Lab Activity II

MANUFACTURE AND EVALUATION OF ANALYSIS ON

FISH SPERM QUALITY

Day : Thursday
Date : 20 September 2018

Name : Isnaeni Rachmawati

Student ID : B1B017036
Group :2
Subgroup : VIII
Assistant : Monica Widianti

LABORATORY OF ANIMAL STRUCTURE AND DEVELOPMENT

FACULTY OF BIOLOGY
JENDERAL SOEDIRMAN UNIVERSITY
PURWOKERTO

2018
 I. INTRODUCTION

A. Aims

The aims of this practical class are to be able doing sperm analysis and determine
the spermatozoa quality of tested animal.

B. Benefits

The benefits of this practical class are we will be able to identify the factors
impacting success of fertilization, and be able to do sperm analysis also to know the
quality of sperm itself.
 II. MATERIALS AND WORK PROCEDURE

A. Materials

The tools that used in this practical class are object glass, cover glass, cavity
slide, dropping pipettes, microscope, tray, paper tissue, toothpick, stopwatch,
haemocytometer, micrometer, 1 mL syringe, 50 mL beaker glass, well plate, and hand
counter.
The materials that used in this practical class are milt of Nilem Fish
(Osteochillus hasselti), physiological NaCl solution or Ringer solution, giemsa or
eosin stain, and aquades.

B. Work Procedures

The work procedures that used in this practical class are:

1. All the tools and materials needed are prepared.
2. Mature male Nilem Fish is lifted from the aquarium, abdomen wall is cleaned
mainly the area around genital pore using paper tissue.
3. Wall of abdomen is massaged gently started from the front of abdominal fin to
genital pore, until thick white liquid like coconut milk is released. That liquid is
called milt (spermatozoa in seminal plasma).
4. The milt obtained is taken out using a needleless syringe which works as a gauge
tool for milt volume. Then milt volume is measured.
5. A drop of milt is put upon the object glass for viscosity testing, a drop on the cavity
slide for sperm motility observation, and the remaining milt is diluted gradually to
calculate the concentration of spermatozoa and for morphological evaluation of
spermatozoa.
6. Milt viscosity testing is done by testing the viscosity of milt using a toothpick, with
spooning-like movements; and changes of milt viscosity is noted.
7. Spermatozoa motility is done by placing milt on the cavity slide, closed with cover
glass and observed under the microscope so clear focus can be obtained, then
aquades is dropped between the cover glass and cavity slide to activate movement
of spermatozoa. This happen because aquades caused the osmolality of seminal
plasma reduced and sperm movement is spurred.
8. The movement pattern of spermatozoa is observed and length of motility and
proportion of motile spermatozoa is noted.
 9. Milt dilution is done inside the well plate. 0,1 mL milt is droppen on the first well
and 0.9 mL of physiological NaCl is added, homogenized by pipetting them
together several times so that a 10x dilution is obtained. Then 0.1 mL of milt from
the first dilution is taken and put it inside the second well, 0.9 NaCl physiological
is added again. Homogenous them to get 100x dilution. Dilution can be done 1000
or 10000x depending on the density level of spermatozoa.
10. Haemocytometer is prepared and cleaned from dust or other dirt. Haemocytometer
is put under the microscope, focused on the calculation chamber that will be used
then covered with cover glass.
11. Diluted milt is dropped on boundary between haemocytometer and cover glass
using dropping pipette. The milt will be spreading to calculation chamber through
capillary movements.
12. Spermatozoa inside the calculation chamber is observed and focus it well. If there
are still solid spermatozoa then haemocytometer include the cover glass are washed.
Then step no 11 is repeated in hinger dilution.
13. The total amount of spermatozoa inside calculation chamber is counted manually.
14. For the morphological observation, diluted milt is dropped on one of the object
glass’s tip, place the object glass vertically creating sharp angle causing the dropped
milt located on the edge of angle. The second object glass is neared until touching
the dropped milt, the second object glass is pushed further away from milt drop so
the milt drop is smeared on the first object glass. Giemsa or eosin tint is given upon
the smeared milt and even it by changing the object glass position. Object glass is
placed on the hot plate, so the rest of stain is poured out and let it dry.
15. Morphology of spermatozoa is evaluated under the microscope, head shape and tail
is observed, also noted on your work sheet.
 III. RESULT AND DISCUSSION

A. Result
a. Macroscopic Parameter
1. Volume : 0.36 mL
2. Odor : fishy
3. Color : milky white
4. pH : 9.0
5. Viscosity : 4 minutes 12 seconds
Tabel 1. Accumulation Data of Spermatozoa Viscosity Observation Entourage
VIII/ D2
K1 K2 K3 K4 Average

4m 4m 8m
Viscosity 9m 1s
12s 16s 44s

b. Microscopic Parameter :
1. Motility :
Tabel 2. Accumulation Data of Spermatozoa Motility Observation Entourage
VIII/D2
K1 K2 K3 K4 Rata-rata

Presentation of Motile
100 70 40 100 77.5
Sperm (%)

Presentation of Non
0 30 60 0 22.5
Motile Sperm (%)

2. Total Amount Of Spermatozoa

Tabel 3. Accumulation Data of ∑ Total Spermatozoa Observation Entourage
VIII/D2
K1 K2 K3 K4 Average
∑ Total
Spermatozoa 16.8x1010 3.28x1010 4x1010 24x1010 12.02x1010
(cell/ml)
Calculation :
Known :
Square 1 = 8 Squara 3 = 6 Square 5 = 12
Square 2 = 6 Square 4 = 9
Answer :
∑ Total of Sperm = average of 5 squares x 4.106 x dilution factor (cell/ml)
= (8 + 6 + 6 + 9 + 12 ) x 4 x 106 x 103
2
= 8,2 x 4 x 109
= 32,8 x 109
= 3.28 x 1010 cell/mL

Figure 1. Calculation Chamber of Haemocytometer

3. Morphology of Spermatozoa

1
2

Figure 2. Microscopic of Nilem Fish Sperm (Osteochilus hasselti)

Magnification 400X

Figure Details :
1. Head
2. Tail

Figure 3. Schematic of Nilem Fish Sperm (Osteochilus hasselti)

Figure Details :
1. Head
2. Tail

Conclusion/diagnose :
Based on the observation, there are two parameters we can observe;
macroscopic and microscopic. For macroscopic there are color, volume, odor,
viscosity, and pH. The milt color we observe is milky white, has volume 0.36 mL,
smells fishy, viscosity 4 minutes and 12 seconds and pH = 9. Meanwhile for the
microscopic observation we determine that the motility is 70%, with total of sperm
3.28x1010 cell/mL and has morphology consist of head and tail.
B. Discussion

Osteochillus hasselti or known as Nilem fish is one of freshwater fishes that

lives either in the river, ponds, swamps, or even lake. Reproductive organ of male
Nilem fish is matured at age + 8 months. Both of their testes can produce 1 to 1.5 mL
milt (in natural ejaculation), but with stripping process commonly only 1 mL can be
released. In this observation obtained 0.36 ml volume of milt. If the volume is less than
1 ml, there is the possibility of abnormalities of the prostate and seminal vesicles is a
major producer of seminal plasma, or because many sperm are wasted during taking
the milt (Yatim, 1992)
The sperm of Nilem fish (Osteochillus hasselti) will be used as experimental
animal in this practical lab activity. That’s because the analysis of fish sperm can be
done with the same procedures as human sperm analysis. Nilem fish itself is very easy
to obtain, and has relatively cheap stain (Soeminto, 2007).
Sperm analysis is the observation about characteristics, quality and quantity of
spermatozoa. Sperm analysis is functioned as determining factors of embryo
development resulted. The sperm itself is cell that produced by male reproductive
organ which responsible to carry genetic information of the male gamete to ovum in
female body. Structurally spermatozoa are adapted to work both function as carrier for
1 set haploid of genetic information and also activate development program in the egg
cell (Sistina, 2000). Milt of fish is taken out using stripping method. stripping process
is done by massaging the abdomen part in front of genital papilla until semen liquid
comes out of the fish (Novianto et al, 2014).
The spermatozoa are released from the spermatocyte into the sperm ducts
where maturation–the process that renders them capable of vigorous motility and
fertilization–takes place prior to sperm release, during the spawning season. Fish
ejaculate sperm spontaneously during spawning, and with the exception of catfishes.
Sperm can also express easily from the sperm ducts after application of gentle
abdominal pressure (referred as stripping). Sperm release can be synchronized with
female spawning via tactile of pheromonal communication (Mylonas et al, 2017).
The color of sperm from stripping on Osteochillus hasselti is milky white, this
indicates that the sperm of the Nilem fish used in the lab is healthy. Based on reference,
Milt has whitish cream or milky white color. Its whiteness or turbidity depends on the
concentration of its sperm. The more feculent is usually the more sperm/mL milt
produced. Milt that are yellowish green usually contain a lot of Pseudomonas
auroginosa bacteria which indicates chronic inflammation in their reproductive tract.
Milt that is red or reddish indicates that it contains little or a lot of blood (Partodiharjo,
1990).
Milt of Nilem fish used has pH 9.0, means it is base. Based on the reference,
Milt has a normal pH between 7.2-7.8. The pH, if more than 8.0 indicates acute
inflammation of the sexual gland or epididymis. A pH of less than 7.2 gives sign to
the presence of a disease. But if the milt shows a very low pH then it means disruption
or aplasia of the seminal vesicles or ejaculatory ducts. pH can change one hour after
ejaculation (Yatim, 1992).
A spermatozoon is an example of a highly specialized cell that has lost most
regular cell organelles during its differentiation. The structure remaining include the
nucleus, mitochondria and some specific structures, such as the flagellum–basal body
complex and, in some species, the acrosome. Flagellum has two main functions; as a
propeller and a rudder, providing motility and orientation respectively. The dimension
of fish spermatozoa is generally smaller than those of spermatozoa of other group of
animals, and most of fish has no acrosome. In most fish species, spermatozoa are
immotile in the seminal fluid. Simple dispersion in an aqueous external environment
or a special activating solution is the only requirement to fully activate the motility of
their flagella. During the period after spermatozoa come in contact with an activating
medium, the ion concentration inside the sperm cell are rebalanced and osmotic
pressure affecting the sperm membranes harmful for sperm integrity, liming the period
of motility to a short interval (Boryshpolets et al, 2018).
Meanwhile milt are spermatozoa that has flagella and no acrosome.
Spermatozoa from testicular suppression are the same with spermatozoa from striping
method. The head is round, diameter about 2.86–0.16 micro meters, a length of about
25.86 micro meters. At the base of the flagella there are buildings such as rings, called
annulus (Jamieson, 1991). The sperm plasma membranes are inevitably damaged, but
motility activation is caused by rapid changes in protein phosphorylation status (Holt
et al, 2018). Sperm shape below is a form of abnormal sperm, according to Jauhari
(2005) :
1. Macro: head sizes larger than normal head size.
2. Micro: the size of the head is smaller than normal head size.
 3. Taper: sperm skinny, wide head half of a normal head, it is not clear acrosome
limit.
4. Piri: no real clear their heads, looked midpiece and tail only.
5. Amorphous: odd head shape, uneven surfaces, no clear boundaries acrosome.
6. Round: head shapes such as circles, showed no acrosome.
7. Cytoplasmic droplet: stick to the head, brighter colors.
8. Abnormal tail: short tail / spiral / surface is not smooth / double.
Solutions commonly used for dilution of sperm fish is NaCl as a buffer,
maintaining the pH of the milt and extend the life (viability) of spermatozoa but milt
storage with physiological saline only can be used for 60 minutes, for the addition of
other materials is needed to be energizing or nutritive, so it can extend the life of
spermatozoa (Barozha, 2015).
Total milt resulted from my group’s observation is 3,28x1010. This amount is
different with other groups because many things can affect the viability of milt. Such
as the way of doing dilution and temperature. Normally the average total sperm of
Nilem fish supposed to be around 3,33x1010/ml. This means that the total spermatozoa
in the fish tested is under average. The number of spermatozoa/mL can be done with
counting chamber (Haemocytometer). The use of Haemocytometer is to determine the
number of spermatozoa in milt. Nilem fish that has total of spermatozoa per ejaculation
is 3,33x1010/ml, compared to total sperm of Goldfish (Cyprinus caprio L.) which has
9,7x109 cell/ml, Nilem fish is proven to produce more than Goldfish (Yatim, 1992).
There are several factors affecting quality and quantity of spermatozoa:
1. Temperature
Metabolic activity and sperm motility will be normal at body temperature. The
optimal temperature for spermatozoa metabolism is at body temperature but even
at lower temperature will give longer sperm motility (Effendy, 1997).
2. Content of Food Substances (Carbohydrates)
Food content substances such as fructose produced by vesicular glands.
Fructose can be used as a source of energy to support the movement and resistance
of spermatozoa because fructose increases the activity of proteins that located in
the sperm tail. Spermatozoa tails are composed of microtubules which contain fiber
substances arranged by dynein protein. Dynein protein is important because it has
ATP-ase activity, which will smooth out the ATP process and also causes sperm
motility increase (Hidayaturrahmah, 2008).
3. The Use of Physiological Solution
Fertilization can be supported by good quality of spermatozoa. Physiological
solutions can increase sperm viability and motility. the use of NaCl physiological
solution can maintain spermatozoa survival between 20–25 minutes
(Hidayaturrahmah, 2008)
 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the result and discussion, it can be concluded that sperm analysis is the
observation about characteristics, quality and quantity of spermatozoa. Sperm analysis
can be done through series of dilution. After dilution, the calculation of sperm using
haemocytometer is needed to count total sperm/ml. Several things affecting the quality
and quantity of spermatozoa such as temperature, food contents and physiological
solution used. From the practical activity, the milt is in healthy condition because
colored milky white, indicating there’s no infection or bleeding, and also the milt is in
average normal quantity.

B. Suggestion

In this practical lab activity, it would be better if students are let to take the milt
from fish directly and not just watching. So the students will understand the procedure
of stripping better.
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