Journal of Coldwater Fisheries 1(1):65-73, 2018 Morphological changes during early development of endangered golden mahseer Tor putitora KISHOR KUNAL, DEBAJIT SARMA, DEEPJYOTI BARUAH, PARVAIZ AHMAD GANIE ICAR-Directorate of Coldwater Fisheries Research, Bhimtal-263136, Nainital, Uttarakhand, India E-mail: [email protected] ABSTRACT Embryonic development of golden mahseer eggs were completed within 94-120 hours after fertilization at an ambient temperature of 20o C ± 2o C. The first cleavage occurs about 3hours post fertilization, with cleavage interval of about 35 minutes. The blastula period last from 6h 45m to 12h 10m post fertilization. During this period; the embryo enters midblastula transition (MBT), the yolk syncytial layer (YSL) forms, and epiboly begins, which continues till gastrulation period. The gastrulation period generally lasts from 16h 30m to 36h. Embryonic development was completed within 94-120 hours post-fertilization at 20º C ± 2º C. The present study provides preliminary insight about the early development progression in Golden mahseer, which can help in developing better rearing strategies for early larval rearing to achieve better survival and eventually leading to population enhancement in natural environment. Keywords: Embryo, Larvae, Development, Golden mahseer Introduction eggs enclosed in a hard and complex eggshell (chorion), which is formed by protein components organized into a Golden mahseer (Tor putitora) is one of the well-known complex structure. This structure plays an essential role in large freshwater game fish of Mountain Rivers and lakes control of the relation between the external and the internal of most Trans-Himalayan countries. It is one of the most- egg environments. It allows for respiratory gas diffusion, sought after species providing the main fishery in the uplands provides mechanical protection and thermal insulation, and all along the Himalayan belt extending from Kashmir in the permits sperm entry (Hamodrakas, 1992). north-west to Sadiya in the north-east. The fish is also known as Greyhound or the thick-lipped mahseer and has been Ultrastructural characteristics of the chorion and the observed to attain the weight of 70-80 kg. (Misra, 1962). micropyle of teleost eggs differ in different species, and have Anglers regard golden mahseer as one of the finest sport-fish recently been considered as a criterion for identification of and it is a source of recreation to innumerable sportsmen eggs (Ohta et al., 1983; Chen et al., 2007). Shape, number both Indian & foreigners. Thomas, 1897 in his famous book and size of micropyle and also reinforcement type of the “The Rod in India” stated that pound for pound mahseer is micropyle canal; the number and length of the longest far superior to ‘lordly salmon’ in sporting qualities. To the and shortest ridge in the micropyle region; the diameter, local fishermen too, mahseers have been of considerable number, and arrangement of the accessory openings; the importance because of their large size, hardy texture, high adhesive structures of egg and mode of them to the substrate; commercial value and longer shelf life. ornamentation and the thickness of the membrane have been used for taxonomic purposes (Hirai and Yamamoto, 1986; An important reason of teleost evolutionary success Riehl, 1993; Giulianini et al., 1994; Chen et al., 1999; Li et is their reproductive system, which has been functionally al., 2000; Bless and Riehl, 2002; Esmaeili and Johal, 2005; acclimated in all aquatic environmental conditions (Koc, Huysentruyt and Adriaens, 2005; Chen et al., 2007; Costa 2010). Reproduction represents one of the most important and Leal, 2009). aspects of the different species biology and the maintenance of viable populations depending on its success (Suzuki Some characters such as shape of the external surface and Agostinho, 1997). Different fish species live in of the egg membrane, number and shape of micropyles, the special ecological conditions; therefore, they have unique branching pattern of the tubules in the primary membrane, the reproductive strategy, with special anatomical, behavioural, width of the membrane and the degree of egg adhesiveness physiological, and energetic adaptations (Moyle and are species typical, although they represent a convergent Cech, 2004). The envelope of teleost fish’s egg has a key similarity in some species (Vorobyeva and Markov, 1999; role in this reproductive success. Teleosts develop mature Huysentruyt and Adriaens, 2005; Costa and Leal, 2009). K. Kunal et al. 66 The chorion surface has also been analysed using Morphological development of embryos was followed scanning electron microscopy (Ohta et al., 1983; Johnson and with the use of Olympus stereomicroscope with bottom and Werner, 1986; Costa and Leal, 2009; Koç, 2010). Johnson top lighting to enable clear observation of organogenesis and and Werner (1986) described the external morphology of to determine the time sequence of different developmental the chorion of five freshwater fishes and concluded that stages. For S.E.M. examination of the embryos, samples of scanning electron microscopy was a powerful tool for each stage of development were fixed in a solution of 23% identifying fish eggs. On the other hand, the micropyle and its glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 and microstructures in unfertilized eggs are important characters stored at 4” C until processing for S.E.M. (Glauert, 1980). in gamete recognition and fish egg identification, therefore Egg samples were also preserved in buffered formalin for its morphology may be species specific (Ginsburg, 1968; measuring size at a later time using profile projector. Kobayashi and Yamamoto, 1981; Chen et al., 2007). The microstructure of micropyles and surface ornamentations, Results however, may change during fertilization. A brief description of the characteristics and time sequence of the stages of embryonic development in Tor Materials and Methods putitora follows: Healthy running male (average total length 30 ± 3.0 cm and average weight 550 ± 10 g; n=6) and gravid female Zygote period (0 - 3h) (average total length 35 ± 4.0 cm and average weight 750 The newly fertilized egg can be considered to be in the ± 15 g; n=6) broodstock of T. putitora were collected zygote period until the first cleavage occurs, about 3 hours during spawning season (May, 2016) from Bhimtal Lake 15minutes post fertilization. The egg and yolk is about 2.6 Uttarakhand, India. Brooders were stripped for eggs and ± 0.1 mm and 2.3 ± 0.1 mm respectively in diameter at the semen and brought to ICAR-DCFR hatchery. Eggs were time of fertilization. fertilized by gently mixing them with semen/sperm cells. Stages during the zygote period Fertilized eggs were kept in hatching tray under flowing water condition. One-cell stage (0-3 h): The chorion swells and lifts away from the newly fertilized egg (Fig. 1 Fertilization also The incubation and hatching unit comprised a set of activates cytoplasmic movements, easily evident within meshed trays (50 ×30 ×10 cm with mesh size of 1.0mm) about 2 hrs. Nonyolky cytoplasm begins to stream toward placed on fibreglass-reinforced plastic troughs (200 ×60×30 the animal pole, segregating the blastodisc from the clearer cm) having a water flow of 1.0-1.2 litre per minute. The yolk granule-rich vegetal cytoplasm (Fig. 2 & 3). This eggs were uniformly spread in a monolayer on the meshed segregation continues during early cleavage stages. trays at a densityof 4000 - 5000 eggs per tray. Water quality parameters were monitored daily and maintained constant Cleavage period (3h 15m – 8h) over the entire incubation and hatching period (temperature: 20o C ± 2o C, pH: 7.2 ± 0.5, ammonia: 0.007 ±0 .002 mg L-1, After the first cleavage the cells, or blastomeres, divide nitrite: 0.02 ± 0.001 mg L-1, dissolved oxygen: 7.3 ± 0.5 mg at about 35 mins intervals (Figs. 4-10). The cytoplasmic L-1). From each developmental stage, random samples were divisions are meroblastic; they only incompletely undercut taken from different trays for further analysis. A particular the blastodisc, and the blastomeres, or a specific subset of development stage was determined when more than 50% of them according to the stage (Kimme1 and Law, 1985a), all specimens reached that stage. remain interconnected by cytoplasmic bridges. The six cleavages that comprise this period frequently occur at Fig 1: 2 hrs, Zygote Period Fig 2: 2 hrs 30 mins, Zygote Period Fig 3: 3 hrs, Zygote Period Early developmental morphology of golden mahseer 67 regular orientations (Figs. 4-10) so that one can see how first one, and on either side of it. They cut the blastodisc into many blastomeres are present are by their arrangement; a 2 x 4 array of blastomeres. As the dechorionated embryo counting them is unneccessary. usually lies in a dish, the four-cell aspect, rather than the two-cell aspect, faces the observer. This “face” view is Stages during the cleavage period along the odd-numbered cleavage planes (furrows 1 and 3 Two-cell stage (3h 15m): The first cleavage furrow, are visible; Fig. 6). The dechorionated embryo tends to lie in ending the first zygotic cell cycle, is vertically oriented, as the same orientation through late blastula stages. is usual until the 32-cell stage. The furrow arises near the 16-cell stage (5 h): The fourth set of cleavages also animal pole and progresses rapidly toward the vegetal pole, occurs along two planes, parallel to and on either side of the passing through only the blastodisc and not the yolky region second one, and produces a 4 x 4 array of cells. Use care to of the egg (Fig. 4). Near the bottom of the blastodisc the distinguish this stage from the eight cell stage, because they furrow changes to a horizontal orientation to undercut the look similar in face view (Fig. 7). blastodisc. 32-cell stage (5 h 35m): The cleavages ending cycle 5 often occur along four parallel planes, rather than two, lying between those of the first and third cycles.