40 MARCH 2025 • WORLD AQUACULTURE • WWW.WAS.ORG selection against EHP + WFD in two consecutive generations of the breeding program for P. vannamei. Methods Animals from two consecutive batches (48 and 49) from the Benchmark Genetics shrimp breeding program were used in this study. In batch 48, a total of 47 families were produced over 2 days using breeders from batch 47, while batch 49 was composed of 67 families produced in 4 days using breeders from batch 48. All families were produced using artificial insemination (AI). Inseminated females were individually placed in spawning tanks. To minimize the effect of environmental influences during early phases, all families were mixed at nauplii stage for the animals participating in the growth tests. However, a different protocol was used for the challenge test, as final survival needed to be determined. In this case, individual larviculture was performed for each family, and the same number of animals per family was used. Challenge tests Upon arrival at the challenge test facilities, shrimp were acclimated in separate small tanks until they reached an average size of over 0.5 grams. The same number of animals were stocked in replicate tanks (10 tanks for the batch 48 trial and 6 tanks for the batch 49 trial). After the pre-challenge phase, animals were challenged with EHP using a cohabitation protocol for seven days. This was followed by a per os WFS challenge using the V. algynolyticus strain, feeding the animals feed coated with bacterial broth for 3 days. Negative control tanks (2 tanks in batch 48 and 1 tank in batch 49) were not challenged with EHP, and in the Vibrio challenge phase, these tanks were fed with feed coated with the culture medium without bacteria. A post-challenge period was conducted for 5 to 6 weeks. Dead animals were recovered on an hourly basis, and tissue samples were collected for genotyping during the duration of the test. At the end of the test, all tanks were harvested. Survivors were individually weighed, tissue sampled, and recorded. Infection was confirmed by both histology and qPCR. Growth tests Growth tests using the same batches and families were also conducted in Colombia and Vietnam. In Colombia, larvae from all families were stocked in 50 T tanks at a density of 180 animals/m² for batch 48 and 200 animals/m² for batch 49. In Vietnam, siblings of the same families were stocked in commercial ponds of 150 m² at 86 animals/m² for batch 48 and 153 animals/m² for batch 49. At harvest, one thousand animals per tank/pond were randomly collected, weighed, sexed, and tissue sampled for genotyping to be used as a training population. Genotyping All tissue samples from the challenge and growth tests were sent to IdentiGEN in Dublin for genotyping using a 43K BMK Shrimp SNP-array developed by Benchmark Genetics. This panel was designed using markers identified in several shrimp populations with diverse genetic backgrounds, including all Benchmark lines, with informative SNPs distributed throughout the genome. The SNP array is available for use via Benchmark Genetics’ genotyping service, and users are provided with all the relevant map and SNP details. In addition, the SNP array contains a SNP marker located on chromosome 12, described by Pérez-Enriquez et al. (2024), which showed over ~97% concordance with phenotypic sex in our population (Peñaloza et al., 2024, unpublished data). This SNP was used to assign sex to the animals in the growth and challenge tests in Vietnam. Selection of Breeders Based on EHP Indexes To evaluate the effect of genomic selection on progeny response to EHP + WFD resistance, the Genomically Estimated Breeding Value (GEBV) for EHP+WFD survival was calculated for both males and females from Batch 48 based on the challenge test results. These values were then used to classify all families produced in Batch 49 into 4 FIGURE 1. Survival at harvest of the infected tanks and the negative control tanks in EHP+WFD challenge tests from batches 48 and 49. FIGURE 2. Final survival in the EHP+WFD challenge test in the families from: (a) batch 48 and (b) batch 49.
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