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DOI https://doi.org/10.18551/rjoas.2021-02.05
THE EFFECT OF FERTILIZATION MEDIA pH ON SPERMATOZOA FERTILIZATION PROCESS AS THE RESULTS OF SEXING ON PANGASIUS (PANGASIONODON HYPOPHTHALMUS)
Adhitomo Yudho*, Astuti Indri
Freshwater Aquaculture Center of Sungai Gelam, Ministry of Marine Affairs and Fisheries,
Jambi, Indonesia
Laishevtcev Alexey
Federal Scientific Center — All-Russian Research Institute of Experimental Veterinary Medicine named after K.I. Skryabin and Y.R. Kovalenko of the Russian Academy of Sciences, Moscow & Laboratory of Biological Control and Antimicrobial Resistance, Orel State University, named after I.S. Turgenev, Orel City, Russia
*E-mail: yudhoadhitomo02@gmail.com
ABSTRACT
Pangasius (P. hypophthalmus) is one of the freshwater commodities of an important economic value. The pH on fertilization media can influence fertilization and affect sex determination (Sandra and Norma, 2009). Sexing with Percoll Density Gradient Centrifugation (PDGC) method can separate X and Y spermatozoa based on their size (Rustidja, 1999). The study analyzed the results of X and Y spermatozoa sexing on Pangasius using the PDGC method and specified the ability of pH in influencing X and Y spermatozoa in the fertilization process. The results showed that, at stage 1, the spermatozoa head on the top layer (La) was larger than that on the bottom layer. The spermatozoa head length (HL) on the top layer was 2.59 ± 0.19 jm with a wide length (WL) of 1.62 ± 0.11 |jm. Meanwhile, on the bottom layer, the HL was 2.27 ± 0.12 |jm with a WL of 1.44 ± 0.07 jm. Therefore, the top layer was identified as X (female) and the bottom layer as Y (male). The average value of spermatozoa concentration after PDGC at the top layer was 7.11 ± 1.743 x 107 cells/mL and at the bottom layer was 3.57 ± 1.075 x 107 cells/mL. The average value of motility at the top layer was 61.67 ± 7.637% and at the bottom layer was 46.67 ± 5.773%, and the average value of viability at the top layer was 61.04 ± 7.27% and at the bottom layer was 55.17 ± 2.53%. This study revealed that the effect of pH on the fertilization rate (FR) with Y control spermatozoa was -177.8 + 68.377 x -4.234 x2, R2 = 0.603. The finding confirmed that the best treatment for this control was pH 8.07 and FR 98.27%. The assessment on Y top layer spermatozoa resulted in of -100.27 + 40.836 x - 2.568 x2, R2 0.637. The finding showed that the best treatment for the top layer spermatozoa was pH 7.95 and FR 62.07%. The examination on Y bottom layer spermatozoa generated an equation of -32.195 + 22.746 x -1.4619 x2, R2 = 0.523. It can be said that the best treatment in the bottom layer was pH 7.78 and FR 56.28%. However, the hatching rate (HR) to pH treatment on each treatment did not show significant differences (P >0.05). To sum up, the PDGC method on Pangasius spermatozoa could separate the spermatozoa based on the top and bottom layer size. The PDGC method could produce female seeds on the top layer by 85% and male seeds on the bottom layer by 68%.
KEY WORDS
Fertilization, Pangasius, pH, spermatozoa.
Pangasius (Pangasionodon hypophthalmus) is a freshwater commodity that has a significant economic value in Indonesia and other Asian countries (Legendre et al., 2008). A method that can produce specific sex from the early stage of rearing (larvae/seed) will create cost-efficiency in feed, ponds, and cultivation labor.
Producing specific sex is crucial and is the main target in aquaculture either for commercial or efficiency purposes because it affects reproduction, growth, and product quality (Alyssa et al ., 2015). It has been noted that the growth rate of female Pangasius is 25-30% faster than the male Pangasius (both P. hypopthalmus and Pangasius djambal) (Arfah and Carman, 2008; Slembrouck et al., 2003). The results from a previous study pointed out that sex determination in teleost fish depends on genetic and environmental factors (Sandra and Norma, 2009). In this matter, genetic factors are influenced by Chromosomal Sex Determination/CSD (Alyssa et al., 2015).
Besides genetic factors, an environmental factor, the acid-base atmosphere (pH) at the time of fertilization, also influences sex determination (Sandra and Norma, 2009). X and Y chromosomes have different characters (X chromosomes are resistant to acidic conditions, while Y chromosomes are more resistant to alkaline conditions). If the pH is acidic at the time of fertilization, the seeds will be dominated by males, and if the pH is alkaline, the seeds will be dominated by females. One of the techniques to produce such specific sex is known as sexing. Sexing is a method of separating X and Y spermatozoa (Susilawaty, 1994) done through Percoll Density Gradient Centrifugation (PDGC) (Kaneko et al., 1984). PDGC is performed to make a density gradient/media concentration and precipitate or separate the spermatozoa through a centrifuge. X and Y spermatozoa have different mass and size. Therefore, the Y spermatozoa, which are smaller than the X spermatozoa, can move faster since they have a higher penetration power to enter a solution. The Y spermatozoa will move downwards, while the X spermatozoa will stay on the top layer.
The objectives of this study are as follows:
• To know the sexing results of X and Y spermatozoa through the PDGC method on Pangasius (P. hypophthalmus);
• To determine the ability of pH in affecting X and Y spermatozoa in the fertilization process.
METHODS OF RESEARCH
This study took place in February 2016 at the Laboratory of Fish Reproduction, Faculty of Fisheries and Marine Sciences, Universitas Brawijaya. The spermatozoa separation was done using the PDGC method at the Laboratory of Artificial Insemination Center, Singosari, as one of the Ministry of Agriculture work units. The observation of spermatozoa morphology was carried out at the Zoology-Biology Research Center, Indonesian Institute of Sciences (Lembaga Ilmu Pengetahuan Indonesia or LIPI) in Cibinong, Bogor. The fish seed gonads were observed at the Laboratory of Fish Health and Environment, Freshwater Aquaculture Center, Sungai Gelam, Jambi.
This study used Pangasius broodfish consisting of 6 females and 11 males from the National Pangasius Development Center (Pusat Pengembangan Induk Patin Nasional or PUSTINA), Freshwater Aquaculture Center, Sungai Gelam, Jambi, Ministry of Marine Affairs and Fisheries.
The hormone used in this study was ovaprim (Syndel Laboratories, Canada), a combination of domperidone and salmon gonadotropin-releasing hormone analog. 1 mL of ovaprim contains 0.02 mg of salmon gonadotropin-releasing hormone analog and 10 mg of domperidone (Subagja et al., 2003).
The fertilization media solution was set at a pH of 6, 7, 8, 9, and 10. A mixture of citric acid (C6H807) with distilled water was made to create a pH 6 solution. Whereas, CaOH was mixed with distilled water to make pH 8, 9, and 10 solutions.
The material used to separate spermatozoa through PDGC was tris aminomethane solution. It is a fresh semen diluent that functions as a living medium for spermatozoa because it contains nutrients and energy sources. The composition of tris aminomethane-egg yolk is tris aminomethane 1.363 g, citric acid 0.762 g, lactose 1.5 g, raffinose 2.7 g, fructose 0.5 g, penicillin 0.1 g, streptomycin 0.1 g, egg yolk 20 mL, and NaCl 0.9% as much as 80 mL. Male Pangasius broodfish used in this study weighed 1.5 - 2 kg/fish and aged >1.5
years. An excellent male broodfish will release white spermatozoa from its genital if it is slowly massaged. The spermatozoa obtained were used as material for further examination.
The spermatozoa were retrieved from 3 male Pangasius broodfish using a 1 mL syringe and then mixed and stirred slowly until homogeneous.
The separation of X and Y spermatozoa in this study was done using the PDGC method. According to Rustidja (1999), the PDGC method is used to create a density gradient/media concentration and to precipitate or separate spermatozoa using a centrifuge. There were 6 levels of Percoll Density Gradient taken in this study (10%, 20%, 30%, 40%, 50%, and 60%) centrifuged at a speed of 1500 rpm for 15 minutes. The making of the Percoll media gradient was completed using tris aminomethane-egg yolk.
In this study, the female Pangasius broodfish had mature gonad and weighed 2 - 2.5 kg/fish aged >2 years. The female broodfish to be spawned was selected for its gonad maturity by observing the diameter of the oocyte through biopsy/cannulation.
The hormone-injected dose was 2 x 0.5 mL/kg of fish weight (the first injection was 1/3 of the dose, and the second injection was 2/3 of the dose). The interval between the first injection and the second injection was 6 hours. Ovulation would occur 6 hours after the second injection depending on the temperature of the water (Baidya and Senoo, 2002). The hormone was injected at the right/left dorsal (intramuscular) with an injection angle of 450. Stripping of the female Pangasius is a technique of removing eggs from the fish's stomach by gently massaging (by hand) the abdomen from the head to the genital opening. Before stripping, the fish were given an anesthetic to facilitate the handling and avoid stress on the fish.
Fish eggs were fertilized with top and bottom layers of spermatozoa that had passed PDGC and spermatozoa without Percoll/control (referring to the analysis results in Stage I) on different fertilization media pH (pH 6, 7, 8, 9, and 10). The results were then analyzed further for the fertilization rate (FR) and the hatching rate (HR).
The larvae and seeds were maintained separately on each treatment. The larvae were reared for 15 days and fed with Artemia nauplii 5 times a day (07:00 UTC+7, 11:00 UTC+7, 15:00 UTC+7, 19:00 UTC+7, and 23:00 UTC+7) from the 2nd to 7th day after hatching. After the 8th day, the larvae would be fed with Tubifex sp. until the 15th day. After that, the seeds were reared for 45 days and fed with worm seeds and Tubifex through an ad libitum manner. During this rearing, 50% of the water was removed. The water quality was maintained at 300C with a DO of >50mg/l and pH 7.
The comparison of male and female larvae was implemented using the acetocarmine squash method. The process was started with an acetocarmine solution that was 0.6 grams of carmine powder added with 100 mL of 45% acetic acid. Then, the mixture of carmine powder and acetic acid was heated for 2-4 minutes. After the mixture was cooled down, it was brought to be filtered to separate the coarse particles (Kurniasih et al., 2006). The observation of the sex ratio was carried out on Pangasisus seeds aged +45 days (D45). In this study, 10 samples were taken for each treatment. The stomach of those samples was dissected to get a gonad candidate. The prospective fish gonads were then compared with the whole samples to obtain the sex ratio of the seeds.
RESULTS AND DISCUSSION
In this study, the average body weight of male Pangasius was 1628 gr/fish. This result was different from the research of Slembrouck (2000) that the bodyweight of male Pangasius broodfish (mature gonads) was >2 kg. Besides that, the average testicular weight of the fish was 74.93 gr. This genital weight analysis aimed to calculate the Testicular Somatic Index (TSI) value to describe the level of maturity of the male broodfish.
That would become the weight ratio of the testis/gonad to the total weight of fish. Jamieson and Barrie (2009) mentioned that the ratio of testicular weight/size to body weight was an important variable to determine the level of gamete cell maturity on male fish. In this study, the mean value of TSI was 4.74. The highest TSI value was found to be 7.45 at a bodyweight of 1.525 grams. It was revealed that a heavier bodyweight of 1.911 grams had
the lowest TSI value of 2.98. The average total length and the standard length of male Pangasius broodfish were 53.33 cm and 44 cm.
Table 1 - The Results of Male Pangasius Broodfish Spermatozoa Analysis
Parameter A Broodfish B C Mean
Body Weight (g) 1450 1525 1911 1628.67
TSI (g) 54.30 113.60 56.90 74.93
Somatic Index (%) 3.74 7.45 2.98 4.72
Total Length (cm) 58 59 43 53.33
Standard Length (cm) 43 44 46 44.33
3.34 x 10 9 cells/mL and the lowest was 3.17 x 10 9 cells/mL with an average of 3.07 ± 0.34 x 10 9 cells/mL. In this study, the spermatozoa density value was directly proportional to the value of TSI and motility.
Table 2 - The Results of Male Pangasius Broodfish Spermatozoa Analysis
Parameter A Broodfish B C Mean
Density (cells x 109 cells/mL) 3.17 3.34 2.69 3.07 + 0.34
Motility (%) 80.77 90.13 85.41 85.44 + 4.68
pH 7.6 7.2 7.4 7.40 + 0.20
Viability (%) 78.7 85.43 80.12 81.42 + 3.54
The oocyte observation in this study was finished using sera solution and analysis of oocyte diameter distribution from 5 female Pangasius broodfish. The oocytes of broodfish number 3 were given the sera solution and showed no clot with an even diameter. The oocyte diameter's distribution pattern was between 0.80 - 1.00 mm, meaning that 84.84% of the oocytes were in the state of final gonad maturation. From this result, the spawning could be predicted to succeed after it was administered with hormone by 60 - 80%. Nevertheless, there were only 15.15% of the oocytes with a diameter between 0.55 - 0.75 mm. To sum up, Pangasius number three, with a bodyweight of 4.60 kg, was the best broodfish based on oocyte analysis.
The centrifugation results in this study could be seen on the clear white top layer/1st layer. The spermatozoa were obvious on the 2nd to the 6th layer and started to look clear on the 7th layer.
Figure 1 - Percoll density gradient A. Before centrifugation B. After centrifugation
In this study, the morphometric analysis of Pangasius spermatozoa was carried out by measuring the length and width of the spermatozoa head, both from PDGC treatment and non-PDGC treatment, through "JEOL" JSM-5310LV Scanning Electron Microscope (SEM). The digital data from SEM were further processed using image-J digital data processing software version 1.44 to analyze the size distribution of the spermatozoa.
Table 3 - The Results of Spermatozoa Size Analysis After Centrifugation without PDGC
Layer Length (|jm) Width (jm)
1st 2.53 + 0.21 1.61 + 0.13
2nd (Top) 2.59 + 0.17 1.65 + 0.14
3rd 2.49 + 0.19 1.57 + 0.10
4th 2.52 + 0.17 1.58 + 0.11
5th 2.54 + 0.21 1.59 + 0.11
6th (Bottom) 2.51 + 0.18 1.59 + 0.11
7th 2.56 + 0.21 1.64 + 0.13
The results of spermatozoa head size analysis after centrifugation without PDGC pointed out that the length of the top layer was 2.59 ± 0.17 jm with a width of 1.65 ± 0.14 jm. Meanwhile, the length of the bottom layer was 2.51. ± 0.18 jm with a width of 1.59 ± 0.11 jm.
Table 4 - The Results of Spermatozoa Size Analysis after Centrifugation with PDGC
Layer Length (jm) Width (jm)
1st 2.56 + 0.14 1.58 + 0.10
2nd (Top) 2.59 + 0.19 1.62 + 0.11
3rd 2.75 + 0.21 1.73 + 0.13
4th 2.50 + 0.13 1.58 + 0.09
5th 2.53 + 0.19 1.56 + 0.10
6th (Bottom) 2.27 + 0.12 1.44 + 0.07
7th nd Nd
*nd: spermatozoa not found.
Table 4 above depict that the length of the top layer is 2.59 ± 0.19 jm with a width of 1.62 ± 0.11 |jm while the length of the bottom layer is 2.27 ± 0.12 |jm with a width of 1.44 ± 0.07 jm.
• Top layer spermatozoa
• Bottom layer spermatozoa
3.50
1.00
2.50
3 JX
Figure 2 - Distribution of the spermatozoa head size between the top and bottom layer without PDGC
Figure 2 depicts no significant difference in the distribution pattern between the size of the top and lower layer spermatozoa. On the other hand, the post-PDGC treatment results (Figure 3) below show a highly significant difference. The mean value of spermatozoa concentration in control spermatozoa was 7.34 ± x 108 cells/mL and decreased in the top
layer by 10.32% with a value of 7.11 x 107 cells/mL. As for the bottom layer, the spermatozoa concentration was 3.57 x 107 cells/mL.
In this study, the control spermatozoa had the highest viability of 70.27 ± 5.87%. The viability value of the top layer spermatozoa was 61.04 ± 7.27%. That was higher than the bottom layer spermatozoa, which had a value of 55.17 ± 2.53%.
ojo
0.00
¿¿¡fi*.
• Top layer spermatozoa
# Bottom layer spermatozoa
0,00
0,50
:,oo
2^0
3,00
Figure 3 - Distribution of the spermatozoa head size between the top and bottom layer after PDGC Table 5 - Spermatozoa Concentration after PDGC
Treatment Mean + Std. Dev.
Control 7.34 + 0.144 x 10s
Top Layer 7.11 + 1.743 x 107
Bottom Layer 3.57 + 1.075 x107
The average value of motility in the top layer was 62.67 ± 7.63%, while the bottom layer was 46.67 ± 5.77%.
Table 6 - Spermatozoa Motility after PDGC
Treatment Mean + Std. Dev. (%)
Control 88.33 + 2.88
Top Layer 61.67 + 7.63
Bottom Layer 46.67 + 5.77
Table 7 - Spermatozoa Viability after PDGC
Treatment Mean + Std. Dev. (%)
Control 70.26 + 5.87
Top Layer 61.67 + 7.27
Bottom Layer 55.17 + 2.53
Figure 4 - The results of damage analysis of Pangasius spermatozoa A. Observation of damage
on spermatozoa using SEM with 5000x enlargement. a: The damage of the head membrane. b: The damage of the broken tail. B. Observation of damage on spermatozoa with acetocarmine.
c: Living/motile spermatozoa.
The effect of pH on the fertilization rate of Pangasius eggs on control spermatozoa and post-PDGC spermatozoa treatment in the top and bottom layers is presented in Table 8 below:
Table 8 - Fertilization Rate of Pangasius Eggs on Control Spermatozoa and post-PDGC Spermatozoa
Treatment Spermatozoa
Control Top Layer Bottom Layer
pH 6 81.21+9.72a 52.59+1.28a 51.92+1.68a
pH 7 90.78+0.72a 58.60+2.88a 54.11+1.86a
pH 8 98.99+1.74c 63.76+7.12c 58.46+3.14b
pH 9 96.24+3.26b 58.13+2.72b 52.39+2.12a
pH 10 81.65+11.15a 51.56+1.67a 49.55+3.32a
pH dramatically affects the fertilization rate of Pangasius eggs because it is very influential in regulating spermatozoa motility. It is known that intracellular pH signals pathways in various spermatozoa cell activities. Meanwhile, extracellular pH affects intracellular pH, thereby interfering with spermatozoa activity in the ability to fertilize eggs. The effect of extracellular pH can increase the H+ ions of intracellular pH to bother ATP metabolic enzyme activity. The inhibition of HCO3- and Ca+ influx is essential in regulating spermatozoa motility. Acidic pH will interrupt the intracellular metabolism, which indirectly inhibits ATP production in spermatozoa cells and the signaling pathway of spermatozoa motility. A pH that is too extreme can cause protein denaturation and thus damaging spermatozoa cells. An alkaline pH will trigger spermatozoa motility because HCO3- is needed in the signaling pathway of spermatozoa motility. HCO3- can activate cAMP in activating spermatozoa motility, but extreme pH will certainly inhibit spermatozoa motility.
Based on orthogonal polynomial analysis of control spermatozoa (Figure 5), a quadratic curve was obtained with the equation of y = -177.8 + 68.377 x -4.234x2 with an R2 = 0.6034. From this equation, the best treatment is obtained at pH 8.07 and a fertilization rate of 98.27%.
110
106
Ferti 155 • *
lizat S5
ion a: * 1
Rate 65
(%) so v = ss.3??«-i,>3i>:!
If -O.WM
fS
to *
55
S3
&
Acidity Level (pH)
Figure 5 - Graph of the relationship between pH and fertilization rate of Pangasius eggs
in control spermatozoa
Ferti ■ lizat
Rate
(%)
5. 6 7 & 9 10 11
Acidity Level (pH)
Figure 6 - Graph of the relationship between pH and fertilization rate of Pangasius eggs
in the to p layer spermatozoa
In the top layer of centrifuged spermatozoa (Figure 6), it is displayed a quadratic curve with the equation y = - 100.27 + 40.836 x -2.568x2 with an R2 = 0.637. The results pointed out that the best treatment was obtained at pH 7.95 and an FR of 62.07%.
Seen in the bottom layer of spermatozoa (Figure 7), a quadratic curve is obtained with the equation y = - 32.195 + 22.746 x -1.4619x2 with an R2 = 0.523.
Hatc hing Rate
(%)
y - • 12,195 * 22,746« -1.4619k* fla * 0,523
Acidity Level (pH)
Figure 7 - The relationship between pH and fertilization rate of Pangasius eggs
in bottom layer spermatozoa
The best treatment in this layer is pH 7.78 and an FR of 56.28%. This study showed that the hatching rate of Pangasius eggs on each pH treatment did not have a significant difference (P >0.05).
Table 9 - Hatching Rate of Control Spermatozoa and post-PDGC Spermatozoa
Treatment Spermatozoa
Control Top Layer Bottom Layer
pH 6 31.31 +1.64ns 28.67+1.28a 51.92+1.68a
pH 7 44.39+16.71ns 30.58+2.88a 54.11+1.86a
pH 8 47.75+15.13ns 63.76+7.12c 58.46+3.14b
pH 9 34.77+16.34ns 58.13+2.72b 52.39+2.12a
pH 10 29.14+8.49ns 51.56+1.67a 49.55+3.32a
The PDGC method on Pangasius spermatozoa can separate spermatozoa based on the size in the top and bottom layers. This study discovered that female seeds dominate the top layer by 85%, while male seeds dominate the bottom layer by 68%.
Table 10 - Prospective Gonad Observation
Treatment Female Ratio (%) Male Ratio (%)
Control 59 + 9.90 41 + 9.90
Top Layer 85 + 7.07 21 + 12.73
Bottom Layer 32 + 11.31 68 + 11.31
CONCLUSION AND SUGGESTIONS
Findings confirmed that the Percoll Density Gradient Centrifugation (PDGC) method successfully separated the Pangasius spermatozoa based on the head size. The top layer spermatozoa (P = 2.59 ± 0.17 ^m and L = 1.62 ± 0.14 ^m) was larger than the bottom layer spermatozoa (p = 2.51 ± 0.18 ^m and L = 1.59 ± 0.11 ^m). PDGC method could influence the concentration, motility, viability, fertilization rate, and hatching rate of top and bottom layer spermatozoa and the sex ratio of the seeds produced. Findings proved that the possibility to produce female seeds was larger in the top layer treatment (85%). In contrast, male seeds were found to dominate the bottom layer treatment by 68%.
The effect of fertilization media pH on the fertilization rate with control spermatozoa was described in the equation y = -177.8 + 68.377 x -4.234 x2 with an R2 = 0.6034. As a result, the best treatment, in this case, was pH 8.07 with an FR of 98.27%. In the top layer of spermatozoa, an equation of y = -100.27 + 40.836 x -2.568 x2 with an R2 = 0.637 was
obtained. The findings implied that the best treatment for top layer spermatozoa was pH 7.95, with an FR of 62.07%. On the other hand, the equation for bottom layer spermatozoa was found to be y = -32.195 + 22.746 x -1.4619 x2 with an R2 = 0,523. Thus, pH 7.78 and an FR of 56.28% were the best treatments for bottom layer spermatozoa.
Further studies are suggested to examine this Pangasius cultivation on production scale/mass scale to determine its field performance tests. It is also recommended to explore the specific protein content in X and Y spermatozoa as biomarkers more deeply. Based on the effect of pH on Pangasius eggs' fertilization, the optimum pH for fertilization is at pH 8. Pangasius cultivation centers are advised to find a location with a water source with a pH range of 8, or as an alternative, the water can be treated first with specific techniques to obtain pH 8. This study explains that the success of a fertilization process is determined by the spermatozoa quality and the optimum pH of the fertilization media, and the quality of eggs from broodfish ready to spawn. In this study, the failures occurred several times during the fertilization process because the broodfish were not ready to spawn, so it is advisable to pay attention to the maturity of the gonads by analyzing the oocytes.
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