Lameness, characterized by alterations in gait or posture, is commonly observed in sows. It is associated with reduced animal welfare and economic losses, including those associated with increased labour and veterinary costs, decreased slaughter value, and animals that are culled prior to reaching optimal breeding efficiency (Dagorn and Aumaitre, 1979; Anil et al., 2005; Anil et al., 2009; Pluym et al., 2013). Recently, lameness was ranked as the most important measure of welfare in pigs (Whay et al., 2003). Estimates of lameness prevalence vary geographically, but range from 8.8% to 15% for many European countries (Gjein & Larssen, 1995; Bonde et al., 2004; Heinonen et al., 2006; Kilbride et al., 2009; Pluym et al., 2011), 15% in the United States (Lay et al., 2008), and 20% in Canada (Seddon et al., 2013). Rates of lameness may also vary with the productive cycle, with the greatest prevalence of lameness reported after sows were moved to the gestation unit (8.1%) versus 4.1% and 5.5% after being moved to the farrowing pens and insemination cages, respectively (Pluym et al., 2013). Lameness in sows can be attributed to several risk factors, including mixing of individuals resulting in increased aggression (EFSA, 2007), lack of exercise (Fredeen and Sather, 1978), and housing type (stalled or group) coupled with floor/bedding type, such as slatted floors (Heinonen et al., 2006; Anil et al., 2007; Karlen et al., 2007; Spoolder et al., 2017). Causes of lameness include claw and skin lesions, infectious and metabolic disorders, trauma, fractures, osteochondrosis, osteomalacia, and osteoarthritis (Fredeen and Sather, 1978; Wells, 1984; Dewey et al., 1992; Kroneman et al., 1993; Bonde et al., 2004; Kilbride et al., 2009).

Lameness typically results in reduced activity, which may impact social, explorative, and feeding behaviours, including increased lying time and decreased water intake (Madec et al., 1986; Cornou et al., 2008; Weary et al., 2009; Ala-Kurikka et al., 2017). These problems may be exacerbated by group-housing, where lame sows may experience greater degrees of social pressure, be unable to compete for resources, and/or be involved in fighting (Gjein and Larssen, 1995; Heinonen et al., 2013). The stress associated with lameness may also limit the abilities of the immune system to fight infection and disease, which can result in the development of secondary diseases, as well as weight loss and reduced reproductive performance (Bonde et al., 2004; Anil et al., 2009). Inactivity is positively associated with urinary and reproductive infections (Madec et al., 1986; Dee, 1992), which have been reported to account for 6.6 to 8% of all deaths (Chagnon et al., 1991).

Lameness directly impacts longevity, with increased culling and euthanasia or mortality rates of lame sows (Anil et al., 2009), leading to reduced welfare and increased economic costs (Jensen et al., 2010). It is estimated that 6 to 35% of sows are culled due to lameness and claw lesions (Anil et al., 2009). Non-lame sows have a survival time that is more than double that of lame sows after the first farrowing, with a greater reproductive success (lower rates of stillborn or mummified fetuses) in non-lame sows (Anil et al., 2009; Pluym et al., 2011). Furthermore, lame sows tend to have lower litter weights (Fitzgerald et al., 2012) and greater piglet mortality caused by crushing due to increased lying time (Anil et al., 2009). However, lameness itself is not associated with lower rates of pregnancy (Heinonen et al., 2006), instead, lame sows are more likely to be removed from the herd (either due to culling or mortality) earlier than their non-lame counterparts, resulting in fewer overall pregnancies. A study conducted on ten different Danish herds found that of euthanized sows, 72% of cases of euthanasia were associated with the locomotory system, most commonly osteoarthritis (OA; 24%) and fractures (16%), with arthrosis as a secondary diagnosis in 88% of cases (Kirk et al., 2005). Furthermore, OA impacts millions of people worldwide and poses a significant public health concern (Maetzel et al., 2004). Porcine models are considered to be a highly translational model of human OA due to similarities between human and porcine joint sizes, weight-bearing, and cartilage thickness (Cruz et al., 2016). Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in pain management of animals, including swine (Keita et al., 2010; Reiner et al., 2012; Kluivers-Poodt et al., 2013). Using a chemical synovitis model, Pairis-Garcia et al. observed that sows treated with flunixin meglumine and meloxicam had reduced lying posture frequency compared to sows treated with saline, and that pain was mitigated at both 48 and 72 h after lameness induction (Pairis-Garcia et al., 2015). Owing to the absence of a pharmaceutical ‘cure’ for arthritis, and the well-established adverse effects associated with common pharmaceutical pain remedies (Ghanem et al., 2016), feed ingredients targeting maintenance of joint health have emerged on the front-line of OA management in other species. The vast majority of these ‘functional’ feed ingredients with do not have research to support their safety or efficacy in modifying the pathophysiology of OA. A feed ingredient with considerable potential to positively influence clinical outcomes in arthritic animals is wheatgrass (WG; Triticum aestivum). WG is a hardy perennial grass that is a rich source of antioxidant compounds (Durairaj et al., 2014; Parit et al., 2018). Its contemporary popularity as a ‘superfood’ arose initially from a patent in 1930’s which claimed to capture the superior nutritive of sprouted wheatgrass for the purpose of detoxifying the body and providing a concentrated abundance of nutrients. Antioxidant products have demonstrated significant benefit in cases of OA (Ansari et al., 2020), as have extracts rich in polyphenols (Oliviero et al., 2018) such as WG (Durairaj et al., 2014; Benincasa et al., 2015). Therefore, the objective of this study was to characterize the effect of WG on porcine cartilage responses to an inflammatory stimulus (lipopolysaccharide; LPS).

For each outcome measure, C and B explants were initially compared, in order to demonstrate that the vehicle in which WG was extracted did not alter baseline responses of cartilage to LPS. Provided that no differences were identified between C and B explants, effects of WG were only compared with B explants.

The purpose of this experiment was to determine the effects of an extract of WG (5 or 15 μg/mL) on porcine cartilage responses to pro-inflammatory stimulation with LPS. We have previously shown that LPS causes an inflammatory response in cartilage, as evidenced by increased production of PGE₂, GAG and NO (Pearson et al., 2007; Pearson et al., 2010; Pearson and Kott, 2019; Pearson et al., 2020). The main findings were that conditioning of cartilage explants with both doses of WG extract (5 and 15 μg/mL) for 24 h prior to and 48 h after stimulation with LPS resulted in significantly reduced breakdown of cartilage, as measured by significantly lower GAG release following exposure to LPS in WG-conditioned explants. Furthermore, LPS-induced inflammation was significantly reduced by WG as measured by a significant decrease in LPS-induced PGE₂ (5 μg/mL) and NO (5 and 15 μg/mL). These findings provide evidence for a protective effect of WG on cartilage structure and inflammation in the presence of a pro-inflammatory stimulus. Furthermore, WG was found to be safe to cells within cartilage explants, as evidenced by a lack of decline in cell viability after 72 hours of exposure.

Wheatgrass has been suggested as part of the treatment for a variety of degenerative diseases due to its anti-inflammatory and antioxidant properties (Kulkarni et al., 2006; Watzl, 2008; Urbonavičiute et al., 2009; Parit et al., 2018). The effects of WG have primarily been tested in vitro, where it has been observed to be capable of countering all major types of excessive radicals (Watzl, 2008; Durairaj et al., 2014). In rodents, WG supplementation has been found to be beneficial in the treatment and prevention of obesity (Im et al., 2015), protect the liver against alcohol and oxidative stress (Durairaj et al., 2015), and prevent or treat cancer (Zalatnai et al., 2001). In rats with glucocorticoid-induced osteoporosis, 30 days of WG extract, coupled with bisphosphonate and risedronate, were found to increase bone mineral content and decrease bone resporption (Banji et al., 2014). In humans, supplementation of WG alongside standard therapy was found to improve symptoms of rheumatoid arthritis (Bálint et al., 2006). However, to the authors’ knowledge, no evidence exists to date to indicate that WG may play a role in OA specifically.

PGE₂ is a key biomarker for pain and inflammation and is the compound that is targeted for inhibition by NSAIDs. PGE₂ is directly associated with the pain and inflammation that is the hallmark of arthritis, and its inhibition by WG suggests that sows with clinical signs of OA supplemented with WG will experience a reduction in pain. Similarly, NO is a key biochemical in the pain signaling cascade, as well as a pyrogenic mediator of the periarticular swelling. The strong inhibition of NO by both doses of WG in the current study support a marked anti-inflammatory potential of the plant.

GAGs are highly polarized structural sidechains of aggregating proteoglycans within cartilage. Their high polarity causes them to strongly attract and retain water within cartilage, contributing to the ability of cartilage to resist compressive and shear forces (Volpi, 2006). In the early stages of arthritis, GAGs are cleaved from proteoglycans, contributing to progressive thinning of articular cartilage and impairment of the tissue’s ability to buffer catabolic effects of forces travelling across the articulating joint (Zhou et al., 2018). The mechanism of action of some useful disease modifying agents such as n-acetyl glucosamine derivatives (Cao et al., 2016) and bioactive plant extracts (Pearson et al., 2010; Liu et al., 2015) involves preventing GAG loss during inflammation, resulting in protraction of the disease process and prolonging healthy function of cartilage. Results from the current study provide evidence for a protective effect of WG on cartilage structure during inflammatory stress. The molecular mechanism by which WG exerts this effect is not known but may be associated with the reported inhibitory effect of polyphenols on proteolytic aggrecanases (Cudic et al., 2009) which catalyze breakdown of proteoglycan molecules during catabolic events such as inflammation and degenerative articular diseases. This hypothesis should be explored in future research.

Safety of WG on cartilage cells was determined using fluorescent dye method. We have previously validated and applied this technique for use in cartilage explants to obtain an estimate of the effect of dietary supplements on cartilage explants for which the actual number of cells within the explants is not known (Pearson et al., 2007; Pearson et al., 2010). This technique utilizes Calcein-AM (C-AM) and Ethidium Homodimer-1 (EthD1) to differentially fluoresce in live and dead chondrocytes, respectively. C-AM is transported into live cells across the cell membrane. Once inside, esterases within the cytosol cleave the ‘AM’ portion from calcein, trapping the calcein molecule inside the cell and causing it to fluoresce green. Conversely, EthD1 will only fluoresce when it is able to bind to DNA. Because intact cell membranes are impermeable to EthD1 this binding can only occur when the cell membrane is dead. When explants are submerged in fluid containing C-AM and EthD1 at the appropriate concentration and for the appropriate duration, simultaneous measurements of fluorescence of both C-AM and EthD1 can be obtained, which gives a good estimation of the percent viability of cells within the individual explants. The current study demonstrates that WG is not cytotoxic at the doses evaluated, which approximated 18 and 54 g for a 300 kg sow.

It is concluded that conditioning cartilage explants in the presence of a wheatgrass extract results in protection of cartilage against LPS-stimulated decline in cartilage structure, reduces mediators of inflammation, and is non-cytotoxic. Nutrition of sows has previously been noted as an important predisposing factor of sow lameness (van Riet et al., 2013). Nutritional strategies to support bone remodeling, cartilage metabolism, and horn production are not yet fully understood (van Riet et al., 2013). In addition to ensuring adequate availability of nutrients and appropriate feed intake, there is potential for functional feed ingredients to also aid in supporting musculoskeletal health.

Due to the similarities in joint anatomy, physiology, and biomechanics between humans and pigs, pigs are an excellent translational model to better understanding human OA. Additionally, pigs have large sized litters that mature quickly, making them an excellent non-rodent model (Cruz et al., 2016). Pigs have previously been used as an animal model to study the physiopathology of OA and cartilage repair (Murray and Fleming, 2012; Schlichting et al., 2014; Sieker et al., 2018; Kremen et al., 2020; Zhao et al., 2021). It is possible that similar chondroprotective and anti-inflammatory effects may be associated with WG supplementation in humans. Further research into this area is warranted.

The results of this study provide evidence for a potentially valuable role of WG in preventing joint dysfunction and pain associated with OA, and supports its use for prolonging healthy cartilage in sows. Further research on the effects of this supplementation in live sows is warranted.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s.

KC - conducted all experiments; analyzed data; critical review and approval of final draft. AG - analyzed data; critical review and approval of final draft. PM - wrote manuscript, critical review and approval of final draft. WP - designed experiment; analyzed data; critical review and approval of final draft. All authors contributed to the article and approved the submitted version.