Chilled storage of white shrimp (Litopenaeus vannamei) spermatophores

Chilled storage of spermatophores from white shrimp (Litopenaeus vannamei) is needed to generate a consistent and reliable supply of spermatozoa for domestication purposes. The objective of this study was to develop a protocol for the chilled storage of white shrimp spermatophores and to evaluate bacterial propagation during such storage. In the first experiment, spermatophores were immersed in four extenders, mineral oil, Ringer's solution, phosphate buffer and 0.85% NaCl, and stored at low temperature (2-4 °C) for 35 days. Characteristics of preserved spermatophores changed the least and viable sperm was highest when spermatophores were stored in mineral oil. Spermatophores preserved with mineral oil appeared morphologically normal. Bacillus circulans, Staphylococcus hominis and S. lugdunensis, S. sciuri, S. xylosus and Micrococcus spp. were identified as the predominant bacteria during chilled storage, and total bacterial counts gradually increased during the experiment. A second experiment investigated the effect of antibiotic on chilled storage. Spermatophores were preserved in only mineral oil or mineral oil with 0.1% penicillin-streptomycin. These were evaluated for changes in external morphology of spermatophores, sperm viability and total bacteria count every week during a 35-day experimental period. Percentages of viable sperm (69.5 ± 3.9%) were significantly higher (P < 0.05) among spermatophores preserved in mineral oil with 0.1% antibiotic compared with those preserved only in mineral oil (57.7 ± 3.4%) over 35 days. The number of total bacteria in the treatment with mineral oil ranged between 28.3 ± 4.8 and 2416.7 ± 299.4 CFU/g, but in mineral oil containing antibiotic bacteria were undetectable. This study suggests that chilled storage of spermatophores is a feasible approach for the management and spawning of white shrimp broodstock.     

Detailed monitoring of white spot syndrome virus (WSSV) in shrimp commercial ponds in Sinaloa, Mexico by nested PCR

WSSV has caused great losses to the global shrimp industry in recent years. This virus can infect shrimps asymptomatically. However, once the clinical signs are developed, mortalities can reach 100% in 3-10 days. PCR has been extensively used to detect WSSV in a specific and sensitive manner. Nested PCR is even more sensitive than single-step PCR and had been used for the detection of WSSV in asymptomatic populations. In this work, a detailed monitoring of WSSV by nested PCR in shrimp commercial ponds in Guasave County, State of Sinaloa, Mexico, is presented. Five ponds from two different farms were monitored for growth and presence of WSSV. At the beginning of the culture, ponds from both farms showed no or very slight WSSV presence. A 3-day period of rain occurred at both farms 10 and 14 weeks of culture for farms 1 and 2, respectively. At this time, WSSV was widely distributed in the shrimp populations of farm 1 according to nested-PCR data, although no visual symptoms were observed. In ponds of farm 2, WSSV was present at low level. However, the number of PCR-positive groups was drastically increased in both farms by nested and single-step PCR. Abrupt fluctuations in temperature and salinity were documented in farm 2 after the rain, which may have contributed to the increasing of viral load in the pond's shrimp populations. Twelve days after the rain period, estimated mortalities of 80% occurred in farm 1. Nevertheless, the study ponds at farm 2 culture continued normally for three more weeks and were harvested successfully (52% and 67% of survival for ponds 1 and 2, respectively). The removal of 40% and 50% of shrimp population 2-4 days after the raining period may have contributed to the thriving of the cultures. Analyses of the presence of WSSV in individuals of both sexes indicated that there is no preference for this virus to infect male or female shrimp. Also, no differences in weight were found between WSSV infected and non-infected individual shrimps, as well as nested-PCR positive against single-step PCR positive organisms. Nested PCR is more useful to monitor shrimp cultures than single-step PCR since it allows knowing how widely distributed the virus is in asymptomatically populations.     

Oxytetracycline (OTC) accumulation and elimination in hemolymph, muscle and hepatopancreas of white shrimp Litopenaeus vannamei following an OTC-feed therapeutic treatment

The white shrimp, Litopenaeus vannamei, has become a very important species for the development of shrimp aquaculture in Northwest Mexico. However, viral and bacterial diseases are considered a major threat to the development of this industry. In the present study a trial was conducted to evaluate the tissue distribution, maximum concentration, and elimination of the widely used antibiotic oxytetracycline (OTC) in L. vannamei using indoor tanks under laboratory-controlled conditions. OTC was given to shrimp simulating a therapeutic treatment through medicated feed for 14 days followed by a period of feeding without antibiotic for another 14 days to evaluate the elimination pattern. Samples of hemolymph, muscle, and hepatopancreas were taken from medicated animals every two days for 28 days. All tissues were removed and frozen immediately in liquid nitrogen. OTC levels were analyzed by High Performance Liquid Chromatography (HPLC). Results showed an important OTC increase during consumption of medicated feed in all examined tissues. OTC maximum concentrations were 33.54 ± 11.19, 194.37 ± 16.11, and 18.79 ± 5.87 µg g^− 1 for muscle, hepatopancreas and hemolymph, respectively. Although the highest OTC level was found in the hepatopancreas, it required only two days after the start of dosing to reach this value, whereas the maximum OTC for muscle and hemolymph was detected after eight days of dosing. Ten days after the cessation of medicated feeding, the drug content in the shrimp tail muscle was under the detectable limit for the method (0.01 µg g^− 1 of OTC).     

White spot syndrome virus (WSSV) infection in tiger shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>: A non-lethal histopathological rapid diagnostic method using paraffin and frozen sections

White spot syndrome virus (WSSV) infection was induced in tiger shrimp, Penaeus monodon, under laboratory conditions, and histopathological changes in subcuticular epithelial cells of the eye stalk and pleopod were studied sequentially at different time post-challenge. Routine histological techniques using paraffin embedded tissues, as well as frozen tissues, were used to document WSSV infection. Histological manifestations such as cellular hypertrophy in the subcuticular epithelial cells of the eyestalk and pleopod could be detected as early as 18 h post-infection (p.i.) before the manifestation of clinical signs of the disease. However, no histopathological changes could be detected before 18 h p.i.. Hypertrophy of the nuclei in the epithelial cells was pronounced after 24 h p.i. Marked necrosis, and eosinophilic intranuclear inclusions, characteristic of early stages of WSSV infection were observed between 24–36 h p.i. Clinical signs of the disease appeared at 48 h p.i. The presence of WSSV at early asymptomatic stages of p.i. has been tested in parallel samples using polymerase chain reaction, for further confirmation of WSSV. This paper discusses the potential of a non-lethal and rapid histopathological diagnostic method to document WSSV infection, using the eyestalk or pleopod, when expensive DNA based diagnostics are not available or affordable.