endocrines _ practical

1. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Glands Morophology
Hypothalmus

2. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed

3. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Thyroid gland

4. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Parathyroid gland
Adrenal gland

5. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Pancreas
Hormone Assay Test
• Many hormones can be measured (Assayed) in the blood to give an
indication of metabolic process and conditions, or “hormone imbalance”
• Hormone assays are blood test – a few millimeters of blood from vein is
requrid .
• Hormone test is give a concentration of specific hormone in the blood
stream .

6. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
• These results are compared to a reference range of “normal” values,
obtained testing well people without symptoms .
• The specific hormone level may be therefore be low, normal or high.
• Each of these results needs to be carefully interpreted by a doctor who
understands the complex interaction in endocrine system .
A high or low level may not indicate disease, and similarly, a normal test
does not always exclude abnormality .
The most common hormone test is:
 Thyroid function tests:
• Thyroid function test (TFT) are a biochemical measure of the amount of
thyroid hormones { Thyroxine (T4) and tri-iodothyronine (T3) } which can
be directly measured, both in their total blood Concentration in the blood
stream, to determine whether the thyroid gland is overactive or
underactive .
• The most commonly performed test is the Thyroid Stimulating Hormone
(TSH), an indicate measure of thyroid hormone levels.
• The secration of TSH is regulated by thyroid hormone levels.
- Thus a high TSH usually means that Thyroid hormone levels are low -
TSH is trying to stimulate the underactive Thyroid gland.
- Low TSH usually means that high thyroid hormone levels from an
overactive gland are suppressing TSH production .

7. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Female Infertility Test:
Female hormonal testing is recommended when irregularities are identified
in menstrual cycle or physical examination. These including estrogen and
progesterone levels, FSH and LH levels, Inhibin, Testosterone and other
androgens, thyroid tests, and Prolactin levels.
• The results of these hormonal tests help determine the best course of
treatment for each individual patient.
Radioimmunoassay
• Radioimmunoassay (RIA) is a very sensitive in vitro assay technique
used to measure concentrations of antigens (for example, hormone levels in
the blood) by use of antibodies. As such, it can be seen as the inverse of a
radiobinding assay, which quantifies an antibody by use of corresponding
antigens.

8. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
• Although the RIA technique is extremely sensitive and extremely specific,
requiring specialized equipment, it remains among the least expensive
methods to perform such measurements.
• It requires special precautions and licensing, since radioactive substances
are used.
• It used usually for the measurement of Blood TSH, LH,FSH and Growth
Hormones .
ELISA
• The enzyme-linked immunosorbent assay (ELISA) is a test that uses
antibodies and color change to identify a substance.
• ELISA is a popular format of biochemistry assay that uses a solid-
phase enzyme immunoassay (EIA) to detect the presence of a
substance, usually an antigen, in a liquid sample or wet sample.
• The ELISA has been used as a diagnostic tool in medicine and plant
pathology, as well as a quality-control check in various industries.
Types of ELISA :
 Indirect ELISA
 Sandwich ELISA
 Competitive ELISA
 Multiple and portable ELISA
 Hormone Assay Techniques ELISA :

9. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed

10. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed

11. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
ELISA PLATE
ELISA Reader

12. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Estrous Detection
Estrus Cycle
•It is the reproductive cycle of female animal other than primates.
It has 4 phases:
1. Proestrus.
2. Estrus.
3. Metesrus.
4. Diestrus.
5. Anestrus.
 Proestrus
• One or several Follicles of the ovary start to grow. Their number is species
specific .
• Typically this phase can last as little as one day or as long as three weeks.
depending on the species.
• Under the Influence of estrogen the lining In the uterus (endometrium)
starts to develop.
• Some animals may experience vaginal secretions that could be bloody.
• The female is not yet sexually Receptive.

13. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Estrus :
• Estrus refers to the phase when the female is Sexually receptive
("in heat”).
• Under regulation by gonadotropic hormones, ovarian follicles mature, and
estrogen secretions exert their biggest influence.
• The female then exhibits sexually receptive behavior, a situation that may
be signaled by
visible physiologic changes.
• Ovulation may occur spontaneously in some species.
 Metestrus :
• This is phase is characterized by the activity of the corpus luteum, which
produces progesterone.
• The signs of estrogen stimulation subside and the corpus luteum starts to
form.
• The uterine lining begins to appear .
 Diestrus :
In the absence of pregnancy the diestrus phase (also termed pseudo-
pregnancy) terminates with the regression of the corpus luteum.
The lining in the uterus is not shed, but is reorganized for the next cycle.

14. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Anestrus :
• Anestrus refers to the phase when the sexual cycle rests.
• This is typically a seasonal event and controlled light exposure through
the pineal gland that releases melatonin .
• Melatonin may repress stimulation of reproduction in long-day breeders
and stimulate reproduction in short-day breeders.
• Melatonin is thought to act by regulating the hypothalamic pulse activity of
the gonadotropin-releasing hormone.
• Anestrus is induced by time of year, pregnancy, lactation, significant illness,
Chronic energy deficit and possibly age.
Estrus Detection
l-Personal observtltion .
•2 times dally for 30 min. )in the morning & In the evening) is essential
 Sings of heat :
• Physical mounting, or "standing heat," occurs within the first 12-18 hours
after the onset of heat(cow)
• Roughened tail-head; if a cow has been ridden by other animals, the half
on her tall-head could be standing up or completely missing
• Dirty streaks and marks on lower hips, sides, or shoulders, In wet Weather
or areas, the forefeet of rider animals may leave muddy traces on the sides
and hips of cows in heat.
• Nervousness and restlessness
• Grouping together; animals coming into heat will usually congregate in
small groups (three to five animals)

15. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
• Swollen vulva; reddening (bright cherry pink), and mucus discharge as an
indicator for estrus
• Bloody discharge at the end of estrus usually indicates a missed heat;
observe this animal for return to heat in 18-24 days .
2- Records
3-Closed circus television (Video Cameras)
4-Temperature Measurement
• There is a drop in temp. 1- 2 days before estrus then it rise again
5-Pedometer
•This device is used along with a computer to
determine how far an animal has walked.
• Animals in heat are usually restless and may
walk long distances searching for bulls.
6- Heat mount detectors (KAMAR).
• These are devices loaded with dye attached above the cow's tail head.
• They are sensitive to pressure and are activated when one animal
mounts another. (dis adv.: may not be suitable because herd management
conditions occasionally may result in false readings, complete loss of the
device and missed heat periods).

16. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
8-Mesarment of vaginal resistance (probe):
• Falling in vaginal electric resistance during estrus (inflammation)
9- Teaser animal (mount (lipido) without conception )
• Treated males (estrogen)
• (Young bulls-treated steers (testosterone)
• treated females ( Estrogen)
• Vasectomized male.
 Vaginal Smear :
• Aim :
• Vaginal smears may be taken to provide information on estrous cyclicity
at various intervals.
• Ovulation occurs at approximately midnight after the pro-oestrous stage,
when the females become receptive to the male.
• The classic stages of the rat oestrous cycle can be designated as estrous
(E), metestrous (M), di-estrous (D) and pro-estrous (P).

17. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Estrous :

18. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Metestrus :

19. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Pro-estrus :

20. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Di-estrus:

21. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Semen Evaluation
 semen Collection:
• Semen was collected from all animals at a frequency
• Frequency of semen collection
- Bull Buffalo. twice a day, 2 days per week.
-Ram, Buck 3-4 times a day for several weeks
-Stallion: one ejaculate every 2-3 days
• Collection of semen was carried out by using artificial vagina.
• Anestrous female was used as Teaser.
•Semen evaluation :
• Immediately after collection, the ejaculates were transferred to the
laboratory and were placed in the incubator adjusted at 37 0
c.
• All surfaces with which the semen get in contact were kept warmed at 37
0
c.
• Semen quality was measured by: volume of the ejaculate, individual
sperm motility, sperm cell concentration, alive and abnormal sperm percent
and biochemical analysis:
I- Physical evaluation:
 Volume of the ejaculate:
it is measured to the nearest 0.1 ml Using a graduated collection cup.

22. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Evolution of sperm density :
Scales Gross appearance Approximate sperm
concentration ( 10
sperm / ml )
0 Clear to cloudy 0 to 200
1 Cloudy to milky 200 to 400
2 Milky 400 to 800
3 Thick milky 800 to 1200
4 creamy 1200 to 1800
5 Thick creamy 1800+
II- Microscopic evaluation:
 Mass motility :
A tiny drop of Semen sample was placed on clean, dry, warm slide and
examined microscopically using thermostatically controlled hot stage
adjusted at 38-40 C .
• Mass of activity of spermatozoa was Scored (0 - 5) according to the
Intensity of the movement as follows:
0= no sperm movement
1= slight tail undulation without forward motion
2= slow tail undulation with slow ot stop and start forward motion
3: forward progression at a moderate Speed
4= rapid forward progression
5= very rapid progression In which cells are difficult to follow visually

23. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
 Alive and abnormal sperm percent :
• Duplicate smears from each freshly collected ejaculate were stained with
Eosin-Nigrosin stain to determine alive (unstained) and morphologically
abnormal sperm percent.
• A total of 200 sperm cell in were examined randomly, 100 in each of two
smears.
• Sperm cell abnormalities were classified into major sperm defects (are
those that have been correlated to impaired fertility) and minor sperm
defects (are those that seem to be of minor importance .
 Sperm concentration :
Sperm-cell concentration per ml was measured by counting the number of
spermatozoa present on both sides of a Neubauer improved
haemocytometer after the semen had been diluted 1:100 in formalin and
then multiplying by dilution factor.

24. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Normal Bull sperms :
Sperms Abnormality:

25. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Two tails – bent tail – coiled tail - Folded tail with pyriform head

26. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Pregnancy Diagnosis
4. Detection of the fetus and fetal membranes:
a. By rectal examination ( large animal)
b. abdominal palpation (small animal e.g. rabbit ,cat ,dog)
c. Ultrasonography: rapid, accurate, determination of fetal gender
Ultrasonography : Picture from lecture

27. Practical endocrine
28 – 4 – 2014
By / Abdalla Ahmed
Abdominal palpation :
Rectal palpation :