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Fishery Bulletin 104(2) 



cold room of a laboratory for several hours at an air 

 temperature of 16-18"C, bled for a period of time, and 

 then held in the coldroom or in a truck until transport 

 back to the dock (Grogan-^; Walls'*). Horseshoe crabs 

 that are returned to the ocean are transferred onto a 

 boat and returned to their approximate point of capture 

 within 72 hours of their collection (HCTC'). The numer- 

 ous stressors to which the horseshoe crabs are exposed 

 throughout the biomedical bleeding process likely have 

 an effect on their mortality rates, both for horseshoe 

 crabs used for bleeding and for those crabs collected, 

 but rejected for use. 



Additional sources of stress also may arise from the 

 bleeding methods used by some biomedical companies. 

 The bleeding protocols at some facilities involve gravi- 

 tationally withdrawing blood from the heart (punctured 

 with a large gauge needle) until blood flow slows to an 

 intermittent drip (Grogan-^; Walls'*). One study reported 

 a range of extracted blood to be from 8.4 mL to 218.7 

 mL in male horseshoe crabs (Walls, 2001) and up to 

 267.8 mL in females (Walls"). Unfortunately, biomedical 

 companies do not know how much blood horseshoe crabs 

 of a given size possess and how much can be safely ex- 

 tracted. Thus, extracting the reported upper ranges of 

 blood volume from males and females may cause stress 

 or mortality. 



Several studies have estimated mortality associated 

 with the biomedical bleeding process to be between 8% 

 and 20% (Rudloe, 1983; Thompson, 1998; Kurz and 

 James-Pirri, 2002; Walls and Berkson, 2003). These 

 studies either simulated the biomedical bleeding process 

 (Rudloe, 1983; Kurz and James-Pirri, 2002) or moni- 

 tored mortality of a given number of horseshoe crabs 

 bled by a biomedical company (Thompson. 1998; Walls 

 and Berkson, 2003). Although the results provide infor- 

 mation on mortality rates associated with a biomedical 

 companies' procedures, they provide no guidance on how 

 biomedical companies may reduce mortality rates. 



Rather than quantifying mortality rates associated 

 with the biomedical bleeding process as a whole, we 

 isolated and tested certain elements of the bleeding 

 process to gain information on the potential causes of 

 mortality for horseshoe crabs used in the biomedical in- 

 dustry. The objectives of this study were 1) to quantify 

 mortality associated with blood extraction at set levels 

 and 2 ) to quantify mortality associated with stress from 

 simulated transport and holding procedures, combined 

 with the stress of blood extraction at the same levels as 

 in the first objective. 



■'' Grogan, W. 2003. Personal commun. Department of Fish- 

 eries and Wildlife Sciences, Virginia Polytechnic Institute 

 and State Univ., Blacksburg, VA 24061. " 



■* Walls, B. 2001. Personal commun. Center for Environ- 

 mental Studies, Virginia Commonwealth Univ., 1000 West 

 Cary Street, Box 843050, Richmond, VA 23284. 



•'' Walls, B. 2001. Unpubl. data. Center for Environmental 

 Studies, Virginia Commonwealth Univ., 1000 West Cary 

 Street, Box 843050, Richmond, VA 23284. 



Methods 



Adult horseshoe crabs were obtained from Cambrex 

 BioScience Walkersville, Inc., a commercial biomedical 

 company on 9 July and 28 August 2003. Horseshoe crabs 

 were captured by using a standard trawling procedure 

 off the coast of Ocean City, Maryland (Hata and Berk- 

 son, 2003). After capture, horseshoe crabs were trans- 

 ported in an air-conditioned van to the Horseshoe Crab 

 Research Center (HCRC) at Virginia Polytechnic Insti- 

 tute and State University in Blacksburg, Virginia. The 

 animals were maintained in a recirculating aquaculture 

 system in appropriate environmental conditions (Brown 

 and Clapper, 1981) and monitored daily, but not fed. The 

 animals were acclimated to the aquaculture conditions 

 for one week, during which time they were tagged, sexed, 

 and measured. Tagging involved drilling two 3/32" holes 

 into the prosoma at its thinnest point and attaching a 

 laminated oval fish tag (Floy Tag, Seattle, WA) with two 

 3/32" wide cable ties. Measurement consisted of record- 

 ing the inter-ocular width (10), the distance between the 

 inside margins of the horseshoe crab's compound eyes, 

 and the prosomal width (P), which is the distance across 

 the horseshoe crab's carapace. Horseshoe crabs used for 

 both bleeding mortality studies had a mean 10 width of 

 14.0 cm and ranged from 10.5 to 19.0 cm; as well as, an 

 average P width of 23.5 cm that ranged from 17.5 to 35.0 

 cm. Only animals that were uninjured (i.e., no cracked 

 carapaces or missing legs, etc.) and free of epibionts were 

 used in the two mortality experiments. 



Experiment 1 



Horseshoe crabs from the July collection were used in 

 experiment 1. From this collection, 100 males and 100 

 females were selected to test only the effects of various 

 levels of blood extraction on mortality. This sample of 

 horseshoe crabs was termed the "lower-stressed" group 

 because they were not exposed to external stressors 

 associated with simulated holding and transport. The 

 selected horseshoe crabs ranged in size to provide a 

 representative sample of animals bled by some bio- 

 medical companies (Grogan^; Walls^). Horseshoe crabs 

 were arbitrarily assigned to one of five bleeding treat- 

 ments: 1) unbled crabs (control), 2) crabs bled lO'/r of 

 their predicted total blood volume, 3) crabs bled 20% 

 of total blood volume, 4) crabs bled 30% of total blood 

 volume, and 5) crabs bled 40% of their predicted total 

 blood volume. Each bleeding subgroup comprised equal 

 numbers of males and females. Predicted blood volume 

 was calculated by using the relationship between blood 

 volume and 10 width (Hurton et al., 2005): 



H = 25.7e 



0.1928 I/O) 



where H = hemolymph volume in mL; and 

 10 = interocular width in cm. 



The bleeding process of experiment 1 involved remov- 

 ing horseshoe crabs from their holding tank, positioning 



