WWW.WAS.ORG • WORLD AQUACULTURE • DECEMBER 2012 53 (CONTINUED ON PAGE 52) In addressing the need for improved reproductive efficiency and the advent of hybrid catfish production in the US commercial sector, innovative technologies were adapted and procedures were developed for 1) use of temperature-controlled ponds to condition broodstock to enable early out-of-season spawning, 2) determining degreeday heating requirements of fish spawning in earthen ponds (Lang et al. 2003, Lang and Tiersch 2007, Pawiroredjo et al. 2008), 3) high-throughput cryopreservation of blue catfish sperm (Hu et al. 2011), and 4) highthroughput ultrasound imaging of channel catfish females (Novelo et al. 2011, Guitreau et al. 2012). Assessment of reproductive condition is of critical importance in all these procedures, and in hybrid catfish production in particular. The selection of spawning-ready females is vital for induced ovulation. Females with mature, ripe eggs that are ready for hormonal induction of final maturation are most valuable for fertilization with sperm from blue catfish. To improve selection of female broodstock for egg production, ultrasound imaging and handling techniques were developed at the LSU Agricultural Center - Aquaculture Research Station over the past eight years to enable direct, noninvasive viewing of the ovary and oocytes. Ultrasound imaging was first accomplished with unrestrained, free-swimming fish in a holding system (Fig. 1). However, the ability to rapidly assess the reproductive condition of female broodstock — for example, 650 catfish in 6 hours with an average imaging duration of 30 seconds for each fish by use of portable equipment — makes ultrasound imaging for assessment of ovarian development feasible in research and commercial settings. The instrument we used for viewing of channel catfish ovaries consisted of 1) a laptop ultrasound unit2 and a linear probe3 designed for imaging of farmed livestock in veterinary diagnostics that together cost US$10,000, 2) a portable cooler for holding 40-50 L of water for positioning of female catfish in an upright swimming position (Fig. 1), 3) a mobile cart to provide power to the ultrasound unit and for positioning at recirculating systems in the laboratory or at ponds and raceways in the facilities of commercial collaborators, and 4) baskets and dip nets for moving single fish for ultrasound imaging. The probe is waterproof and can be safely submersed for imaging. This combination of equipment and procedures meant that additional handling for application of ultrasound gel, restraint of fish in a holding apparatus, use of anesthesia, and exposure to air were not necessary for channel catfish. Possible injury to valuable fish was avoided by minimizing handling within and next to holding systems, providing the full potential of non-invasive ultrasound imaging assessment in fish reproduction. The basic instrument settings used for viewing of channel catfish ovaries consisted of Real-time Brightness-Mode (B-Mode) which provides a moving cross-sectional grey-scale image displayed in a rectangular field of view created by a multifrequency (5 to 8 MHz) linear probe set at 5 MHz. The depth of ultrasound penetration was set at 80 and 110 mm, and the acoustic power control of the ultrasound beam and overall gain control were set at 100% to produce uniform amplification of returning echoes. FIGURE 2. The orientation of the probe, and the ultrasound medium (water) in relation to the external anatomical positioning of the probe (in Figure 1) is illustrated in ultrasound images of early (A), developing (B, C) and atretic (D) ovaries of channel catfish obtained during the natural spawning season in Louisiana. Solid white lines represent the skin, the closest anatomical structure to the probe, the dashed lines represent the ovarian wall, and the double-headed arrows indicate the thickness of the muscle tissue in each image. Adapted from Novelo and Tiersch, 2011. FIGURE 1. Completely submersed, non-anesthetized, unrestrained channel catfish held only for positioning using the caudal peduncle (A) in a portable container (40-50 L of water), and free-swimming catfish (B) in a recirculating system tank (80 L of water) with the probe aligned alongside the dorsal fin for ultrasound imaging of reproductive condition.
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