OPTIMAL RATIO OF MALE AND SEXUAL FEMALE IN THE MATING OF MOINA MACROCOPA (CRUSTACEA, MONIDAE)

Moina macrocopa is a natural feed that can be cultured to producing ephippia as a bioproduct for fish and shrimp larvae feed. The number of males in the population affects the quality and quantity of ephippia produced. This study was conducted with the purpose of examining the female age in M. macrocopa mating and examining the ratio male-female M. macrocopa in the mating on the quantity and quality of ephippia produced. The treatment in this research was the ratio male-female sex of 1:30, 3:30, 5:30, 7: 30. 9:30., 12:30, 15: 30. Male and female sexual offspring M macrocopa were produced from cultured using a combination of induction factors such as density, feed concentration, kairomones and dissolved oxygen. male and female offspring produced were cultured with a density of 1000 ind/ L. This mating culture was using a container with a volume of water of 2 ml per individual. The results of this study were indicated that mating M. macrocopa using 70-hour old sexual females resulted in the highest ephippia production. Mating M. macrocopa with a sex ratio of 9:30 (male and female sexual) ware resulted in ephippia containing two eggs of 100%, with ephippia hatching degree of 35.438.3%. Keyword : culture, hatching, ephippia, eggs, ricebran ةيقارعلا ةيعارزلا مولعلا ةلجم 2021 : 52 ) 2 :) 471 478 نورخَاو كرابم ةبسنلا ىلثملا روكذلل ثانلإاو يف جوا زت انيوم ابوكوركام ( تايرشق ، يادينوم ) كرابم يدسجلا دز روينوج يدويربوس 1 مسق . ةيرحبلا ، ةيلك دياصملا ةيرحبلاو ، ةعماج Airlangga ، مرحلا يعماجلا C Mulyorejo Surabaya 60115 ، ةواج ةيقرشلا ، ايسينودنإ 2 مسق شاوكأ ، ةيلك دياصملا مولعو راحبلا ، روجوب كيرجأ . ةعماجلا ، Kampus IPB Dramaga. روجوب 16680 ، ةواج ةيبرغلا ، يسينودنإ صلختسملا Moina macrocopa وه فلع يعيبط نكمي هعا رزتسا جاتنلإ تابن ايبيفلإا جتنمك يويح ةيذغتل تاقري كامسلأا نايبورلاو . رثؤي ددع روكذلا يف ناكسلا ىلع ةدوج ةيمكو ايبيفلإا ةجتنملا . تيرجأ هذه ةسا ردلا فدهب صحف رمع ىثنلأا يف جوا زت M. macrocopa و صحف ةبسن روكذلا ىلإ ثانلإا M. macrocopa يف جوا زتلا ىلع ةيمك ةيعونو ephippia جتنملا . ناك جلاعلا يف اذه ثحبلا وه ةبسن سنجلا نيب روكذلا ثانلإاو نم 1:30 ، 3:30 ، 5:30 ، 7 : 30 . 9:30 . ، 12:30 ، 15:30 . جتني نم تاتبنتسملا مادختساب ةعومجم نم لماوعلا ةيثحبلا لثم ةفاثكلا زيكرتو فلعلا تانوموريكلاو نيجسكلأاو باذملا . مت عا رزتسا روكذ ثان او ةيرذ ةفاثكب 1000 نودنإ / رتل . مت مادختسا ءاعو هب ءام مجحب 2 لم لكل درف . تراشأ جئاتن هذه ةسا ردلا ىلإ نأ جوا زت M. macrocopa مادختساب ثانإ ةيسنج رمعب 70 ةعاس ىدأ ىلإ ىلعأ جاتنإ ايبيفلإل . جوا زت M. macrocopa ةبسنب سنج 9:30 ( روكذ ثان او ) جتن هنع ءاوتحا ايبيفلاا ىلع نيتضيب ةبسنب 100 ? ، ةجردو سقف ايبيفلاا 35.4 38.3 ?. ةملكلا ةيحاتفملا : ةفاقثلا ، سقفلا ، ايبيفلاا ، ضيبلا ، ةلاخن زرلأا Received:12/3/2020, Accepted:26/6/2020 Iraqi Journal of Agricultural Sciences –2021:52(2):471-478 Mubarak &et al. 473 INTRODUCTION The rising price of Artemia cysts makes Moina one of the natural food choices for fish and shrimp larvae, due to its body size (± 400 um) and nutritional value comparable to Artemia. Ephippia M. macrocopa can be stored for up to 70 years and hatched at any time in a relatively short time (18). The production of ephippia M. macrocopa is still constrained in terms of both quantity and quality ephippia, which is currently produced has low hatchability. One effort to increase the quantity and quality of ephippia is by regulating the sex ratio of male females in the mating of M. macrocopa. Male and female sex ratios in Moina mating affect the success of conception, the number of ephippia containing an embryo and the number of ephippia containing two embryos. Ephippia M. macrocopa only produced by female sexual offspring who do not undergo reproductive parthenogenesis (2). Production of male offspring and ephippia cladosera can be induced by the quality and quantity of feed (10), population density (22) , temperature, water quality and fish kairomone. Our previous results showed that male and female sexual M. macrocopa can be produced from culture by combining several induction factors simultaneously including density, the concentration of ricebran suspension and carp feces (kairomone) (15). Cultutre of M. Macrocopa without aeration with a parent density of 660 ind/L using ricebrain suspension of 45-54 μg/ parent resulted in the production of M. macrocopa male offspring (253-263 ind/L.) M. macrocopa culture used aeration with a parent density of 660 ind/L, ricebrain suspension of 56-80 μg/parent, goldfish feces 3.86 mg/L was produced the females with the highest ephippia production (6069 grains/ L). Conde et al. (5) stated that if the egg cells (resting eggs) from the sexual female M. macrocopa are not fertilized by the males than it will be described as ephippia without eggs or (the eggs is) damaged. The quantity and quality of males affected the amount of ephippium fertilized in ephippia or the number of ephippia containing two eggs (4). Higly number male in population will increases the number of sexual females being married (21). According to Leung, (12), he reported that the sex ratio of male and female M. australiensis in mating is 4: 5. With this research, an optimal ratio of sexual males and females will be obtained in the mating of M. macrocopa which is useful for increasing the quantity and quality of M. macrocopa ephippia that being produced. This study aims to examine the sex ratio of male and female age on the quantity and quality of ephippia M. macrocopa produced. MATERIALS AND METHODS Research design This study uses a completely randomized design (CRD), which consists of two substudies. First are the effective age of female sexual offspring induced in M. macrocopa mating and the second is the sex ratio of males and sexual females which is as follows: A). 1:30, B). 3:30, C). 5:30, D). 7: 30 E). 9:30. F). 12:30 and G). 15:30. Production of the male and sexual female offspring M. macrocopa. M. macrocopa sexual females were produced from a culture with a parent density of 660 ind/L using aeration (28 ml/min), ricebran 5680 μg/parent suspension, 3.86 mg/L goldfish feces. M. macrocopa male offspring were produced from a culture with a parent density of 660 ind/L and using a ricebran-45-54 μg/parent suspension. The males and females are obtained on the third day and separated from the mother by screening. Offspring was cultured with a density of 1000 ind/L with ricebran suspension feed that supports the production of ephippia (22). During the rearing period, M. macrocopa fed with ricebran suspension concentrations, as in Table 1. The cultured M. macrocopa was conducted in a confined space with daytime lighting of 700-900 lux and 50 -100 lux nights. On the second day (24-hour age) identification and separation of males, especially in the culture for the provision of sexual females. Male are kept until 3 days (72 hours) before being used in mating culture. Iraqi Journal of Agricultural Sciences –2021:52(2):471-478 Mubarak &et al. 473 Table 1. Ricebran suspension concentrations for induction of production of males, sexual females and the culture of M. macrocopa offspring Day Ricebran Suspension (mg/L)


INTRODUCTION
The rising price of Artemia cysts makes Moina one of the natural food choices for fish and shrimp larvae, due to its body size (± 400 um) and nutritional value comparable to Artemia. Ephippia M. macrocopa can be stored for up to 70 years and hatched at any time in a relatively short time (18). The production of ephippia M. macrocopa is still constrained in terms of both quantity and quality ephippia, which is currently produced has low hatchability. One effort to increase the quantity and quality of ephippia is by regulating the sex ratio of male females in the mating of M. macrocopa. Male and female sex ratios in Moina mating affect the success of conception, the number of ephippia containing an embryo and the number of ephippia containing two embryos. Ephippia M. macrocopa only produced by female sexual offspring who do not undergo reproductive parthenogenesis (2). Production of male offspring and ephippia cladosera can be induced by the quality and quantity of feed (10), population density (22) , temperature, water quality and fish kairomone. Our previous results showed that male and female sexual M. macrocopa can be produced from culture by combining several induction factors simultaneously including density, the concentration of ricebran suspension and carp feces (kairomone) (15). Cultutre of M. Macrocopa without aeration with a parent density of 660 ind/L using ricebrain suspension of 45-54 µg/ parent resulted in the production of M. macrocopa male offspring (253-263 ind/L.) M. macrocopa culture used aeration with a parent density of 660 ind/L, ricebrain suspension of 56-80 µg/parent, goldfish feces 3.86 mg/L was produced the females with the highest ephippia production (6069 grains/ L). Conde et al. (5) stated that if the egg cells (resting eggs) from the sexual female M. macrocopa are not fertilized by the males than it will be described as ephippia without eggs or (the eggs is) damaged. The quantity and quality of males affected the amount of ephippium fertilized in ephippia or the number of ephippia containing two eggs (4). Higly number male in population will increases the number of sexual females being married (21). According to Leung,(12), he reported that the sex ratio of male and female M. australiensis in mating is 4: 5. With this research, an optimal ratio of sexual males and females will be obtained in the mating of M. macrocopa which is useful for increasing the quantity and quality of M. macrocopa ephippia that being produced. This study aims to examine the sex ratio of male and female age on the quantity and quality of ephippia M. macrocopa produced.

Research design
This study uses a completely randomized design (CRD), which consists of two substudies. First are the effective age of female sexual offspring induced in M. macrocopa mating and the second is the sex ratio of males and sexual females which is as follows: A). 1:30, B). 3:30, C). 5:30, D). 7: 30 E). 9:30. F). 12:30 and G). 15:30. Production of the male and sexual female offspring M. macrocopa. M. macrocopa sexual females were produced from a culture with a parent density of 660 ind/L using aeration (28 ml/min), ricebran 56-80 μg/parent suspension, 3.86 mg/L goldfish feces. M. macrocopa male offspring were produced from a culture with a parent density of 660 ind/L and using a ricebran-45-54 μg/parent suspension. The males and females are obtained on the third day and separated from the mother by screening. Offspring was cultured with a density of 1000 ind/L with ricebran suspension feed that supports the production of ephippia (22). During the rearing period, M. macrocopa fed with ricebran suspension concentrations, as in Table  1. The cultured M. macrocopa was conducted in a confined space with daytime lighting of 700-900 lux and 50 -100 lux nights. On the second day (24-hour age) identification and separation of males, especially in the culture for the provision of sexual females. Male are kept until 3 days (72 hours) before being used in mating culture. Ricebran suspension made using an amount of 100 grams of the ricebran was suspended in 500 ml of water using a blender at a speed of 2000 rpm for 5 minutes twice. The second suspension was done 30 minutes after the first suspension. Then the suspensions were filtered using 2 mm, 0.1 mm and 40 µm filters. The suspensions that passed the filtration then had more water added to reach a volume of 500 ml. The results of proximate analysis of the ricebran suspension contained 74 mg/ml organic materials, 0,83% proteine, and 0,79% fat (14). Note: n is the number of eggs in the ephippia During the culturing period, the water quality was assessed for its dissolved oxygen, pH, temperature and total ammonia and hardness Ephippia hatching rate assessment using ephippia M. macrocopa containing two eggs was stored wet in a microtube containing of distilled water at a density of 200 eggs/ml. Ephippia from each treatment after being stored for two months, incubated in a glass container containing 300 ml of water with a lighting intensity of 1800 lux (10). The hatching M. macrocopa was removed and counted on the second and third days. The degree of hatching ephippia was calculated based on the equation in Haghparast et al. (12) where Ii is the hatching index and Ni is the number of larvae that hatched.

Data Analysis
The observational data did not show good homogeneity so that Kruskal Wallace was tested (nonparametric) to determine the treatment with the best response at a 95% confidence level.

RESULTS AND DISCUSSION
Age and percentage of sexual females/ephippia Nurture of female offspring of 70-hour-old female stock in a marital container produces the highest percentage of ephippia production in the total female population of 89%. Nurture of female directly induced into the mating container produces females with a percentage of ephippia production of a total female of 2% (Figure 1.). Ephippia is produced by M. macrocopa at the age of four days to six days with the most production at the age of five days. The number of males in mating does not affect the number of sexual females that produce eggs but does affect the number of fertilized ephippia (Fig. 3A). The mating of M. macrocopa using male and female sex ratios of less than 7:30 results in ephippia containing two eggs of less than 60%. The mating of M. macrocopa with a male and female sex ratio of more than 9:30 produces ephippia containing two eggs at 100% (Fig. 3B). Male and female sex ratios in M. macrocopa mating affect the degree of hatching of ephippia being produced. The degree of hatching of ephippia from a mating using a 70hour-old female at the mating ratio of 9:30, 12:30 and 15:30 was not significantly different by 36,17%, 38,26% and 36,67% respectively. The degree of hatching of the lowest ephippia came from matings with a 1:30 sex ratio of 9.92% (Figure 4.). Water quality during the study which included temperatures ranging from 28-30 ºC, water pH 7,5-8, with hardness of 55 mg/L. Dissolved oxygen at the beginning of the study was 4 mg/L and decreased at the end of the study to 2,5 mg/L. Total ammonia after 24 hours of maintenance is less than 0,250 mg/L as shown in Table 3.A Female offspring of M. macrocopa induced by sexual females remain as sexual females and producing ephippia while maintained at low densities (30 ind/60 ml) when they are 70 hours old. Our observations also show that the sexual female M. macrocopa begins to synthesize its sexual eggs at 75-80 hours of age. M. macrocopa cannot transition from asexual reproduction into direct sexual reproduction, but female parthenogenesis M. macrocopa can produce sexual females and males from the development of eggs parthenogenesis to producing ephippia (6). Female child M. macrocopa becomes sexually reproductive when it fails to reproduce the first parthenogenesis (2). M. macrocopa used in this study was first given birth at 65 hours.
Efforts to increase the production of sexual females can be made by maintaining M macrocopa broodstock by regulating feed concentration and density and using kairomone (15). Furthermore, the female child was raised with high density (1000 ind/L) (22) with the regulation of feed concentration.. The results of this study indicate that M macrocopa females transitioned as sexual females at around 70 hours of age so that they can be used for marital testing. Moina mating consists of three phases beginning with the male capturing sexual females, then followed by male movements to position themselves perpendicular to sexual females, then copulation lasts for 16-25 seconds (8). The behavior of male M. macrocopa in mating differs from Daphnia, which only attacks sexual females in the condition of synthesizing eggs in the ovaries and has not been released in the incubation chamber (9). The duration of mating for daphnia and Moina lasts for 8-10 minutes (9) The results of this study show that the number of males in mating does not affect the number of sexual females that produce ephippia, but does affect the number of perfectly fertilized ephippia (containing two eggs) (Figure 5.). Conde et al. (5) state that, egg cells (ephippium) from M. macrocopa sexual females, if eggs are not fertilized, will be released as ephippia without eggs or damaged. Mating M. macrocopa with a low male sex ratio produces ephippia without eggs or high damaged eggs. Mating of M. macrocopa with male and female sex ratio 9:30 -15:30 produces ephippia containing two eggs at ± 100%. The sex ratio of male and female sexual M. macrocopa 9:30 is the lowest mating sex ratio that produces ephippia containing two eggs at 100%. According to Leung (12), the sex ratio of male-female M. australiensis is 4: 5. In our study, the sexual females used began to produce ephippia at the age of four days (96 hours) to six days. The production of ephippia that is not simultaneous causes the ratio of male and female sexual in this study is lower than the results of research Leung (120 on M. australiensis. The quality and quantity of feed affect the ability of male sexual activity, among others, the length of copulation so that it affects the probability of fertilization is higher (4). Male and female sex ratios in M. macrocopa mating affected the degree of hatching of ephippia produced. In matings with female sex ratios of 9:30, 12:30 and 15:30 produce no different degree of hatching (36,17-38,26%). The hatchability of Moina's ephippia is influenced by several factors, including irradiation, temperature, pH, and duration of storage (Stross, 1966). Light intensity and pigment concentration in ephippia skin can cause variations in the value of hatching degrees, where pigments in ephippia skin function to protect the embryo against UV radiation, so a decrease in pigment concentration during storage will increase sensitivity to light at the time of hatching (16 ). In this study, M macrocopa was given ricebran suspension feed. The low hatchability of ephippia M. macrocopa with bran suspension feed is due to the low content of EPA fatty acids and α-linolenic acid (0,20 -0,27%) in ricebran (7). The concentration of EPA in feed affects the production of ephippia (1) and also the degree of hatching (4 , 19).. Some species of cladoceran are reported to be able to convert α-linolenic acid to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with varying abilities between species (13). Bran also contains high linoleic acid (6,35 -6,85%). Linoleic acid can be converted to arachidonic acid and docosapentaenoic acid which can be oxidized to DHA (3). The results of the research of the female-male sex ratio in M macrocopa mating will encourage further research, especially food engineering in order to improve the quality and quantity of ephippia Moina. The availability of Ephippia Moina will improve business sustainability in aquaculture.

Conclusion
The results of this study were indicated that mating M. macrocopa using 70-hour old sexual females resulted in the highest ephippia production. The mating of M. macrocopa with sex ratio male-female of 9:30 (male and female sexual) have resulted of ephippia containing two eggs of 100%, with ephippia hatching degree of 35,4-38,3%.