1. Introduction
American bison (
Bison bison) once flourished in North America, numbering in the tens of millions [
1,
2]. However, great numbers of bison were slaughtered during the peak of the hide trade in the late 1800s, and the species was driven to near extinction. By the early 1900s, the remaining bison survived as small herds on five private ranches and within a small wild herd in Yellowstone National Park, which had less than 25 animals in 1902 [
1,
3]. Bison on the remaining ranches were crossbred with domestic cattle (
Bos taurus) in an attempt to improve the traits of cattle, including meat quality, quantity, hardiness, feed efficiency, and disease resistance [
4,
5,
6]. Although the crossbreeding events were discontinued, they resulted in the incorporation of cattle DNA into American bison populations. In 1905, the American Bison Society was formed, and its lobbying efforts led to the creation of several public conservation herds within the United States [
7,
8]. Currently, there are approximately 400,000 bison in commercial herds in North America and some 31,000 bison managed within conservation herds [
9,
10]. The International Union for Conservation of Nature (IUCN) Red List considers American bison to be “Near Threatened”; however, the population is considered stable and there is a harvest management plan in place [
9].
The North American bison industry experienced steady growth from 2010 to 2020, with sales of bison meat in restaurants and retail stores reaching USD 350 million per year [
11]. The majority of bison producers (80%) sell directly to consumers and restaurants rather than grocery stores [
12]. Bison sales fell in 2020 due to restaurant closures associated with the coronavirus pandemic; however, the industry is receiving support from the US Department of Agriculture (USDA), which agreed to purchase USD 17 million (~1.1 million kg) of bison meat from processors. Bison, which is sometimes mistakenly referred to as “buffalo,” is low in fat, calories, and cholesterol and contains high amounts of protein, iron, and vitamin B-12 [
11]. Bison spend the majority of their lives on grasslands, with little or no time in the feedlot, and they are generally not given antibiotics or growth hormones. Bison meat is highly valued compared to beef from domestic cattle. For example, the average direct-to-consumer price for ground lean bison in March 2020 was USD 30.14/kg [
13] compared to an average retail price of USD 12.40/kg for lean and extra lean ground beef [
14]. During the same time period, the average direct-to-consumer price for bison ribeye steaks was USD 80.90/kg [
13], which is almost five times that of the average retail price of beef steaks at USD 16.89/kg [
14]. These price differences combined with the similar appearance of meat from bison and domestic cattle introduce the potential for intentional mislabeling for the purpose of economic gain.
Mislabeling species of meat products is commonly detected using DNA-based techniques, such as DNA barcoding and real-time polymerase chain reaction (real-time PCR) [
15,
16]. DNA barcoding is a widely used sequencing method in which universal primers target a short, standardized genetic region for the identification of species [
17]. In animals, the most commonly used region is a ~650 base-pair (bp) fragment of the mitochondrial gene coding for cytochrome
c oxidase subunit I (COI). Previous US market surveys using DNA-based techniques such as DNA barcoding or real-time polymerase chain reaction (PCR) have reported the identification of cattle in commercial bison or buffalo products [
18,
19,
20]. For example, a market survey conducted on game meats sold in the US reported that two products labeled as “stewed bison meat” and “rib-eye bison steak” were identified as domestic cattle with DNA barcoding [
19]. Another US market survey detected a mixture of beef, pork, and horse in a product labeled as “ground bison” using real-time PCR [
18].
A shortcoming of previous market surveys involving bison products was that the analytical methods used for the detection of bison were based on mitochondrial DNA (mtDNA), which is inherited through the maternal line [
19]. This is problematic when testing for the presence of bison because historical crossbreeding of the two species was reliant on breeding male bison with female cattle, and backcrosses of male bison with female offspring [
6]. Although the cross-breeding programs were halted over a century ago, some American bison populations still carry ancestral cattle DNA, with an average of 13.9% mtDNA cattle ancestry and 0.6% autosomal cattle ancestry across 22 herds studied [
6]. Additionally, according to US regulations, the term bison may refer to American bison or cattalo, which is a result of direct crossbreeding between American bison and domestic cattle (Exotic Animals and Horses, 9 Code of Federal Regulations (CFR) § 352). As a result, DNA-based testing of bison products has the potential to give a false-positive result for cattle due to the presence of ancestral cattle DNA in bison or the use of cattalo in a bison product. However, previous studies reporting the detection of cattle DNA in bison products did not perform additional testing to verify the identity of the product. A method that could be used to confirm the species in these situations is polymerase chain reaction (PCR)-satellite fragment length polymorphism (SFLP), which is a variation of PCR-restriction fragment length polymorphism (RFLP) that targets centromeric satellite DNA. A PCR-SFLP method was previously developed for the differentiation of bovine species, including animals of hybrid origin such as bison and cattle [
21]; however, it has not been used to verify species labeling of bison products.
The objective of this study was to perform a market survey of bison meat products and verify the species using a combination of molecular methods. DNA barcoding was used as an initial test for species and any bison products that were identified as domestic cattle were then analyzed using PCR-SFLP to verify the species.