Lab activity II

ANALYSIS OF THE QUALITY OF SPERM FISH

Day : Thursday

Date : September 20th 2018

Name : Mellya Rizki Pitriani

Student ID : B1B017031
Group : VIII
Subgroup :1
Assistant : Nur Hidayati

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 :

1. To analyze sperm fish.

2. To determine the quality of test animal spermatozoa.

B. Benefits
The benefits of this practical class are students can identify factors that
influence the success of fertilization.

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 II. MATERIAL AND WORK PROCEDURES

A. Material
The tools that used in this practice are object glass, cover glass, cavity
slide, drop pipette, microscope, tissue paper, toothpick, stopwatch,
haemocytometer, micrometer, spuit 1 mL, beaker glass 50 mL, well plate, and
hand counter.
The materials that used in this practice are milt of nilem fish, NaCl
physiologis solution / Ringer solution, Giemsa dyes, Eosin dyes, and aquades.

B. Work Procedures

The methods that used in this activity are:

1. Male gonadal mature nilem fish is held then stretched, so that the abdominal
area is above, while the dorsal area is below. The urogenital porous area is
cleaned using tissue.
2. The abdomen is sorted from the front of the abdominal fins (pinna
abdominal) to the urogenital pores so that the milt exits.
3. Milt is accommodated in a 1 ml needle-less syringe, then the recorded
volume of milt is recorded.
4. Milts are dripped on the object glass, the color and smell are observed, then
the pH is measured using a universal pH. After that, milt was tested for
viscosity using a toothpick attached to the milt, then removed, affixed, lifted
again until the viscosity changed. Changes in milt viscosity are measured
using a stopwatch.
5. A total of 1 ml milt was diluted on the first well plate, added 9 ml of
physiological NaCl solution, then homogenized, so that a dilution was
obtained 10 times. As much as 1 ml of milt was diluted 10 times into a
second well plate, 9 ml of physiological NaCl were added, then
homogenized, so that a dilution of 100 times was obtained, and so on until the
milt was obtained 10000 times.
6. A total of 1 ml milt was diluted with 100 times the powder on the cavity
slide, closed using a cover glass, observed under a microscope until a clear
focus was obtained. The drops of water in the border between the cover glass
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 and the cavity slide to activate sperm motility or motility. The addition of
distilled water causes the seminal plasma osmolarity to decrease so that it
stimulates sperm movement. Sperm motility is observed and proportion is
determined.
7. Haemocytometer is placed on a microscope and focused on the counting
chamber and then closed using a cover glass. As much as 1 ml of milt
dilution results 1000 times squeezed at the border of the haemocytometer
with a glass cover. Milt will spread to the counting chamber through capillary
motion. Spermatozoa in the calculated booth were observed, then calculated
using a hand counter.
8. Sperm nilem preparations are placed on a microscope, observed from weak
magnification to strong magnification and focused. After being seen clearly,
the results of the observation in the photo, then drawn, given a description,
and recorded the enlargement.

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 III. RESULT AND DISCUSSION

A. Result
a. Macroscopic Parameters

1. Volume : 0,36 ml
2. Odor : Fishy
3. Color : Milky White
4. pH : 9,0
5. Viscosity : 9 minutes 1 second

Table 1. Accumulation of Spermatozoa Viscosity Observation Data Group VIII

G1 G2 G3 G4 Average

Viscosity 00.09.01 00.04.12 00.04.16 00.08.44 00.06.43

b. Microscopic Parameters :

1. Motility:

Table 2. Accumulation of Spermatozoa Motility Observation Data Group VIII

G1 G2 G3 G4 Average

Percentage of motile
100 70 40 100 77,5
sperm (%)

Percentage of non-
0 30 60 0 22,5
motile sperm (%)

2. Total Spermatozoa

Table 3. Accumulation of Observation Data ∑ total Spermatozoa Group VIII

G1 G2 G3 G4 Average
 ∑ Total
Spermato
16,8x1010 3,28x 1010 4x1010 24x1010 12,02x1010
zoa
(sel/ml)

Calculation :

Is known :

Box 1 = 4 Box 3 = 5 Box 5 = 9

Box 2 = 1 Box 4 = 2

Answer :

∑ Total Sperma = average of 5 boxes x 4 x 106 x dilution factor (sel/ml)

= ((4+1+5+2+9)/5)x 4 x 106 x 103

= 16,8 x 1010

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 . . .

. .

. . .

. . . .

. .

. .

Figure 1. The Haemocytometer Calculate Chamber

3. Morphology of spermatozoa 1

6 2

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

 400X magnification

Details :
Figure 2 : Microscopic Figure of Nilem Fish Sperm (Osteochillus hasselti)
Magnification : 40 X 10
Figure details:

1. Head of Sperm
2. Tail of Sperm

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

Drawing Details :

1. Head

2. Tail

Conclusion/Diagnosis:

Based on the results of observations and calculations milt, it can be concluded that
the pH obtained is 9, milt is milky white, has a fishy odor, and the viscosity value is
9 minutes 1 second. In addition, the accumulation of spermatozoa motility data with
a percentage of motile sperm had an average of 77.5% and a percentage of non-
motile sperm 22.5%. Then, the average total spermatozoa obtained is 12.02 X 10 10
mL so that the sperm obtained is categorized as good.

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B. Discussion
 The fish used by his sperm for practicum analysis of sperm is nilem fish
which has a sperm pH of 9. The pH 7.5 size is normal pH, this is in not accordance
with the results of data from Maulana (2014) who found that sperm from lan fish
were in the range of 7. For example, betok fish with sperm ranges between 7 or 8,
then salmon with pH it's 7.5. The color of the fish used in the lab analysis of fish
sperm is a milky white color and a long shape. These colors and shapes indicate that
the sperm is normal, this is in accordance with Fahmi's (2015) opinion, namely, the
calculation of the number of sperm using Neubauer haemometer and observed under
a microscope with weak and strong magnification. The percentage of normal and
abnormal sperm is obtained by calculating the number of normal or abnormal sperm
compared to the total sperm count. Normal sperm cells are characterized by the body
of spheries and long tails, while abnormal sperm cells are characterized by body
defects such as twisted, short and missing tails. Other fish also have the color of milk
white sperm, for example Tawes (Rizal, M., 2015). Goldfish also have sperm with a
milky white color and thick (Linayati, 2015).

Fish sperm consists of three main components namely head, neck and tail.
The head consists mainly of a solid nucleus crowned with a small crescent shaped
acrosome. The acrosome contains a number of hydrolytic enzymes and is thought to
play a role in the penetration of eggs by spermatozoa (Paxton, 1986). Spermatozoa
heads vary in shape. Its contents are the core (inside it contains genetic material)
haploid in the form of a bag containing hydrolytic enzyme secretions. Spermatozoa
that come into contact with eggs, the contents of the acrosome are released by
exocytosis which is called the acrosome reaction (Sistina, 2000).

Calculation of the number of spermatozoa per mL can be done with a

counting chamber (haemocytometer). Spermatozoa calculations in our group got 16,8
X 1010. The number of sperm obtained by groups2, 3, and 4 is quite far from the one
we got, there are 3,28 X 1010, 4 X 1010, and 2,4 X 1010 . The difference is due to the
less homogeneous NaCl and sperm. NaCl is used to optimize the period of use and
maintain sperm quality after being removed from the fish's body, so retailers can

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maintain the life of spermatozoa. NaCl solution gives buffer properties, maintains
semen pH at room temperature, isotonic with cell fluid, protects spermatozoa against
coldshock and suitable electron balancing (Marthin, L et al, 2018).

Then the difference in sperm counts can also be due to the dirty device, this is
in accordance with Partodiharjo (1990), which is that using a haemocytometer to
determine the number of spermatozoa in Milt is considered to be less practical in the
latest opinion, because it requires a little skill in sucking also takes time in
calculating with a microscope. With the lack of mixing of NaCl and sperm, it can
affect the results. Sperm dripped over the haemocytometer box is closed and
calculated, the results are recorded for example y. This is the number of spermatozoa
cells that die and which look immovable in boxes. Immobile Spermatozoa are not
necessarily dead. It is difficult to determine whether the counts are spermatozoa or
garbage / dirt contained in the haemocytometer.

Visual observation was carried out to the fresh semen/sperm color and sperm volume
was measured by the cryotube with scale. The standardized pH paper (pH range 5-
10) was used to measure sperm/semen pH. The spermatozoa viability, abnormality,
and motility were observed under microscope. The method was used to analyze the
spermatozoa motility, the spermatozoa viability and spermatozoa motility
(Abinawanto et al, 2015).

In fish used in the sperm analysis practicum, it can be produced around 1-1.5
ml milt (in a state of natural ejaculation), but on striping most are obtained 1 ml milt.
This amount is the general number of sperm of some other fish (Soeminto, 2002).

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 IV. CONCLUSION AND SUGGESTION

A. Conclusion

Based on the results and discussion above, it can be concluded that fish used
as test animals have good sperm. Because the color is milky white, the smell is fishy,
and the average motility is greater than the non-motile one.
B. Suggestion
Nilem fish should be used as a new nilem fish, so that the fish used by their
sperm are not stressed and die.

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 REFFERENCES

Abinawanto., Nisa, F., Retno, L., Agung, S., Rita, R., & Yushinta., F, 2015. The
Intracellular Cryoptectant Effects in Preserving Goramy Spermatozoa after
To Days Sub-Zero Freezing. Journal of Aquacultura Indonesiana. 16 (1), pp.
16-21.
Fahmi, A., Agus, O. S., Siti, S., 2015. Kualitas Sperma Induk Litopenaeus
vannamei  Yang Disuntik PMSG Dan Antidopamin. Jurnal Akuakultur
Indonesia. 14(2), pp. 98–103.
Linayati, Basuki, F., Pinandyo, 2015. Efektivitas Penambahan Glyersol Dalam Susu
Pengencer Terhadap Prosentase Sperma Hidup Dan Penetasan Telur Ikan
Mas (Cyprinus carpio Linn). PENA Akuatika. 12(1), pp. 49.
Marthin, L., Ginting, E. L., dkk, 2018. Penambahan Madu Dalam Pengenceran
Sperma Terhadap Motilitas Spermatozoa, Fertilisasi Dan Daya Tetas Telur
Ikan Patin Siam, Pangasius hipophthalmus. Budidaya Perairan. 6(2), pp. 45-
52.
Maulana, F., Alimuddin, Junior, M. Z., 2014. Morfologi, fisiologi, preservasi sel
sperma ikan betok, Anabas testudineus Bloch 1792 dan ketahanannya
terhadap kejut listrik. Jurnal Iktiologi Indonesia. 14(3), pp. 211-223.
Partodiharjo, Soebadi. 1990. Ilmu Reproduksi Hewan. Mutiara Sumber Widya,
Surabaya.
Paxton, M. J. W., 1986. Endocrinology, Biological and Medical Perspetives.
Wm.C.Brown Publishers, Lowa.
Rizal, M., Diana. F., Mariani, D., 2015. PENGARUH PENGGUNAAN
KONSENTRASI AIR KELAPA MUDA PADA PENGENCER NACL
FISIOLOGIS TERHADAP KUALITAS SPERMATOZOA IKAN TAWES (
Puntius javanicus). Jurnal Perikanan Tropis. 2(2), pp.2355-5572.
Sistina, Yulia. 2000. Biologi Reproduksi. Fakultas Biologi Unsoed, Purwokerto
Soeminto. 2002. Pembentukan Ikan Jantan Homogamet (XX) lewat Ginosenis dan
Pemberian Andriol pada Ikan Nilem (Osteocillus hasselti CV). Jurnal
Permberdayaan Pedesaan  6(2) : pp. 1-6.

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 Font 14

REFFERENCES
2 spasi
Pocock, G. & Richards, C. D., 2006. Human Physiology: The Basis of Medicine. 3rd
ed. Oxford: Oxford University Press. 1,5 spasi
Choudhary, P., Sudhamani, S., Pandit, A. & Kiri, V., 2012. Comparison of Modified
1 spasi Ultrafast Papanicolaou Stain with The Standard Rapid Papanicolaou Stain in
Cytology of Various Organs. Journal of Cytology/Indian Academy of
Cytologists, 29(4), pp.241-245.

NOTES :
1. Times New Roman font size 12 except the title and chapter (14).
2. The margin left 4, right, down, up 2.5.
3. Spacing between chapters to Section 2 spaces, spacing between last word
in subbab to next subbab is 2 space, spaces between sentences to the first
paragraph of section 1,5 and the spacing between lines of 1.5 spaces.
4. Paper A4 80 grams
5. The background contains practical reason for the event, when quoting
from journals or books do not forget listed the author and that included
in the reference list.
6. Background consist of at least 3 paragraphs, and each paragraph
consists of at least 3 sentences.
7. In preparing the report using at least 5 text book and 2 journals last 5
years (2014-2018).
8. Discussion contains a comparison between the theoretical and the
practical results of existing research results in journals that are relevant
to practical events.
9. Conclusions based on the results and discussion that refers to the goal.
10. All theories taken from the quote should be in included in the reference
list.