Pertemuan ke-1

Male infertility factor

Obstructive diseases as well as varicocele to be effected to quality of spermatozoa decresing >

repair surgically
Hormonal mainly prolactinemia and testosterone deficiency

Androgen receptor defects

1. Testicular feminization (insensitivity) of androgen
Karyotype 46 XY
Bilateral testes
Absent of hypoplastic wolfian duct
Appearing female external genital but hypoplastic
Common occur since embryonal 8-12 weeks gestation
All masculine symptom wont be occur
Location androgen recept, at x-chrome between Xq11 TO Xq13
2. Reifensteins syndrome (incomplete androgen resisten)
Incomplate and hypoplastic male external genital tract and absent female external

genital
Mullerian duct absent, but present derivated wolfian

Samples management

How to collect the sample

What kind of the sample needed
How to prepare the sample
When the sample to be collected
Process and store
What kind analysis to be used

Endocrine analysis

What kind of the sample to be used

How to collect the sample
How to prepare the sample
When the sampe to be collected
Process and keeping
What kind analysis to be used

Reference

Chard T, 1992. Laboratory techniquein biochestry and molecular biology, an introduction to

radioimmunoassay and related techniques. Elsivier biomedical press, Amsterdam

Greenspan FS and DG Gardner 2004. Basic and clinical endocrinology 7 th ed. Lange med

book/ mc grow hil Comp NY

International atomic energy agency (IAEA), 1984. Laboratory trainingg kefoto..
Manual on radioimmunoassay in anim reprod. Join FAO-IAEA Vienna Autria
Mahaputra et al, 1990. Study on reproductive efficiency of cattle using radioimmunoassay

techniques. Proc final research coordination meeting, join FAO-IAEA vieena Austria
Mahaputra l, dan K. suhadi 1993. Kadar estosteronurin pagi dan siang haripada pria menikah
dan belum. Media IDI. 18

Preparing sample and sampling technique

Preparing samples
Plasma
Serum
Sperm
Urin
faeces
Product cells
Milk

The others

Fruits
leafs
Organs
Hormonal quantitative analysis (RIA, IRMA, ELISA)
Blood plasma/serum/milk/semen
Product cells, Organ bodyfluid, Feaces, Urine,All species including plant or fruit

Blood plasma/serum

Blood plasma: how to get?

Mix with 1-2% EDTA or heparin (w/v,v/v)
Centrifuge 100xg for 20 mnts
Separates it out the upper part with the transf. yellowyis collourand pellet at the bottom part
Fibrinogen included in this fluid

Blood serum, how to get?

Fresh blood put into plain tube

Following 2 hrs collection puncturing 4-5 point facing with around glass wall
1-2 hrs after puncturing, centrifuge 1000Xg for 20mnts

Centrifugate need a not clarified?

Collect all transf yellowyis fluid as serum at upper and pellet at bottom part

Milk sample

5ml fresh milk from mamma

5 drops 11 % potassium bechromate
Mixe vigorously for homogeny
Centrifuge 15mnts 1000Xg
Upper part is cream unused
Bottom part is defected milk kept frozen pending assay

Why skim milk

Much moresimilar withblood serum hormones concentration

Hormones soluble or nor soluble water existing in skim milk
The skim milk more difuse particle content
Independent on fatty variation individual or breed milk product production

Why not whole milk

Difficult to make difuse solution , because of two different texture

Difficult to prepareduring assay because quite often quagulationin the pippet tip
Those make wide variation concentration eventhought the same sample

Why not cream milk

As well as known individually, breed and species having a lot variation fat content in milk
Event in the same reproductive phase in order have to behormone concentration almost the
same, but found much variation is due to cetainsoluble hormone in cream

Semen sample

1-2ml semen sample centrifuged 1000XG for 15 mnt

0,5-1ml pick up upper part by pipetting into 1,5 vial
Kept frozen pending assay
The bottom part/pellet not used
Mainly for detection of testosterone and PGF2@
Hyperprolactinemia is not sensitive into this sample

Sperm evaluation

This evaluation involving on

Semen volume
Semen PH
Concentration of spermatozoa
Mass movement
Individual movement
Live percentage
And resistency

Product cells

Cumulus cells, how to prepare?

Aspirates the cell by puncturing ovary connected tissue at around target follicle with 17-19G

needle that connected 5ml syrine consisted with 1 ml OWS

Repeat 3-5 follicle aspirate then collect into glass tube in water bath 39C
Rinse with TCM199 and cells selection by repipetting the cells
At each rinsing stage centrifugation1000Xg is done for 20mnt
Adjust the cells concentrations
10UL pellet +90 UL TCM199 and then
by pipetting 100 UL this mixture +900 UL TCM 199>>> found 1.5x 10 6 /ul
pipette into petri disk or polyesterene TC bottlewith or without mineral oil
let the cells in 5% CO2 incubator 38,5 C for 2-3 days and change media
product cells settle as sample depand on cells age porposed (3,6,9 days or passage method)
vesicular seminalis gland for PGF2@
prepare same with oviductal cells
using enzyme trypsin 0.025% for 10-15mnt
wash with TCM199 twice and to obtaind certain concentration
diluted with the same technique already mention at preparing urine and faeces samples
urine for quantitative analysis
extracted using 1 vol samples: 5 vol. petroleum benzene
vortex for minm 3 mnts
allow frozen the pellet at the freezing mechine for 15mnts
paw the filtrate consisted hormone into glass assay tube
the filtrate put into waterbath 380 C and blow with air to each hole surface of the sample in the

tube to arise evaporation

hormone tube attached inside wall of the assay tube
at day of assay dilute the sample with zeroserum of porposing hormone in the same volume at

commencement
faeces sample

homogenized I,0g normal faeces consistency with 1 ml NAcl physiol. >>>> centrifuge 1000Xg

15
pick up supernatant and add with petroleum benzene with proportion 1:5
vortex for 3MNM
put into freezer for 15
the pellet freezing at bottom
paw it out filtrate into assay tube
evaporation the filtrate by using waterbath 38 0c and air blowing pump into each hole surface of

the assay tube

the hormone contents to be attached inside wall of the assay tube
add the sample with the same volume at commencement extraction with serum in zero
concentration of purposing hormone

if the faeces too solid, increase the dilution in proportion 1G:5ml during homogenizing sample
in the calculation, the result should be multiply by 5

meat, organ or fruits and leafs

meat or organ should be directed balanced 1 Gram in fresh condition

slice to small portion and homogenized into uncontaminated marbel bowl
still in homogenizing process step added with 4ml physiol saline
then centrifuged 100XG for 15
filtrate pawed into assay tube and pallet through it out
the filtrate ready as a sample hormone analysis
the concentration IN gram/ml must be multiply by 4, was due to 25% solution was made
for fruit and leaf

the sample can be done with two way

as fresh sample, straightly processed
dried first under indirect sun light up to dry more less 90% drop the weight
techniques, duration andsampling time

to monitoring of cycle
progesterone depend on species
twice a weeks for duration twice cycle
in cows 2x21 D
primate and women 28X2 D

to monitoring ovulation

progesterone 5th to 10 D following estrus/fertile period

estrogen at the time of starting estrus day 0 day 14 of women cycle repeat 12 hrs
LH starting at estrus every 20 to 30 minutes for 6-12 hrs

To monitoring of axis hypothalamus hypophyisis test

Following treatment the sample should be withdrawn at least every 30, much better in 20

interval
Therefore the minimal and maximal fluctuation concentration can be differentiated
Let say GnRH against LH or FSH

Be phasic hormones concentrations

Endorphine as stress indicator

The sample should be withdrawn at early in the morning and at noon
As evaphoria indicator the same schedule sample should be withdrawn
Dont withdrawn sample beyond that schedule in same purpose

Prolactine, testosterone, cortisol, ACTH

All this mentioned hormone above classified into biphasic concentration

Dont collect the samplefor the same purpose in different time extrame

Because gradually the concentration low at noon and high early morning time

Impact of bephasic testosterone

Most of adult man will be felt and occur erection in early morning much frequent than at noon

In bull that why semen collection in al staion performed at morning time

That mean lobido much strong at morning than at noon

Impact of bephasic prolactine concentration

At morning milking time always collected more quantitatively milk than at noon milking time
Physiologically this hormone will be decrease in order of postpartum period
Over lower than cut off point concentration will be releasing of gonadotropin

Prolactine impact effect and defect (1)

Effect positive felt by dairy farmer because arise this hormone mainly in early morning effected

increasing of milk production

This positif effect also felt for fresh mother just interm, expected the milk production increasing
for baby nursing

Prolactine impact effect and defect (2)

In couple just now marrie this increasing to be effect on sperm production. High concentration

in long time to be depress of gonadotropin hormone well as FSH and LH >>> infertile
Contrary for fertile male. By increasing of prolactin as a technique of contraceptic

Impact of cortisol biphasic concentration

At morning this hormone higher concentration than at noon
This evident shown that during activity and at working time this cortisol increase depend tensity
of work the concentration than freshly after weak up at morning much higher
Testosterone norm value (ng/ml)
Species
times
Morningnoon
th
Man 20-49
absolute 24hrs
265mol
g keliatan
1. Stallion
24
g keliatan
2. Bull
g keliatan
g keliatan
3. Goat
g keliatan
g keliatan
(1,2,3 Mahaputra dkk, 1999)
Urine testosterone

Morningafternoon
Bachelor
350 ng/dl
360ng/dl
(20-27 y)
Married 265ng/dl
206ng/dl
(25-35y)
Mahaputra &soehadi (madia IDI 1993)

Norm values of estrogen (pg/ml)

Female
Early folic
30-100
Late folic
100-400
Pregnant up to
35000
Postmenopause <18
Prepubertas
<10
LH normal values
hFemale
mIU/ml
Folic phase
0.6-6.2
Midcycle
12-50
Postmenop
11-50
Adult hmale
0.4-5.7
Cow estrus
50-100
Adult bull
10-50
FSH normal values
Hfemale
mIU/ml
Folic phase
3,3-8.8
Midcycle
5.4-20
Luteal
1.6-8.7
Postmenop
42-126
Progesterone normal values
Human
ng/ml
Female
Folic phase
0.15-1.4
Luteal phase
1.6-2.1
Midluteal phase 5.2-21
Pregnant 1st threeM
7-70
Postmenop
0.1-0.9
Progesterone normal values
Mare and cow
ng/ml
Folic phase
0-0.4
Luteal phase
0.75-25
Pregnant
1.5-20 (mare)
1-2M
1.8-3.8
3-4M
8.4-9.5
5-6M
9.5-19
8-9M
6.3-10.6
Prolactin normal values
Female
ng/ml
Npregnt
ND-20
rd
3 threemester
85-232
1d pp
25-319
Male
ND-15#
#careful if higher infertility

Low milk production early.pp

There are related to the oxytocine at commencent let down
2nd ly PRL hormones is the independent variable
In pp.mother a lot of leaf extracts be able and available commercially in drug store
Most of those herbal mechanism of action to be stimulate releasing of PRL hormone
In dairy cattle and mare by administration of tx haloperidol 35mg/D for 3D successfully to arise
milk production
Carefully with the dose (tx/pxychosis, g keliatan, g keliatan)