where
Gy= rate of genetic progress per year (
SD = selection differential
h2
= heritability estimate, and
GI = generation interval in years
Methods of Selection
 Individual and family
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Animal production 111

  • 1. NATURAL RESOURCES DEVELOPMENT COLLEGE ANIMAL PRODUCTION 111 MODULE ANIMAL REPRODUCTIVE PHYSIOLOGHY, BREEDING, GENETICS, FEEDING AND BY- PRODUCTS ELLISON MSIMUKO (MPVS, B.Agric. Dip.Agric) 7/7/2015 Application of animal breeding and genetics through appropriate understanding of animal physiology and reproduction in a key to achieving maximum response to selection that brings value to quality and quantity of livestock products and by products. in a sustainable environment
  • 2. i Contents Introduction..............................................................................................................1 Structure And Function Of The Male Reproductive System.......................................2 Male Macrostructure And Function ...........................................................................2 Penis And Related Structures: ................................................................................2 Testes And Related Structures:...............................................................................3 Epididymis And Related Structures:.......................................................................3 Accessory Glands: .................................................................................................3 Semen.......................................................................................................................4 Male Microstructure And Function .........................................................................4 Hormonal Regulation Of Testicular Function ........................................................6 Structure And Function Of The Female Reproductive System ..............................6 Placenta....................................................................................................................8 Cervix .......................................................................................................................9 Vagina.......................................................................................................................9 Female Microstructure And Function ..................................................................10 Oogenesis ..............................................................................................................10 Prenatatal Maturation........................................................................................ 11 Postnatal Maturation.......................................................................................... 11 The Mature Ovum ..................................................................................................12 Comparison Of Spermatocytogenesis And Ovigoogenesis ...................................13 Ovulation................................................................................................................ 14 Transport Of Gametes, Fertilisation And Implantation ........................................14 References..............................................................................................................16 Chapter Two..........................................................................................................16 Artificial Insemination.......................................................................................... 16 Principles Of Artificial Insemination ....................................................................16 1.0 Introduction .....................................................................................................16 History Of Artificial Insemination .........................................................................16 Aim Of Ai ................................................................................................................ 17 Role In Animal Breeding........................................................................................ 17 Utilization Of Bulls.............................................................................................. 17
  • 3. ii Role With Regard To Health And Sterility ......................................................... 18 Scientific Role .....................................................................................................19 Critics Of Artificial Insemination ...........................................................................19 Advantages Of Artificial Insemination:.............................................................. 19 Disadvantages Of A.I:.......................................................................................... 20 Semen Collection Methods And Evaluation:......................................................... 20 Artificial Vagina Method..................................................................................... 20 Recovery Method................................................................................................ 22 Massage ..............................................................................................................22 Electro-Ejaculator............................................................................................... 22 Semen Analysis ......................................................................................................23 Semen Storage....................................................................................................24 Processing Semen............................................................................................... 26 Packaging............................................................................................................26 Calculation Of Sperm Concentration .....................................................................26 Insemination Methods........................................................................................... 27 Vaginal Method:..................................................................................................32 Symptoms Of Heat .................................................................................................32 Detecting Estrus As Aid To Artificial Insemination..............................................33 The Timing Of Insemination ..............................................................................34 Chapter Three .......................................................................................................34 Embryo Transfer....................................................................................................34 Super-Ovulation .................................................................................................34 Methods Of Embryo Recovery And Transfer......................................................... 35 Methods Of Collection Or Recovery ...................................................................35 Surgical Methods................................................................................................ 35 Site Of Transfer ......................................................................................................36 Transfer Into The Receipient................................................................................. 36 Surgical Method..................................................................................................37 Synchronization.....................................................................................................37 Advantages .........................................................................................................37 Disadvantage ......................................................................................................37 Chapter Four .........................................................................................................38
  • 4. iii Principles Of Animal Genetics................................................................................ 38 Mendel’s Laws........................................................................................................38 Genetic Material.....................................................................................................38 Gene Coding........................................................................................................39 Considering Multiple Traits-Dihybrid Cross ......................................................... 41 Other Concepts In Genetics.................................................................................... 43 Mutations And Other Chromosomal Abnormalities .............................................43 Sex-Linked Traits...................................................................................................44 Quantitative Vs Qualitative Traits .........................................................................44 Measuring Heritable Variation ..............................................................................44 Heritability.............................................................................................................45 Estimating Heritability From Regression .......................................................... 46 Comparison Slope............................................................................................... 46 Role Of Reproduction In Genetic Improvement.................................................... 46 Methods Of Selection ............................................................................................. 47 Factors Affecting Genetic Change And Progress ................................................... 48 Modern Genetics ....................................................................................................49 Cloning................................................................................................................ 49 Chapter Five..........................................................................................................49 Animal Breeding Systems...................................................................................... 49 Objectives Of The Topic ......................................................................................... 49 Breeding Systems............................................................................................... 49 Linebreeding ..........................................................................................................50 Linecrossing .......................................................................................................50 Outcrossing.........................................................................................................50 Grading Up..........................................................................................................51 Crossbreeding (X) ..................................................................................................51 Beef Crossbreeding Systems ..............................................................................51 Two-Breed Rotation........................................................................................... 52 4 And 5 Breed Rotations .................................................................................... 52 Static Terminal Sire System ...............................................................................52 Rotational –Terminal Sire System .....................................................................53 Composite Breeds............................................................................................... 53
  • 5. iv Breed And Crossbred Means (Reciprocal Crosses) For Various Traits Of Cattle In Zambia ................................................................................................................ 53 Extra Beef Production........................................................................................... 54 Intensive Beef Production Systems.......................................................................54 Production Systems............................................................................................ 54 Principles Of Ration Formulation For Livestock................................................... 56 Ration Functions ................................................................................................ 57 Balancing Rations............................................................................................... 58 Summary.............................................................................................................60 Relationship Between 100 Percent Dry Matter Basis And As-Fed Basis ..........60 Rules Of Thumb For Balancing Beef Rations ..................................................... 61 Energy System....................................................................................................61 Protein System.......................................................................................................62 Least Cost Formulation ...................................................................................... 62 Beef Cattle: High-Grain Finishing Rations............................................................ 65 All-Concentrate Rations......................................................................................... 65 Disease Problems Of Animals On All-Concentrate Rations ............................... 66 Application In Zambia............................................................................................ 66 Use Of Other Grains With Or Without Roughage Supplementation.................. 66 Sources Of Roughage In High-Grain Rations ..................................................... 68 Ration Characteristics............................................................................................ 75 The Metabolisable Energy (Me) System................................................................ 75 Introduction:.......................................................................................................75 Evidence Of Energy Balance Regulation............................................................ 75 Responses To Changes In The Environmental Heat Loas :................................ 75 Eefects To Exrcise :............................................................................................. 75 Effect Of Lactation : ............................................................................................ 76 Effect Of Metabolic Rate And Growth :............................................................... 76 Short Term Regulation Of Feed Intake..................................................................76 Chemostattic Theories ....................................................................................... 76 Thermostatic Theory.......................................................................................... 76 Effect Of Gut Digestion :...................................................................................... 77 Role Of Gut Hormones........................................................................................ 77
  • 6. v By-Pass Protein :.................................................................................................77 Regulation Of Energy Balance Through Feed Intake.........................................77 Internal (Or Animal) Factors :...........................................................................77 External (Or Env Iromnental) Factors...................................................................77 The Prediction Of Voluntary Feed Intake.............................................................. 78 Equations Used To Predict The Voluntary Intake Of Feed By Growing Animals .78 The Metabolisable Energy (Me) System................................................................ 79 Feed Energy:.......................................................................................................79 Chapter Six.............................................................................................................80 Flaying .................................................................................................................... 81 Hand Flaying .......................................................................................................81 Machine Flaying ..................................................................................................81 Damage After Flaying ............................................................................................. 81 Preserving ...............................................................................................................81 Methods Of Preservation...................................................................................... 82 Salting ................................................................................................................. 83 Brine Curing ........................................................................................................83 Storing Of Preserved Hides...................................................................................... 83 The Grades ..............................................................................................................85 1st Grade..............................................................................................................85 2nd Grade ............................................................................................................85 3rd Grade.............................................................................................................85 4th Grade.............................................................................................................85 Rejects................................................................................................................. 86 Packing.................................................................................................................... 86
  • 7. Ellison Msimuko (MPVS, B.Agric DipAgric) .Mr. E. Msimuko studied at Natural Resources Development College, University of Zambia and University of Adelalide Australian. He works has been dedicated to improving Livestock in Zambia through handson experience obtain from Zambezi Ranching and Copping Limited for nine years, the government of Republic of Zambia (ZCA-MPIKA and NRDC) as Assistant Farm manager/ Farmer Manager. He was also involved in lecturing animal science and genetic courses at NRDC and Lusangu University for years. Currently, he has dedicated his work in molecular and qunatitative genetics in Zambian Livestock to assist in developing and consrving well and adapted genetic material in Zambia Livestock. He has made several publicastions, attended several internation conferences as invited speaker. Inaddition he is member of ISAG, AAABG, ASGN.
  • 8. Acknowledgements This task was huge and needed help from other colleagues elsewhere. Therefore, I am indebted to numerous people and organisations that contributed significant time and resources to enable me to complete this research. I would like to acknowledge and thank the providers of the open and distance learning, for the considerable financial support I received to undertake this work. In recognition of my principal supervisor Associate Professor Wayne Pitchford, thank you very for your guidance that is exceptional and ever valuable in imparting research knowledge and skills. Your dedication to work has indeed motivated me to take any future challenges without fear or favour. Out of your busy schedule, you always found time for me without that I would not have completed my work as scheduled. You are truly my living example across numerous domains of my life ranging from professional development, teaching to general outlook. A significant appreciation goes to my co supervisor Associate Professor Cynthia DK Bottema (Animal Genetic Technologies, South Australia), for her swift and tremendous feedback on written and oral work that was provided during my work In particular many thanks for your assistance in addressing Mendelian inheritance and genomics notes. Your input really was tremendous special to me. Thank you ‘Cindy’ Finally, I acknowledge several individuals whom I may not include in the lists including from other universities I had contacted for assistance in data analysis and allowing me to use their writtern notes and author of various books
  • 9. 2 Introduction Reproduction is the process by which animals produce offspring for the purpose of continuing the species. The process of reproduction begins with copulation, which is the mating of a male and female of the species. Sperm cells from the male are deposited in the female reproductive tract and try to unite with an egg cell. When fertilization (a sperm cell and egg cell unite) occurs, an embryo begins to develop. The embryo attaches to the wall of the uterus where it is protected, receives nourishment, and develops. When the new offspring reaches the end of the gestation period, it is delivered from the female reproductive tract in a process called parturition. To completely understand the process of reproduction, a basic knowledge of the reproductive tract structures and functions is required. Structure and Function of the Male Reproductive System The male reproductive system is made up of several organs, glands, and muscles; The major functions of the male reproductive system include: Production, storage, and deposition of sperm cells, Production of male sex hormones, Serve as passageway for expelling urine from the urinary bladder. Figure 1. Male reproductive system of a bull Male Macrostructure and Function Penis and Related Structures: Penis an organ that allows for the deposition of semen into the female reproductive tract. The penis of the stallion is vascular, which means that it depends on the engorgement of blood within certain tissues for erections to occur and it forms no sigmoid flexure when relaxed. The penises of the bull, ram, and boar are fibroelastic, which means that they are primarily composed of connective tissue and depend little on blood for erections. The rear portion of
  • 10. 3 the fibroelastic type penis forms an S-shaped curve or sigmoid flexure when relaxed The penis of a bull, muscle contracts to retract the penis and form the sigmoid flexure and relaxes to extend the penis upon sexual excitement Testes and Related Structures: Testes a paired, ovoid shaped organs that produce sperm cells and testosterone the male sex hormone that is responsible for the development of secondary male characteristics and sex behaviour (libido). It has a spermatic cord a protective fibrous sheath consisting of smooth muscle, blood vessels, and nerves; cremaster muscle, primary muscle supporting testes and coursing the length of the spermatic cord and scrotum, a sac outside the body cavity that protects and supports the testes. The spermatic cord extends from the body through the inguinal ring to suspend the testes within the scrotum. The cremaster muscle, spermatic cord, and tunica dartos muscle raise and lower the testes to maintain a constant temperature (4 – 6 degrees below body temperature) for sperm to develop. Sheath is an external portion of the male reproductive tract that serves to protect the penis from injury and infection Epididymis and Related Structures: Figure 2. Epididymis and related structures of a bull Epididymis is a coiled tube connected to each testis that is responsible for the maturation, storage, and transport of sperm cells. Deferent Duct (Vas Deferens) is part of the spermatic cord that is the passageway for sperm from the epididymis to the urethra. Ampulla is an enlargement of the deferent duct that opens into the urethra and may serve as a temporary storage depot for sperm. Urethra that is a passageway for both semen and urine that extends from the ampullae and bladder to the end of the penis. Accessory Glands: The accessory glands are responsible for the production of secretions that contribute to the the liquid non-cellular portion of semen known as seminal plasma. Semen and ejaculate are terms given to the sperm plus the added accessory fluids. Vesicular glands (seminal vesicles) paired accessory glands that secrete seminal fluid that nourishes the sperm and provides protection and transportation medium for sperm upon ejaculation. It adds fructose and citric acid to nourish the sperm. Seminal vesicles empties into ejaculatory duct produces about 60% of semen and contains fibrinogen with high pH
  • 11. 4 Prostate Gland located at the neck of the bladder, secretes thick, milky fluid that mixes with seminal fluid to provide nutrition and substance to the semen. It cleans the urethra prior to and during ejaculation, provides minerals for sperm, acts as medium for sperm transport and provides the characteristic odor of semen. Produces about 30% of semen thin, milky secretion with high pH and contain clotting factors, and fibrinolysin Bulbourethral glands (Cowper’s glands) secretes fluid that cleanses and neutralizes the urine residue that can kill sperm cells in the urethra. it contribute about 5% to semen, mucous secretion just before ejaculation and helps neutralize pH of female vagina. Semen 2-5 mL of fluid expelled during orgasm 60% seminal vesicle fluid, 30% prostatic, 10% sperm normal sperm count 50-120 million/mL Other components of semen fructose energy for sperm motility fibrinogen causes clotting enzymes fibrinolysin liquefies semen within 30 minutes prostaglandins stimulate female peristaltic contractions spermine is a base stabilizing sperm pH at 7.2 to 7.6. Secretions of all three accessory glands plus sperm cells referred to as semen. Emission is the discharge of semen into prostatic urethra while Ejaculation is referred to as a forceful expulsion of semen from urethra caused by peristalsis. Temporary coagulation as fibrinogen becomes fibrin then fibrinolysin breaks up the coagulation. Sperm swim up vagina. Emission: accumulation of sperm cells and secretions of the prostate gland and seminal vesicles in the urethra controlled by sympathetic centers in spinal cord peristaltic contractions of reproductive ducts. Seminal vesicles and prostate release secretions and accumulation in prostatic urethra sends sensory information through pudendal nerve to spinal cord Male Microstructure and Function Microscopic cellular parts within the testes produce sperm cells and testosterone. Spermatogenesis refers to the development of sperm cells (spermatozoa) through a process of cell division and maturation. Firstly, spermtogenic cells form rounded cells called spermatids (Spermatocytogenesis) and secondly, spermatids differentiate into specialized cells known as sperms-Spermiogenesis. It is a process by which spermatogonia are transformed into spermatozoa. It begins at puberty. At birth: germ cells in the male present in the sex cords of the testis are surrounded by supporting cells (Sertoli cells). At this time the primordial germ cell divides to give spermatogonia. At puberty the germ cells awaken and start the actual process of spermatogenesis. These cells increase in number by simple mitosis to form cells known as spermatogonia ; type- A and type- B. Type-B spermatogonia, replicate DNA to have 46 double structured chromosomes to begin meiosis-1 and are called primary spermatocytes. Meiosis 2 follows immediately without DNA replication. Only 23 double structured chromosomes are involved 2 secondary spermatocytes quickly undergo meiosis-2 (Centromeres split in metaphase 2) and end with the formation of 4 sperrmatids each with 23 single structured chromosomes and1N DNA. Two spermatids bear X chromosome complement and other two bear Y chromosome complement.
  • 12. 5 Figure 3. Process of sperm formation As steps of spermatogenesis continue, the spermatocytes progressively move from basement membrane to the luminal side of seminiferous tubule. The cells of Sertoli provide nutrition and pockets of support to developing spermatocytes, support production of spermatozoa, androgen-binding protein intratesticularly (tebg), form blood-testes barrier, produce inhibin and seminiferous tubule fluid (bathing medium, nutrients, capacitation, motility). Spermatids are rounded cells. They modify to assume specific shape of the sperm. The changes include;  Golgi apparatus forms acrosomal cap-proteolitic enzymes  Nucleus is condensed  Centriols: make collar around neck  Microtubules, form flagellum,  Mitochondria arrange as spiral around neck  Excess cytoplasm cast off as residual body  Cytoplasmic bridges break and sperms release from Sertoli cells to lie free in lumen of seminiferous tubules. About 64 days are required to go from a spermatogonium to a sperm. A mature sperm has head, neck and tail. From lumen of seminiferous tubules sperms enter duct of epididymis and they take 20 days to travel this 4-6 meter long tortuous duct. If ejaculation does not occur they die and degenerate.
  • 13. 6 Hormonal Regulation of Testicular Function Testicular regulation involves three sets of hormones: GnRH - gonadotropin-releasing hormone secreted by hypothalamus and stimulates secretion of anterior pituitary secretion hormones (FSH/LH). LH act as Interstitial Cell Stimulating Hormone secreted by anterior pituitary and directly stimulate the testes (interstitial cells) to secrete testosterone while FSH stimulates formation of ABP (androgen binding protein). Testosterone secreted by cells of Leydig (Interstitial cells of seminiferous tubules) exert negative feedback on hypothalamic and ant. pituitary hormones and stimulates spermatogenesis by binding to ABP and development of secondary sex characteristics. QUIZ??? 1. Describe the structure of the testis and the process of spermatogenesis. 2. What is spermatogenesis. 3. Describe the normal and abnormal morphology of the sperm. 4. Appreciate functions of different hormones produced from Male spp 5. Describe cell cycle Structure and Function of the Female Reproductive System The functions of the female reproductive system include: Produce egg cells (ova), serve as receptacle for the penis during copulation, and house and nourish the fetus until parturition as well as production of hormones.
  • 14. 7 Figure 3. Female reproductive system of a cow Ovaries are paired structures that produce eggs (ova) and the female hormones, estrogen and progesterone. Broad ligaments that support the female reproductive tract and arteries, veins, and nerves of the ovaries in the abdominal cavity. The ovary is comparable to the male testicle and is the site of gamete production. A bovine animal has 20,000 potential eggs per ovary. Ova are fully developed at puberty and are not continuously produced as in the male. All species contain two functional ovaries except for the hen that has only a left functioning ovary. The ovaries have three major functions:  Gamete production (oovigenesis)  Secrete estrogen (hormone)  development of mammary glands  development of reproductive systems and external genitalia  fat deposition on hindquarters and belly (source of energy)  triggering of heat  Form the corpus luteum that secrete Progesterone Oviducts (Fallopian tubes), paired tubes that transport the eggs from the ovaries to the uterus and serve as the site where sperm and ova meet and fertilization occurs. Infundibulums two funnel-like openings of the oviducts that pick up the eggs at ovulation and direct them to the body of the oviducts.
  • 15. 8 Uterus a major reproductive organ that consists of the uterine body and two uterine horns. The embryo attaches to uterine body or uterine horn, depending on the species. The uterus varies in shape between livestock species from long uterine horns of the sow to relatively short uterine horns in the mare. This varying degree of sizes reflect the species differences in litter sizes, monotocous species having short horns and polytocous animals having much longer uterine horns. Figure 4. Types of uterus found in various species Functions of the uterus include:  Passageway for sperm during copulation,  Incubation and nourishment of the embryo during pregnancy, and  Expulsion of the fetus during parturition by contractions.  Hormonal production P2G PLACENTA Classification is based on the thppgross shape, the distribution of contact sites between fetal membranes and endometrium, the number of layers of tissue between maternal and fetal vascular systems and type of tissue (figure below)
  • 16. 9 Figure 5. Types of Placentas and common examples CERVIX Cervix is a thick-walled mass of connective tissue with a small tube-like opening that joins the uterus to the vagina; it serves as a passageway for semen during copulation. It also contains glands that secrete a waxy-like substance that seals off the uterus during pregnancy and between heat periods to protect against infection, disease, or foreign matter. Figure 6. Cervix of Cow Vagina Vagina is a reproductive structure that serves as the receptacle for the penis during copulation and the birth canal at parturition; it also serves as a passageway for expelling liquid wastes, as the urethra joins the bladder to the vagina prior to the opening at the vulva. It receives semen (except in sow) while the vulva an external portion of the female reproductive tract that serves to protect the internal system from infection, to initially receive the penis at copulation, and to act as a passageway for urine.
  • 17. 10 Female Microstructure and Function Oogenesis is the process of producing ova (eggs) in the follicles of the ovaries. Oogonia cells develop in the ovaries of the fetus and mature into oocytes by birth. Only a small proportion of oocytes develop into ova or reach ovulation. Follicle which is a blister-like mass on the surface of the ovary that contains a developing ovum and produces and stores estrogen. The follicle secretes estrogen as a signal to the rest of the reproductive tract to prepare for ovulation (release of the ovum from a mature follicle). Corpus hemorrhagicum that develop after ovualation is a small hemorrhage or blood-clotted area that develops at the site of a ruptured follicle and lasts 2 – 3 days. Then later Corpus luteum (CL), a yellow body of cells that develops in place of the corpus hemorrhagicum and produces progesterone. Figure 7. An ovum with different stages of follicle development Progesterone prepares the female reproductive system for pregnancy; it is produced by the corpus luteum and lasts about twelve days, unless the ovum is fertilized. During luteal phase corpus albicans, a white body of connective tissue that is the result of the degeneration and re-absorption of luteal tissue. Oogenesis The number of ova produced per cycle varies with each species. For example a cow or mare normally produces one ovum per cycle, ewe produces two ova per cycle whiles a sow produces eight to fifteen ova per cycle and a hen approx. 28 eggs/month. Oogenesis means differentiation of the primordial germ cells (oogonia) that are present in the cortex of the ovary into mature ova. Oogenesis passes into two stages of maturation: prenatal and postnatal maturation:
  • 18. 11 Prenatatal maturation The primordial germ cells (oogonia) undergo mitotic divisions then they are arranged in clusters surrounded by a layer of flat epithelial cells. The flat cells are called follicular cells and are derived from the epithelium covering the ovary. The development of ova mature, unfertilized eggs cells happens in the ovary. Some oogonia differentiate into large primary oocytes where DNA replication occurs and they enter the prophase of the first meiotic division. The surviving primary oocytes become surrounded by flat epithelial cells. Together they are called primordial follicles. Oogonia (stem cells that give rise to ova) multiply and begin meiosis STOPS at prophase 1. At this phase, the cells are called primary oocytes and remain in this phase until the onset of puberty, when they are activated by hormones LH and FSH. Near birth, all the primary oocytes have started prophase of the first meiotic division then they are arrested till puberty. The number of primary oocytes at birth ranges from 700,000 to 2 million. At puberty only 400,000 primary oocytes remain and about 400 only will be ovulated during the female life time. Postnatal maturation With the onset of puberty, 5-15 primordial follicles begin to maturate with each ovarian cycle. The primary oocyte begins to increase in size and the surrounding flat cells change
  • 19. 12 from flat to cuboid and proliferate to produce a stratified epithelium of granulosa cells. The follicle is now called the primary follicle. The granulosa cells rest on a basement membrane separating them from the stromal cells of the ovary that form the theca folliculi. The granulosa cells and the oocyte secrete a layer of glycoprotein on the surface of the oocyte called the zona pellucida. Fluid-filled spaces appear between the granulosa cells then they coalesce together forming the follicular antrum and the follicle is now called the secondary follicle. The follicle enlarges and is called the Graafian follicle In a 46 diploid individual, the primary oocyte continues the first meiotic division leading to two unequal daughter cells with 23 chromosomes each (22 autosomes + X). The large cell is called the secondary oocyte and the small one (having little cytoplasm) is the first polar body. The secondary oocyte and the first polar body enter the second meiotic division without DNA replication. The second meiotic division is completed only if fertilization occurs to give fertilized oocyte (mature ovum) and a second polar body that soon degenerates. FSH periodically stimulates a follicle to grow and induces its primary oocyte to complete meiosis 1 and start meiosis 2 meiosis then STOPS again and LH stimulates the completion of meiosis. The mature ovum It is a large oval cell that varies from 117 – 142 µ in diameter. It has two membranes; the inner thin one is the vitelline membrane and the outer one is the zona pellucida. The corona radiata is two or three layers of cells surrounding the zona pellucida externally when the ovum is shed from the follicle. .
  • 21. 14 Ovulation Mechanisms of ovulation involves hormonal surge that leads to increase in enzyme activity; collagenase and increased intrafollicular pressure. Ovulation a rupture of ovarian wall and follicle releases gamete. Gamete is transported and fertilised in uterine tube. Granulosa cells and theca interna become luteinised and form corpus luteum(CL), a transitory endocrine organ. In the Luteal phase progesterone is secreted from CL and maintains pregnancy. Life span of CL dependent on luterophic LH and luteolytic PG2a. Formation, maintenance and regression of CL always under hormonal control. Transport of gametes, fertilisation and implantation Ova ovulated from ovary, transported through uterine tube. Sperm deposited in vagina where it is transported and also capacitation occurs. .Fertilisation at ampulla-isthmic junction of the reproductive tract. After fertilisation, the Zygote is transported and get implanted in endometrium. Fertilisation Fertilisation consists of binding of sperm to zona pellucida and acrosome reaction. Thereafter, the sperm head penetrate of Zona Pellucida through cortical reaction that help fusion of plasma membranes of sperm and egg. Sperm nucleus enters oocyte, forms pronucleus, to complete meiosis II.
  • 22. 15 Fertilization that is the union of the haploid sperm and the haploid ovum to form a diploid individual; the actual beginning of pregnancy. Insemination can be natural or artificial. When bred naturally, the male deposits the semen into the vagina of the female. The sperm make their way through the cervix, into the uterine body, both uterine horns, and finally into the oviducts. At first, the embryo floats freely in the uterus obtaining its nourishment from fluids secreted by the uterine wall. The embryo will then attach to the wall of the uterus (20 days in cattle; varies with species) and begin taking a recognizable form, at which time it becomes a fetus. The embryo is encompassed by a fluid filled membrane called the amnion, which protects the embryo from mechanical disturbances. The amnion is surrounded by the chorion, which functions as a protective coat and point of nutrient exchange. In ruminants (cow and ewe), the chorion develops cotyledons (raised button-like nodules) that attach at certain places on the uterus called caruncles. In the mare and sow the placental attachment is made over most of the surface area of the chorion. The fetus receives nourishment from its mother through these attachment sites. The placenta is the term given to the membranes (chorion and uterine mucosa) that surround the embryo and attach to the uterus. Parturition
  • 23. 16 The fetus is expelled from the uterus, passing through the cervix and vagina, out of the reproductive tract. The membranes that had formed around the fetus are also expelled and, collectively, are called afterbirth. After parturition, the corpus luteum on the ovary begins to degenerate, a developing follicle on the ovary begins to release estrogen, estrus occurs, and the estrous cycle begins again. References Alberts, B; Johnson, A; Lewis, J; Martin, R; Roberts, K; & Walter, P(2002). The Molecular Biology of the Cell. 4th.Edition. Garland Science Chapter 20. Dyce, K; M.,Sack, W;O, & Wensing, C;J;G.(2006). 3rd. Edition. Textbook of Veterianry Anatomy.Pages183 – 202, 435 -453 Neill,J;D.(editor-in-Chief). Physiology of Reproduction. 3rd. Edition. Elsevier. London.Volume 1. Page3-113. Chapter two ARTIFICIAL INSEMINATION PRINCIPLES OF ARTIFICIAL INSEMINATION 1.0 introduction This chapter will be devoted solely to technical aspects of artificial insemination (AI). This is a tool for improving livestock production. A productive tool used to bring out the potential of animals by using superior sires. Artificial insemination is the technique in which semen with living sperms is collected from the male and introduced into female reproductive tract at proper time with the help of instruments. It is a breeding tool that involves methods of collecting semen from the bull and its deposition in the female reproductive tract by any insemination technique rather than natural. It is a very modest expedient aimed solely at collection of semen and its insertion into the female genital passages. History of Artificial Insemination Documents from around 1322 A.D. state that an Arab chief wanted to mate his mare to a stallion owned by his rival. Then in 1780, Spallanzani successfully bred two dogs with the use of A.I. Leading to him being named the inventor of artificial insemination. The mass
  • 24. 17 breeding of cattle however didn’t come until later, in 1931. His experiments proved that the fertilizing power reside in the spermatozoa and not in the liquid portion of semen. Few further studies under research station conditions helped this technique to be used commercially all over the world including India. Artificial insemination is not merely a novel method of bringing about impregnation in females. Instead, it is a powerful tool mostly employed for livestock improvement. In artificial insemination the germplasm of the bulls of superior quality can be effectively utilized with the least regard for their location in far away places. By adoption of artificial insemination, there would be considerable reduction in both genital and non-genital diseases in the farm stock. Student Learning Objectives 1. Describe the importance of animal reproduction. 2. List the parts and explain the functions of female and male reproductive systems. 3. List and describe the phases of the estrous cycle. 4. Explain how artificial insemination is performed. 5. Explain the advantages and limitations of artificial insemination. 6. Explain new technologies that are being used in reproductive management of animals. AIM OF AI It is a breeding to tool used to enhance improvement in breeding stock and it can never be sufficiently emphasis that artificial insemination is essentially a method essentially a method whose aim is better livestock breeding ROLE IN ANIMAL BREEDING Utilization of Bulls a. Use of outstanding males by the majority of breeders. Most breeders are able to use best bulls which otherwise would have been impossible for many of them owing to distance, high cost of purchasing such a bull or the prohibitive cost of service. b. Testing of young bulls. It helps produce, test, discover and use new genetic material c. Reduction in the number of bulls. Reducing the number of males reduces direct and indirect costs. With natural insemination a bull giving 100 services per year, or say
  • 25. 18 80 calves, will total 250 descendants in three years: with AI this number has to be multiplied by a coefficient which may be as high as 40:250 = 10,000. d. Withdrawal of bulls from small or average farms. Many farms carry one bull to ten cows. This makes service very expensive for poor semen. Removal of bull will allow a productive cow to be added to the stock e. Use of superior males that are old or already dead. Semen collection methods enable old bulls to be used that for various reasons, such as excess weight, tired legs etc are incapable of service. Use of refrigerated semen enables bulls that have long since dead to be used, facilitates testing. f. Use of bulls in various regions. Bulls of the best breeds are generally delicate and exacting in their demands and it has been hazardous and onerous task to transport them from region to another: sending semen eliminates such journeys. in addition as a results of the rigours of acclimation these animals often provide a poor reproductive service, whereas semen rapidly conveyed to where it is needed and inseminated with all its own qualities and those of the donor into indigenous females well accustomed to the environment will give cross-breed of good quality. g. Use of bulls restricted by international sanitary regulations. AI overcomes this obstacle by sending semen instead of the bull which makes easier to comply with the requirement. h. Use of bulls in cross-breeding. AI solves the problems of the sexual repulsion that exist between breeds and thus facilitates cross-breeding. Role with regard to health and sterility a. Prophylaxis of non- venereal diseases. By avoiding any contact between animals b. Prophylaxis of venereal diseases. Control sexually transmitted diseases. The bulls are tested before use. c. Therapy and sterility. Direct introduction of semen into the uterus may vaginal and cervical spasms to be overcome and the injurious influence of the vaginal environment to be avoided. It overcomes the effect of granular vaginitis d. Recognition of sterility. Renders worth service gynaecologic ally. Tool for investigation of reproductive issues of females
  • 26. 19 Scientific role a. Synchronization of estrus. If the animals are synchronized it is difficult to mate them but AI makes it easier. b. Makes possible the histo-physiological study of semen, study of its fertilizing capacity and examination of factors affecting if especially nutrition, health and sex life of the bull. c. It enables optimum time of coitus to be established. d. The effects of medicaments and the possibilities of disinfection and antiseptics of the semen can be studied Critics of artificial insemination a. It is difficult in developing countries for most farmers to properly detect heat. b. In developing countries, the delivery system is poor such that semen may be received later than the day you request it. c. Very accurate breeding records are needed to know when the animal comes on heat. d. Safety on the farm. In some dairy bulls are very dangerous and aggressive. ADVANTAGES OF ARTIFICIAL INSEMINATION: There are several advantages by artificial insemination over natural mating or servicing. These include: 1. There is no need of maintenance of breeding bull for a herd; hence the cost of maintenance of breeding bull is saved. 2. It prevents the spread of certain diseases and sterility due to genital diseases e.g: contagious abortion, vibriosis. 3. By regular examination of semen after collection and frequent checking on fertility make early detection of interior males and better breeding efficiency is ensured. 4. The progeny testing can be done at an early age. 5. The semen of a desired size can be used even after the death of that particular sire. 6. The semen collected can be taken to the urban areas or rural areas for insemination. 7. It makes possible the mating of animals with great differences in size without injury to either of the animal. 8. It is helpful to inseminate the animals that are refuse to stands or accept the male at the time of oestrum. 9. It helps in maintaining the accurate breeding and cawing records.
  • 27. 20 10. It increases the rate of conception. 11. It helps in better record keeping. 12. Old, heavy and injured sires can be used. Disadvantages of A.I: 1. Requires well-trained operations and special equipment. 2. Requires more time than natural services. 3. Necessitates the knowledge of the structure and function of reproduction on the part of operator. 4. Improper cleaning of instruments and in sanitary conditions may lead to lower fertility. 5. If the bull is not properly tested, the spreading of genital diseases will be increased. 6. Market for bulls will be reduced, while that for superior bull is increased. SEMEN COLLECTION METHODS AND EVALUATION: Various methods of collection of semen have been devised from time to time. The older unsatisfactory methods have gradually replaced by the new modern techniques. There are four common methods.  Use of artificial vagina  Electro-stimulation method.  Massaging the ampulae of the duct us differences through rectal wall.  Recovery method The ideal method of semen collection is use of artificial vagina which is safe for sire and the collector also. ARTIFICIAL VAGINA METHOD The artificial vagina has the following parts: A heavy hard rubber 2" lose, open at both ends with a nostle for air and water in and outlet, inner sleeve of rubber or rubber liner and the semen receiving cone or rubber cone; the semen collection tube made of glass or plastic graduate as well as insulating bag. Before using for semen collection all the parts must be washed thoroughly and sterilized properly, and assembled. As in artificial vagina, the rubber liner is inserted into the hose; inverting both ends back by folding back from either side opening, and fastening with rubber bands. Now the space between the hard rubber hose and
  • 28. 21 inner rubber liner forms a water tight compartment. The nostle at one end of the hose can be fixed . Turning through the threaded nut up or down. The water jacket of the Artificial -vagina is filled with hot water at a temperature of 45°C (113°F) by opening the nostle. The graduated semen collection tube is fixed to the narrow end of the artificial vagina hose, and fastened by a rubber band. The inner side of the rubber liner on the anterior side of the artificial vagina is lubricated with sterile jelly to a length of 3 to 4 inches. Air is blown through the nostle into the water jacket, to create pressure in if, and the same is exerted the rubber linear, to simulate natural vagina. The temperature of the artificial vagina is to be checked, at each collection, and it should simulate natural vagina at mounting time. If the artificial vagina is to mount later. If it is too cold ejaculate may not be there after a thrust, or even if ejaculate is there; it may be contaminated with urine, and becomes unfit for use. The artificial vagina assembled is held at 45° angle from the direction of penis, and the thrust is that angle. The artificial vagina is held with the left hand by a right handed person; and when the bull mounts the cow, the sheath of the bull will be graphed by the operator, directing the gland penis into the artificial vagina, and then the bull gives a thrust to ejaculate (Figure). The operator should evince care so as not to touch the exposed past of the penis. After the bull dismounts, the artificial vagina is taken off from penis and the air vent is opened to release the pressure from the jacket.
  • 29. 22 The water from the jacket is also drained by opening the nostle. This allows the ejaculate to flow from the cone to the semen collection tube. The semen collection tube is detached from the cone, plugged with cotton wool, and taken to the laboratory for examination. The rubber cone and the semen collection tube can be protected from external contamination or heat or higher, by covering with an insulation bag with zip. Recovery Method This method allows the male to mate with the female and you use a spoon to take the sperms that were ejaculated. Some us a spongy in the female reproductive system and just take the spongy out which will have some semen. The main disadvantages is that spermatozoa is of poor quality because they may mix with secretion from the female. it also increases chances of sexually transmitted diseases. Massage The hand is passed in the rectum of the bull (gloves or plastic should be used). If there is a lot of fecal matter it should be removed. The hand is passed over the vesicles glands and ampullae of the vas deferens to induce the female of semen. The cause the penis to protrude the sigmoid flexure can also be massaged so that the semen collected is less contaminated. Disadvantage is semen collected may be contaminated with urine and debris. Electro-ejaculator An electro-ejaculator probe powered ether by battery or battery cum-electric transistorized circuits is used by inserting it into the rectum and stimulating nerves of the reproductive system by gradually increasing voltage(10-15 volts) in a rhythmic fashion with a rheostat for short period. The electricity is passed on in pulses. The change of voltage causes stimulation which causes erection and ejaculation.
  • 30. 23 This type of collection is recommended because; Semen is clean because the area around the penis is cleaned and it is of high concentration. Incapacitated animals (lame) that cannot mount can also be used provided they are genetically fine. Advantage is that it does not require female or a dummy is required, however, the disadvantage of this method require an expensive machine and may affect sciatic nerves. Semen Analysis Sperm concentration- Total sperm is determined by multiplying concentration (sperm per ml) by ejaculate volume (ml). Semen traits include Volume-sperm/g and Density classified as:  Very good(VG)-750-1 billion sperms (Creamy)  Good (G)-400-750 spermatozoa (Milk-like)  Fair (F)-250-400 spermatozoa (Skim milk-Like)  Poor –less than 250 (translucent) Sperm motility is the percentage of sperm that are progressively motile. A progressively motile sperm swims briskly forward in a relatively straight line.  Very good(VG)-rapid dark swirls  Good (G)-slower swirls  Fair (F)- No swirls, but permanent individual cell motion  Poor – little or no individual cell motion Sperm morphology - the percentage of sperm with normal shape and size is determined. Double heads, not tails, double tails, no heads,
  • 31. 24 SEMEN STORAGE The discovery that bull semen could be successfully frozen and stored for indefinite periods has revolutionized AI in cattle. In 1949, British scientists discovered that addition of glycerol to the semen extender improved resistance of sperm to freezing. Glycerol acts to remove water from the sperm cell prior to freezing and prevents the formation of cellular ice crystals which would damage the sperm. There are two methods of freezing and storing semen: dry ice and alcohol (-100 degrees F) and liquid nitrogen (-320 degrees F). Liquid nitrogen is preferred because there is no evidence of fertility deterioration with age. Fertility gradually declines in semen stored in dry ice-alcohol. Frozen semen can be stored indefinitely if proper temperature is maintained. A recent report told of a calf born from frozen semen stored for 16 years. Fresh, liquid semen can be successfully stored for 1 to 4 days at 40 degrees F. Semen is usually stored in glass ampoules. Other methods appear promising, particularly the French-straw. Several AI organizations have gone to this method exclusively. Artificial colouring is frequently added to semen extenders in order to distinguish one breed from another. Complete identification of the bull is required on each individual semen container. Freezing of semen for successful preservation of spermatozoa, for long periods, is of great importance in livestock breeding and farm management. It has made it possible” to make available the use of outstanding proven sizes for larger number of cows, covering larger area, frozen semen shipment has become possible to different continents in the globe to any place connected with any service. Now a day if farmer wants to use of an outstanding size for inheritance of high milk yield, he can go in for frozen semen service provided his area is, covered by Artificial insemination, with supply of frozen semen. At present frozen semen is used in most of the states in India. The technique of semen preservation in straws was developed in France . Freezing of semen is done with a special diluents, which has the following composition.
  • 32. 25 Sodium citrate dihydrate (angular) 2.4 y. 2.0 gm 8.0 ml 25.0%byvolume 50,000 units per 100 ml of semen Fructose Glycerol Egg Yolk Penicillin dilulent. Dihydro-streptomycin 50.0 mg per .100m1 of semen dilulent. Distilled water double glass distilled 100.Om1. The addition of glycerol to the dilulent makes the cells more resistant to the rigours of freezing and icy crystals, which form are smaller and smoother thus creating less damage to the spermatozoa. The addition of fructose to the diluent luprores sperm resistance to glycerol; and also provides nutrition. Frozen semen is packed in single dose glass vials or plastic straws at +5°C. The final level of glycerol should be 7.0 to 7.6% during the freezing process. The antibiotics are added to inhibit bacteria and to kill pathetic organisms. The semen to be diluted in such a way that one ml. of extended semen will contain 20 million motile spermatozoa. The semen must be cooled carefully for spermatozoa to remain with life. The final temperature is lowered to - 79°C or still lower. Quick freezing is done for a period of 3 to 5 minutes to -75°C with the help of atmosphere created by liquid nitrogen. In the slow freezing technique cooling is done at the rate of 1 °C per minute from +5°C to -15°C. From -15°C to -31 °C at the rate of 2°C per minute. From -31°C to 75°C at the rate of 4 to 5°C per minute. Thus taking 40 minutes in total, further cooling to -96°C can be done quickly as it is not critical after freezing. Before freezing the diluted semen in equilibreated for 3 to 5 hours or for the best 16 to 20, hours period in refrigerator at 5°C. Frozen semen facilitates the percent use of the semen diluted and frozen, and thus the delivery price is reduced, and it can be supplied with the gaps of months to the A.I technicians as against the supply of fluid semen every days or alternate days. Liquid nitrogen plays a vital role for storing the frozen semen straws, at a temperature of -196°C for longer periods. The sperms after being ejaculated cannot survive foe a long time outside the female reproductive tract unless various agents are added. The agents added have the following characteristics;  Isotonic with the semen (ions) contained should not be different from those of semen.  Buffering Capacity. The agents should have the buffering capacity to avoid shifts in pH as the semen is being processed.  Protect the semen from cold shock. Lipo-proteins are added to the extender which prevent cold shock as the sperms are being frozen.  Nutrients. These agents should provide nutrients for metabolism of sperm. Milk, egg yolk and simple sugars are added.
  • 33. 26  Microbial Contamination should be controlled by use of antibiotics such as penicillin.  Should be able to protect cells during freezing -Glycerol is added to prevent water crystals forming during freezing. Examples of Diluters (Extenders) Egg Yolk Extender. Add 4 parts egg yolk to 6 parts of Nacitrate (2.9g Nacitrate in 100ml distilled water) add 500 IU penicillin and 1mg streptomycin per ml of the mixture. Skim Milk Extender. Heat skim milk at 95o C for 10 minutes then cool and add glucose at 1g/100ml milk. Then penicillin and streptomycin are added to protect sperm against bacteria PROCESSING SEMEN Extending (or diluting) the semen increases the number of females that can be inseminated from one ejaculation. There are several good semen extenders. Those made from egg yolk or pasteurized, homogenized milk are two of the most widely used. A good extender not only adds volume to the ejaculate, but favors sperm survival and longevity. Dilution rate depends on quality of sperm. Antibiotics such as penicillin and streptomycin are added to semen extenders to inhibit bacterial growth and reduce the spread of diseases. PACKAGING Extended semen frozen as pellets, in straws or in ampules is held at 50o C prior to freezing. Straws are frozen in nitrogen vapour and stored at –196o C. Ampules are frozen at 3o C/minute to –15o C and then the rate of freezing increased to –15o C and then the semen is transferred to liquid N at –196o C in the ampules on canes. Pellets freeze in few seconds to –79o C on a block of ice. Calculation of sperm concentration If 8ml ejaculate containing semen evaluation showed the following Volume of semen 8ml Concentration 900 x 106 Motility 80% Normal cells percentage 95% 1. Estimate the total number of cells in the ejaculate = volume x concentration = 8ml x 900 x 106 = 72 x 108
  • 34. 27 2. Live cells = total number of cells x motility = 72 x 108 x 80/100 = 576 x 107 live cells 3. Live normal cells = live cells x normal cell % = 576 x 107 x 95/100 = 5472 x 106 Therefore live normal cells = volume x concentration x normal cell x motility For bull semen recommended; 10 x 106 live normal cells per insemination (not frozen) 15 x 106 `live normal cells per insemination pre-freezing. INSEMINATION METHODS The recto-vaginal technique is the most commonly used method of artificially inseminating (AI) cattle. The basic skills required to perform this technique can be obtained with about 3 days practice under professional instruction and supervision. Additional proficiency and confidence come with time and practice. Regardless of whether the inseminator is left or right handed, it is recommended that the left hand be used in the rectum to manipulate the reproductive tract and the right hand be used to manipulate the insemination gun. This is because the rumen or stomach of the cow lays on the left side of the abdominal cavity, displacing the reproductive tract slightly to the right. Thus it may be easier to locate and manipulate the tract with the left hand. Step #1: Restrain the animal to be inseminated. There are several things that should be kept in mind when choosing a location for inseminating cattle. Some of these include safety of both the animal and the inseminator, ease of use, and shelter from adverse weather. A gentle pat on the animal’s rump or a soft spoken word as the inseminator approaches will help to avoid startling or surprising the cow. Step #2: Raise the tail with the right hand and gently massage the rectum with the lubricated glove on the left hand. Place the tail on the back side of the left forearm so it will not interfere with the insemination process. Cup the fingers together in a pointed fashion and insert the left hand in the rectum, up to the wrist. Step #3: Gently wipe the vulva with a paper towel to remove excess manure and debris. Be careful not to apply excessive pressure which may smear or push manure into the vulva and
  • 35. 28 vagina. With the left hand, make a fist and press down directly on top of the vulva. This will spread the vulva lips allowing clear access to insert the gun tip several inches into the vagina before contacting the vaginal walls. Step #4: Insert the gun at a 30° upward angle to avoid entering the urethral opening and bladder located on the floor of the vagina. With the gun about 6 to 8 inches inside the vagina, raise the rear of the gun to a somewhat level position and slide it forward. To become a successful inseminator, it is very important to always know where the tip of the insemination gun is located. The walls of the vagina consist of thin layered muscle and loose connective tissue. The insemination gun can be easily felt with the left hand in the rectum. As the breeding gun is inserted into the vagina, keep the gloved hand even with the gun tip (Figure #1). Figure #1: Keeping the gloved hand even with the tip of the inseminator gun. Manure in the rectum can often interfere with the inseminator’s ability to palpate the cervix and gun tip. However, it is seldom necessary to remove all the manure from the bowel. Instead, keep the open hand flat against the floor of the rectum, allowing the manure to pass over the top of the hand and arm (Figure #2). Figure #2: Allowing manure to pass over the top of the hand and arm. With the hand in the rectum, the inseminator may notice colon constrictions or "rings" attempting to force the left arm from the cow. To relax these rings, place two fingers through the center of a ring and massage back and forth. The constriction ring will eventually relax, pass over the hand and arm, and the inseminator can continue the palpation process (Figure #3)
  • 36. 29 Figure #3: Dealing with colon constrictions. Because the reproductive tract is freely movable, cows that have strong rectal and abdominal contractions in response to being palpated may actually push their reproductive tract back into the pelvic cavity. This will cause many folds to form in the vagina. In such cases, the insemination gun can get caught in these folds and little or no progress will be made until they are removed. If the cervix can be located, grasp it and gently push it forward. This will straighten the vagina and the gun should pass freely up to the cervix (Figure #4). The inseminator will note a distinct gristly sensation on the gun when it contacts the cervix. Figure #4: Grasping the cervix and gently moving it forward. In most cows, the cervix will be located on the floor of the pelvic cavity near the anterior (front) end of the pelvis. In older cows, the cervix may rest slightly over the pelvic bone and down into the abdominal cavity. Once the gun is in contact with the external surface of the cervix, the inseminator is ready to begin threading the cervix over the end of the gun. Place the cervix on or over the insemination gun; the gun is not passed through the cervix. Excessive movement or probing with the insemination gun during this step is seldom productive. The key to mastering this step of the insemination process is knowing how to hold and manipulate the cervix and concentrating on doing the work with the hand inside the cow, not the one holding the gun. When the gun first contacts the cervix, the inseminator will usually find that the tip is in the fornix area directly over the top of the opening of the cervix. If this happens, grasp the external opening to the cervix with the thumb on top and forefingers underneath (Figure #5).
  • 37. 30 Figure #5: Finding the opening of the cervix. This closes the fornix at top and bottom. It is also still important to know the location of the gun tip. This is accomplished by contacting the gun tip with the palm and 3rd and 4th fingers of the hand in the rectum. Use the palm and these two fingers to guide the gun tip to the cervical opening located between the thumb and forefingers. With gentle probing, the opening of the cervix should be located. The inseminator will feel the gun slide forward until it contacts the second cervical ring. Maintain gentle but steady forward pressure on the gun and slide the thumb and forefingers just in front of the gun tip and re-grasp the cervix. Because the cervix is composed of dense connective tissue and muscle, it is difficult to clearly distinguish the gun tip when it is located within this structure. However, the inseminator can determine the approximate location by bending the cervix. Using the flexibility of the wrist, gently twist and bend the cervix until the second ring of the cervix slides over the gun tip (Figure #5). Repeat the process until all the rings have been passed over the gun tip. Remember, the cervix is being placed over the gun, not the gun through the cervix. For the most part, gentle forward pressure is all that is necessary and gun movement should be minimal. 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, the inseminator will once again be able to feel the tip of the gun. It is now time to check the gun placement and deposit the semen. Rotate the gloved hand until it lies on top of the cervix. With the index finger of that hand, locate the far end of the cervix (Figure #7). Pull back on the gun until the tip of it is directly underneath the index finger near the internal opening of the cervix. Raise the finger and slowly deposit the semen. Push the plunger slowly so that drops of semen fall directly into the uterine body.
  • 38. 31 Figure #7: Locating the end of the insemination gun and depositing the semen in the body of the uterus. With proper AI technique and gun placement, semen will be deposited in the uterine body. Uterine contractions will then transport spermatozoa forward to the horns and oviducts with a good distribution of both sides (Figure #10). Figure #8: Good distribution of the semen to both uterine horns while Improper distribution of the semen into one horn because the insemination gun is pushed too far forward. When the insemination gun is more than 1 inch through the cervix, all the semen will be deposited in only one horn (Figure #8). Be sure to raise the index finger after checking gun placement. Not doing so may obstruct one horn, creating a situation of uneven semen distribution. When checking gun tip placement, be careful not to apply excessive pressure. The delicate uterine lining is easily damaged, potentially causing infections and reduced fertility. Make sure to push in with the plunger and do not pull back on the gun. Pulling back may result in much of the semen dose being deposited in the cervix and vagina instead of the uterine body. Although the recommended site of semen deposition is in the uterine body, research suggests that when exact gun tip placement is in doubt, depositing semen slightly into one uterine horn is less likely to compromise fertility than depositing it in the cervix. However, if the cervical mucous of a cow that has been previously inseminated feels thick and sticky on the gun, the cow may be pregnant. In this case, deposit the semen about halfway through the cervix.
  • 39. 32 After properly depositing semen, slowly pull the gun from the reproductive tract. Remove the gloved hand from the rectum. Check the gun tip for signs of blood, infection or semen leakage inside the sheath. Make the necessary notes for future reference and for the local veterinarian. Remove the sheath from the gun and hold it in the gloved hand. Check again to see which bull was used. Remove the glove starting at the top of the arm by turning it inside out trapping manure, the sheath, and dirt inside. Dispose of the used glove in a proper receptacle. Wipe the gun clean and dry and return it to the proper storage location. Correct insemination procedures will result in better breeding efficiencies. More selection pressure can then be placed on economic traits such as milk and beef production, enabling a higher return on semen investment dollars. VAGINAL METHOD: Hand is passed through the vagina and the inseminating tube is guided by hand to the site of insemination and semen is deposited. Here there is a risk of contamination and injury of female genitalia. Advantage- The semen collected is of high quality and is highly concentrated. Type of breeding method (AI - artificial insemination vs. live coverage): SYMPTOMS of HEAT The estrous cycle is a series of steps that occur from estrus to estrus. These are the phases of reproductive readiness in the reproductive system of a mature female. The cycle does not occur during pregnancy nor when a female is in anestrus. Anestrus is the absence of cycling. Anestrus may occur due to disease, not being of reproductive age, or other conditions. The estrous cycle is comprised of four phases. The phases occur in a definite sequence unless the female is pregnant. 1. Estrus is the phase when a female is in heat. The animal is receptive to mating and will stand for copulation with a male. Females exhibit signs of heat. An enlarged vulva, restlessness, and a mucus discharge are signs. Some females exhibit behaviours indicating readiness for mating such as when a cow mounts another cow in the mating position. Ewes, sows, mares, and some small animals ovulate at this time. Some animals usually release only one egg while others may release twenty eggs at one time.
  • 40. 33 2. Metestrus is the phase following heat. Ovulation occurs during metestrus in does and cows as do other processes that help maintain a pregnancy should conception occur. During metestrus luteinizing hormone (LH) causes the corpus lutea (CL) to develop. 3. Diestrus is the phase in the estrous cycle when the reproductive system assumes that conception has occurred, even if it has not. Diestrus is several days long depending on the species of animal. High levels of progesterone cause the uterus to begin preparing for pregnancy. 4. Proestrus is the period following diestrus in which preparation is being made by the reproductive system for the next heat period and ovulation. If conception has occurred, the estrous cycle ceases until it is renewed after gestation and parturition. The following table outlines the length of estrous, length of estrus, and when ovulation occurs for a variety of species. Detecting estrus as aid to artificial insemination. 1. Standing heat is one of best indicators of estrus in cows. Cows will stand to be mounted and will try to mount other cows. They will be restless and will oftentimes have an enlarged vulva and discharge.
  • 41. 34 2. Mares will “wink” to expose their clitoris and will urinate frequently. 3. Redness in the vulva is an indicator for sows. 4. Ewes will not show signs of estrus until a ram is present. Producers will use a castrated ram to detect heat. 5. Goats will act aggressively, be noisy and active, stand to be mounted, and have a mucous discharge. The timing of insemination The timing of insemination varies greatly by species and can vary between individuals to a degree. 1. Cows are usually inseminating in the evening after showing signs of estrus in the morning or in the morning after showing signs the evening before. 2. Mares are usually inseminated on the third, fifth, and seventh days of estrus. 3. Turkeys are inseminated three times over the course of several weeks. About one week should pass between the first two inseminations and then a little over a week between the second and third inseminations. A female turkey has a storage gland for semen making this possible. 4. Twenty-four hours after the initiation of estrus sows can be inseminated. Some producers will inseminate the sow after another 24 hour period. CHAPTER THREE EMBRYO TRANSFER The mammalian ovary contains thousands of ova but the number of off-springs produced is small. The number of times an animal can become pregnant is affected by the length of gestation. Under normal conditions the number of ova release per estrus is one or two in non litter bearing animals. The number of offspring that a female can bear can be greatly increased by allowing it to temporally become pregnant recovering the embryo in early pregnancy and transferring them to the reproductive tracts of other females to complete gestation. The process can be amplified if the donor super-ovulates Super-ovulation This is the procedure in which the female is treated with hormones to cause her to produce a large number of ova instead of the one or two that she normally produces at each estrus.
  • 42. 35 This procedure is done so that these ova can be transferred to another female. About 60-70 % of the super-ovulated ova form normal embryos. Reliable super-ovulation has not yet been effected in the mare but can be accomplished in the sow. Superior germplasm could be identified earlier and utilized more. To date response has been variable and fertilization rates have been very low. Methods of Embryo Recovery and Transfer. Embryo transfer refers to the technique by which fertilized ova are collected from a female called the donor and transferred for development to term to another female known as the recipient. The chain of events in the process of embryo transfer includes 1. management of the donor for production of a suitable number of viable ova, 2. mating or A.I, 3. collection, 4. evaluation, 5. short term storage of the embryo from that donor, 6. transfer of these embryos to suitable recipients. Also associated with embryo transfer a number of procedures including maintenance of vigorous health program of donors and recipients and the keeping of a detailed series of records for every aspect of the program. The main objective of embryo transfer is the improvement of animal populations through better utilization of superior females. Methods of Collection or Recovery Early collection techniques involved either slaughtering the females and excising (cutting out part of the body) the oviduct or surgically removing the oviduct from the live females at 72 hours post ovulation so that the embryos could be recovered by flushing. This however defeated the primary purpose of superovulation so other methods were developed. Surgical Methods Laparotomy. This is surgically opening of the abdomen (is performed to expose the reproductive tract). A clamp or thumb and forefinger can be used to block the distal of 1/3 of the uterine horn so that fluid injected into that segment can be forced through the oviduct with a gentle milking acting and collected at the infindibulum. An alternative method is to occlude (block) the uterine horn at the body of the uterus and introducing culture medium through a puncture at the uteral junction or through the
  • 43. 36 oviduct until the uterus is turgid. The uterus is then punctured with a blunt needle attached to a flexible catheter. The pressure causes the medium to gush through the catheter with enough turbulence to carry the embryos into a collection tube. Although these procedures allow for the recovery of a high percentage of the embryos, they can be repeated only a few times because of the surgical tromer and the resulting adhesions. The adhesions make it difficult if not impossible to expose the reproductive tract repeatedly. Now surgical techniques that give results equal to surgical methods have been developed for cows. This involves the use of a Foley catheter which is a two ways or three way flow catheter. It allows flushing fluids to pass into the uterus at the same time allows fluids to be returned from the uterus to a collecting receptacle. A feature in this catheter which can be inflated just inside the uterine horn to prevent the flushing fluid escaping through the cervix. Non surgical techniques for collection of ova are desirable because all surgical technics may lead to the formation of adhesions and because there is less risk to life and health of the donor with non surgical methods. The non surgical methods have been used for repeated recovery from the same donor practically. Ova are being collected from cows without superovulatioin. Each uterine is filled with 30-50ml of medium which is then allowed to flow into the collection vessel while the uterus is gently massaged through the rectum. This is repeated until 300-800ml of medium has been used. The Foley catheter is then inserted into the other uterine horn and the process repeated. After ova are collected (recovered) they are analyzed under the microscope. Some ova are not fertilized and others are deformed. Those that can develop are then frozen for storage. SITE OF TRANSFER If the embryo has less than 8 cells in it then it is transferred to the oviduct because the conditions in the uterus are not conducive for their development. The uterus produce a toxic environment for theses cells. If the embryo has more than 8 cells then the uterine transfer is recommended. TRANSFER INTO THE RECEIPIENT As in embryo collection the method of transfer are similar to those in embryo collection. There are surgical and non-surgical methods.
  • 44. 37 Surgical Method In this method the cow is excised to expose parts of the reproductive system. When embryo is being transferred into the oviduct a capillary pipette is used to drop ova with medium at the infundibulum and ampula of the oviduct. When the transfer is made into the uterus puncture the uterine wall with blunt needle and then expel the embryo from the tip of the capillary pipette inserted into the uterine lumen. Now non surgical methods are used similar to A.I ones to deposit ova in the uterus. Synchronization The recipient should be in the similar period of estrus with the donor either naturally or by synchronization. The recipient should be in estrus within 12 hours as the donor. Pregnancy rates reduce if the difference is more than 24 hours in cows. The recipient for frozen embryos should be in physiological synchrony with the embryo. If the embryo was frozen 3 days then the recipient should have been in estrus 3 days. Advantages Embryo transfer is an experimental technique used to evaluate reproductive problems and improve the genetic performance of the cattle population. Disadvantage There is high variability in the results due to unpredicted response to super ovulation and many ova are unfertilized and abnormal Assignment. Discuss factors that limit the use of AI and ET in Zambia.
  • 45. 38 Chapter four PRINCIPLES OF ANIMAL GENETICS Genetics is the science of heredity and variation. It is the scientific discipline that deals with the differences and similarities among related individuals. All animals have a predetermined genotype that they inherit from their parents. However, an animal’s genotype can be manipulated by breeding and more advanced scientific techniques (genetic engineering and cloning). For many years, managers of agricultural systems have manipulated the genetic makeup of animals to; mprove productivity, increase efficiency and adaptability. Successful manipulation of the genetic composition of animals requires a depth understanding of fundamental principles of genetics Mendel’s Laws Gregory Mendel is recognized as the father of genetics and in the 1850’s and 1860’s, he developed his theories without any knowledge of cell biology or the science of inheritance- he failed his teachers exams. In later years, genes, chromosomes, and DNA were discovered and people began to understand how and why Mendel’s theories worked. Mendel proposed three principles to describe the transfer of genetic material from one generation to the next. To understand Mendel’s principles and the relationships between phenotype and genotype, it is necessary to understand; what makes up the genetic material of animals and how this is transferred from one generation to the next. Genetic Material The body is made up of millions of cells which have a very complicated structure. These cells are made up of many parts that have specialized roles. These include: 1. Nucleolus 5. Rough Endoplasmic Reticulum 9. Mitochondria 2. Nucleus 6. Golgi Apparatus 10. Vacuole 3. Ribosome 7. Cytoskeleton 11. Cytoplasm
  • 46. 39 4. Vesicle 8. Smooth Endoplasmic Reticulum 12.Lysosome13.Centriole The nucleus contains chromosomes that are visible under the microscope as dark-staining, rod-like or rounded bodies. Chromosomes occur in pairs in the body cells. The number of chromosomes in each cell is constant for individual species, but it differs among species. Suis-38, Caprine-60, Carnis 78, Galus-78, Bovine. Chromosomes are made up of tightly- coiled strands of DNA and is a complex molecule composed of deoxyribose, phosphoric acid, and four bases. Individual genes are located in a fixed position (known as the loci) on the strands of DNA. A chromosome is made up of two chromatids and a centromere. The chromatids are formed from tightly coiled strands of DNA. If these strands of DNA are stretched out, individual genes can be identified.eg DT GENE CODING A gene is made up of a specific functional sequence of nucleotides, which code for specific proteins. A specific protein is produced when the appropriate apparatus of the cell (the ribosome) reads the code. In animals, chromosomes are paired and, therefore, genes are paired. These paired genes code for the same trait, but they are not identical and can have different forms, known as alleles. For example, sheep and cattle can be polled or horned. One gene codes for this trait and two possible forms (alleles) of the gene are polled or horned Identified by Mendel as three fundamental principles of genetics. The Principle of Dominance A capital letter is used to denote the dominant form of the gene (P) and a small letter is used to denote the recessive form of the gene (p). In the example, the polled allele is dominant and is, therefore, denoted by P, while the horned allele is recessive
  • 47. 40 and denoted by p. Because genes are paired, an animal can have three different combinations of the two alleles: PP, Pp, or pp. When both genes in a pair take the same form (PP or pp), the animal is referred to as being homozygous for that trait. An animal with a PP genotype is referred to as homozygous dominant. An animal with the pp genotype is referred to as homozygous recessive. If one gene in the pair is the dominant allele (P) and the other gene is the recessive allele (p), the animal is referred to as being heterozygous for that trait and its genotype is denoted as Pp. If an animal has the allele combination PP, it will be polled. If the combination is pp, the animal will be horned. If it is a heterozygote, the animal will have both traits (Pp), but the animal will be polled because the polled allele (P) is the dominant form of the gene. Mendel’s principle of dominance states that in a heterozygote, one allele may conceal the presence of another. The Principle of Segregation When animals reproduce, they only pass on half of their genetic material to their offspring. The offspring will only receive one allele from each parent. The Principle of Segregation explains some of the differences that are observed in successive generations of animals and can be used to predict the probability of different combinations of alleles occurring in offspring. In a heterozygote, two different alleles segregate from each other during the formation of gametes. Aa individual will produce two gametes- A-alleles and a-alleles. Considering these three types of individuals, six combinations of the various genotypes are possible:  PP x PP (both parents are homozygous polled),  PP x Pp (one homozygous polled parent and one heterozygous polled parent),  PP x pp (one homozygous polled parent and one homozygous horned parent),  Pp x Pp (both parents are heterozygous polled),  Pp x pp (one heterozygous polled parent and one homozygous horned parent), and  pp x pp (both parents are homozygous horned) The genotypes of the parents can be used to predict the phenotypes of the offspring.
  • 48. 41 Predicting the Genotypes and Phenotypes of Offspring by A punnett square - grid-like method that is used to display and predict the genotypes and phenotypes of offspring from parents with specific alleles. The male genotype is normally indicated at the top and the female genotype is indicated in the vertical margin (Figure) When crossing homozygous dominant parents (PP x PP) all heterozygous parent with a homozygous dominant parent (Pp x PP), 1:1 two heterozygous parents are crossed (Pp x Pp) hen crossing homozygous recessive parents (pp x pp) This happens because Alleles separate during meiosi. In others words the punnet chart maybe presented in this Considering Multiple Traits-Dihybrid Cross Commonly, there are multiple traits that need to be considered when mating animals. For example, consider that cattle can be horned or polled and white-faced or red-faced.The horns and red-faced coloring are recessive traits. If two individuals with two pairs of heterozygous genes (each affecting a different trait) are mated, the expected genotypic and phenotypic ratios would be:  Genotypes: 1 PPWW, 2 PPWw, 2 PpWW, 4 PpWw, 1 PPww, 2 Ppww, 1 ppWW, 2 ppWw, and 1 ppww;  Phenotypes: 9 polled, white-faced; 3 polled, red-faced; 3 horned, white-faced; and 1 horned, red-faced offspring
  • 49. 42 The Principle of Independent Assortment the alleles of different genes segregate, or assort, independently of each other. When considering multiple traits, Mendel hypothesized that genes for different traits are separated and distributed to gametes independently of one another. Therefore, when considering polled and white-faced traits, Mendel assumed that there was no relationship between how they were distributed to the next generation. In most cases, genes do assort independently. However, advances in genetics have shown that an abnormal situation, called crossing-over, can occur between genes for different traits. Crossing-over is an exchange of genes by homologous chromosomes during the synapses of meiosis prior to the formation of the sex cells or gametes. PpBb x PpBb gives 9:3:3:1
  • 50. 43 Other Concepts in Genetics Non-traditional inheritance involves alleles that are not dominant or recessive. Incomplete, or partial dominance, & co-dominance are two examples of non-traditional inheritance. Recent studies in sheep has indicated another form of inheritance called POLAR DOMINANCE Partial, or incomplete, dominance occurs when the heterozygous organism exhibits a trait in- between the dominant trait and the recessive trait. eg Homozygous mice are black (BB) or white (bb) and the heterozygous mice will be grey (Bb). When a pure, brown-eyed sheep is crossed with a pure, green-eyed sheep, blue-eyed offspring are produced Codominance occurs when a heterozygote offspring exhibits traits found in both associated homozygous individuals. An example of codominance is the feather color of chickens. If a homozygous black rooster is mated to a homozygous white hen, the heterozygous offspring would have both black feathers and white feathers. Roan is a coat color in horses (sometimes dogs and cattle) that is a mixture of base coat colored hairs (ex. black, chestnut) and white hairs. Neither the base coat color or the white hairs are dominant nor do they blend to create an intermediate color. Under these circumstances, neither allele is dominant and neither is recessive. Therefore, each allele is denoted by a capital letter. Epistasis It is possible for more than one gene to control a single trait. This type of interaction between two nonallelic genes is referred to as epistasis. When two or more genes influence a trait, an allele of one of them may have an epistatic, or overriding, effect on the phenotype. Comb shape in chickens is an example of an epistatic relationship. Mutations and Other Chromosomal Abnormalities Genes have the capability of duplicating themselves, but sometimes a mistake is made in the duplication process resulting in a mutation. The new gene created by this mutation will cause a change in the code sent by the gene to the protein formation process. Some mutations cause defects in animals, while others may be beneficial. Mutations are responsible for variations in coat color, size, shape, behavior, and other traits in several species of animals. The beneficial mutations are helpful to breeders trying to improve domestic animals. Changes in chromosomes are reflected in the phenotypes of animals. Some chromosomal changes will result in abnormalities, while others are lethal and result in the death of an animal shortly after fertilization, during prenatal development, or even after birth. Changes that can occur in chromosomes during meiosis include:  Changes in the chromosome number,
  • 51. 44  Translocation or deletion – chromosome breakage, and  Inversion and insertion – the rearrangement of genes on a chromosome. Sex-Linked Traits Sex-linked traits involve genes that are carried only on the X or Y chromosomes, which are involved in determining the sex of animals. The female genotype is XX, while the male genotype is XY. The X chromosome is larger and longer than the Y chromosome, which means a portion of the X chromosome does not pair with genes on the Y chromosome. Additionally, a certain portion of the Y chromosome does not link with the X chromosome. The traits on this portion of the Y chromosome are transmitted only from fathers to sons. Sex-linked traits are often recessive and are covered up in the female mammal by dominant genes. The expression of certain genes, which are carried on the regular body chromosomes of animals, is also affected by the sex of the animal. The sex of an animal may determine whether a gene is dominant or recessive (Ex. Scurs in polled European cattle). In poultry, the male has the genotype XX, while the female has the genotype Xw. An example of a sex-linked trait in poultry is the barring of Barred Plymouth Rock chickens. If barred hens are mated to non-barred males, all of the barred chicks from this cross are males, and the non-barred chicks are females Quantitative vs qualitative traits Quantitative traits are controlled large number of genes. They exhibit normal distribution and phenotypes and show continuous express with additive gene effect. They traits affect more genes with large effect. In selection, quantitative traits are economical traits and expensive to select for (Carcass quality, Milk Yield). Single dominant or recessive genes control qualitative traits. They tend to exhibit Non additive gene and non- continuous e.g (Colour and Polled) Measuring Heritable Variation The value of quantitative traits such a mohair length or size or milk yield is determined by their genes operating within their environment. The size of how a spp grows is affected not only by the genes inherited from their parents, but the conditions under which they grow up. For a given individual the value of its phenotype (P) (e.g. the weight of a broiler in grams) can be considered to consist of two parts genotype (G) and environment (E). P = G + E.
  • 52. 45 The quantitative genetics approach depends on taking a population view and tracking variation in phenotype and whether this variation has a genetic basis. We measure variation in a sample using a statistical measure called the variance. The variance measures how different individuals are from the mean and the spread of the data. Mean and variance are two quantities that describe a normal distribution. For Your Information:  Variance is the average squared deviation from the mean.  Standard deviation is the square root of the variance.  Covariance is correlations among characters or relatives Heritability Heritability is the proportion of the total phenotypic variation controlled by genetic rather than environmental factors. The total phenotypic variance may be decomposed: VP = total phenotypic variance VG = total genetic variance VE = environmental variance VP = VG + VE Estimation heritability (H2 ) = VG/VP (broad-sense) The total genetic variance (VG) may be decomposed: VA = additive genetic variance VD = dominance genetic variance VI = epistatic (interactive) genetic variance Variance Mean
  • 53. 46 Estimation heritability = h2 = VA/VP (narrow sense) h2 = VA / VP = VA / (VG + VE) Heritability ranges from 0 to 1 (Traits with no genetic variation have a heritability of 0). Since heritability is a function of the environment (VE), it is a context dependent measure. It is influenced by both, The environment that organisms are raised in, and the environment that they are measured in ESTIMATING HERITABILITY FROM REGRESSION one common approach is to compare phenotypic scores of parents and their offspring: junco tarsus length (cm) Cross Midparent value Offspring value F1 x M1 4.34 4.73 F2 x M2 5.56 5.31 F3 x M3 3.88 4.02 Slope of a plot of two variables (x,y) = Cov (x,y) / Var (x) Comparison Slope Midparent-offspring h2 Parent-offspring 0.5 h2 Half-sibs 0.25 h2 First cousins 1/8h2 As the groups become less related, the precision of the h2 estimate is reduced Role of reproduction in genetic improvement Heritability important because it allows us to predict a trait’s response to selection Let S = selection differential Let h2 = heritability Let R = response to selection R = h2 S Slope (h2 )
  • 54. 47 Figure 8. Response to selection. The selection differential (S) = mean of selected individuals - mean of the base population. The response to selection,: for a given intensity of selection, the response to selection is determined by the heritability which can either be high or low. The higher the slope, the better offspring resemble their parents. In other words, the higher the heritability, the better offspring trait values are predicted by parental trait values. The response to selection can be predicated for example in the large ground finch, Geospiza magnirostris Mean beak depth of survivors = 10.11 mm Mean beak depth of initial pop = 8.82 mm S = 10.11 – 8.82 = 1.29 h2 = 0.72 R = h2 S = (1.29)(0.72) = 0.93 Beak depth next generation = 10.11 + 0.93 = 11.04 mm. Therefore, the Response to Selection and is defined as the difference between the mean trait value for the offspring generation and the mean trait value for the parental generation i.e. the change in trait value from one generation to the next. Gy = SD * h2 GI where Gy= rate of genetic progress per year ( SD = selection differential h2 = heritability estimate, and GI = generation interval in years Methods of Selection  Individual and family