Utilising cottonseed in animal feeding: A dialectical perspective

As the population expands livestock industry faces challenges including protein inadequacy and infectious diseases. Natural alternatives can help mitigate these issues, for example, by incorporating plant‐derived metabolites containing appropriate concentrations of natural antibiotics and health‐promoting agents into feed supplements. One promising source is cottonseed, a fat‐ and protein‐rich by‐product of cotton plant (Gossypium spp.). During processing, gossypol, a natural substance with potential adverse effects when consumed long‐term, is often eliminated. However, gossypol has broad‐spectrum antiviral and antimicrobial properties, making it a potential solution to feed safety and animal health concerns. Here, we outline the challenges faced in animal husbandry and discuss how cottonseed may address diverse scenarios.


INTRODUCTION
As the population increases and urbanisation progresses, the demand for meat, eggs, milk, and other animal products is also increasing, which directly promotes the rapid development of animal husbandry.However, rapid urbanisation has led to intensive animal farming, which brings about serious problems, such as protein shortages and infectious diseases. 1,2Antibiotics and antivirals are important for the treatment and prevention of infectious diseases; however, their overuse can lead to drug residues, environmental pollution, and pose human health threats, having become a key public health challenge. 3Therefore, an alternative to antibiotics and antivirals is urgently needed for animal disease control.
Plant-based feed additives (PBFAs) are novel green alternatives to antibiotics and antivirals, having the benefits of being safe, nontoxic, and producing no residue. 4PBFAs are natural plant materials or parts that are processed through physical extraction or biological fermentation.They provide rich nutrition, promote animal growth, enhance the feed utilisation rates and improve animal product quality. 5Various types of PBFAs contain abundant active components, such as alkaloids, polyphenols, polysaccharides, terpenoids, flavonoids, and organic acids.Most of these active ingredients possess many biological properties, such as antibacterial, antioxidative, anti-inflammatory, analgesic, and antiviral properties, while also exhibiting bidirectional regulation of the immune function in animals. 6Therefore, they are ideal alternatives to antibiotics and antivirals for the prevention of livestock and poultry diseases.
The cotton plant (Gossypium spp.) is an economically significant crop that belongs to the Malvaceae family.It is one of the important economic crops to be globally cultivated by humans.The main product of the cotton plant is fibre, and by-products include cottonseed, cotton stalks, and various secondary metabolites.Cottonseed meal is a product produced by cottonseed after oil extraction that has a high crude protein content and a diverse range of amino acids, making it a high-quality protein feed ingredient. 7It is widely acknowledged that cottonseeds contain gossypol, a toxic secondary metabolite of the cotton plant, which hinders the efficient utilisation of cottonseed meal in animal husbandry.][13][14][15] Furthermore, gossypol possesses a chiral axis, resulting in two enantiomers: (−)-gossypol and (þ)-gossypol.These enantiomers exhibit different activities and toxicities, offering diverse possibilities for the application of cottonseed under different circumstances.This review provides an overview of the current utilisation of cottonseed as animal feed and explores its potential application scenarios.

Current status of protein feed in animal husbandry
Proteins are essential nutrients for maintaining the cell structure and normal physiological activities.Protein deficiency can lead to malnutrition [16][17][18][19] and death in severe cases. 20Protein feed is an indispensable component of animal husbandry.As the scale of animal husbandry continues to expand, the demand for protein feed in the industry is gradually increasing.The shortage of protein feed is increasingly restricting animal husbandry development. 21Owing to its high crude protein content and balanced amino acid profile, soybean meal is currently one of the most widely used plant-based protein feeds. 22However, in many countries, especially in China, the production of soybean meal is insufficient to meet the animal husbandry demands.Based on the growing demand for soybean meal, fully exploiting existing high-quality protein feed resources such as cottonseed meal, canola meal, corn gluten meal, sunflower meal, pea protein, fish meal, etc, 7,22,23 and adopting advanced feed scientific theories and processing techniques is essential to promote sustainable animal husbandry.

Common animal diseases in animal husbandry and main prevention measures
Modern intensive and large-scale animal breeding has greatly increased the risk of animal infection caused by various diseases.Livestock and poultry are highly susceptible to pathogenic infections during their growth processes, leading to high death rates and huge economic losses in the animal husbandry industry. 24Viral infections are one of the most serious infectious diseases in veterinary clinics, especially those caused by highly pathogenic viruses, such as foot-and-mouth disease virus, 25 avian influenza virus, 26 swine fever virus, 27 bluetongue virus, 28 porcine reproductive and respiratory syndrome virus, 29 and Newcastle disease virus, 29 which often result in significant economic losses to the livestock industry.Therefore, the use of antiviral drugs is particularly important.According to the genome type, viruses can be divided into DNA viruses (including single-and double-stranded DNA viruses), RNA viruses (including double-stranded RNA viruses, and single-stranded positive-and negative-sense RNA viruses), and retroviruses (including retroviral DNA and RNA viruses). 30Depending on the genome type and virus-specific requirements, various viruses possess different polymerases that participate in viral genome synthesis (Figure 1).Antiviral agents can exert their antiviral effects through various mechanisms based on their structural and functional characteristics: 1. Blocking viral entry into the host cells.The first step of the viral infection of a host is the adsorption on a host cell via electrostatic or specific receptors and the injection of the nucleic acids into the host cell.Amantadine can act as antiviral agents by preventing viral proteins from fusing with the host cells or by preventing viral uncoating. 31,32In clinical practice, amantadines are mainly used for the prophylaxis and treatment of avian influenza 33 and swine influenza. 34. Blocking viral nucleic acid replication or transcription.Viral polymerase plays a crucial role in the replication and transcription of the viral genome constituting an ideal target for antiviral drugs. 30Viral polymerase inhibitors include nucleoside and nonnucleoside inhibitors (NNIs).Nucleoside inhibitors (NIs) exert their antiviral activity by binding to the active sites of viral polymerases, thus inhibiting DNA and RNA replication. 35NIs are crucial active components of antiviral drugs, accounting for approximately half of those of the antiviral drugs currently used, such as ribavirin, 36 moroxydine, 37 idoxuridine, 38 acyclovir, 39 and so on.1][42] NNIs have a distinct mechanism of action, high selectivity, and low toxicity, but are currently less used clinically because of their limited application.3. Participating in host immune regulation.Besides the antiviral agents that directly target viruses or their proteins, some antiviral agents, such as interferons, interleukins, transfer factors, and so on, indirectly regulate the host immune response. 43,44Interferons, which are potent immune regulatory factors, are classified into two types: types I and II.Type I interferons are related to antiviral activity, whereas type II interferons play a critical role in immune regulation. 45,46In veterinary clinical practice, interferons can be used to treat piglet diarrhoea, infectious gastroenteritis, and swine fever. 47,48 addition to antiviral drugs, antibiotics are widely used in livestock and poultry production.They are not only used for treating animal diseases caused by pathogens but also as growth promoters, which often leads to their overuse.Commonly used antibiotics include β-lactam antibiotics, tetracyclines, macrolides, and sulfonamides. 49he use of antibiotics and antivirals in animal husbandry plays a significant role in ensuring a large-scale production.However, the long-term and extensive use of these drugs results in drug residues and resistance, posing a serious threat to the sustainable development of animal husbandry and human health.Therefore, developing environmentally friendly and efficient substitutes is urgently needed.

Nutritional value of cottonseed meal
Cottonseed is a cotton production by-product, and for every unit of cotton fibre produced, 1.65 units of cottonseed are produced. 50These are mainly used for animal feed or cottonseed oil extraction. 51The cottonseed contains 22.9% fat, 43.2% carbohydrates, 7.3% water, 3.5% minerals, and 23.1% protein. 52After extracting the cottonseed oil, the remaining cottonseed meal retains a protein content of over 40% and is commonly used as high-protein animal feed. 51,53The cottonseed meal protein fraction exhibited the highest concentration of salt-soluble globulins (33%-63.7%),followed by water-soluble albumins (20.8%-32.2%),and alkali-soluble glutelin (9.2%-28%). 54he amino acid composition and proportion are critical parameters that affect the nutritional value and application of proteins.6][57][58][59][60] The most abundant amino acids in cottonseed meal are glutamine (20.8%), arginine (13%), and aspartic acid (10.3%).Except for histidine, tryptophan, tyrosine, and cysteine, whose contents are lower than 3%, the other amino acids account for approximately 5% of the cottonseed protein content.Compared to soybean meal, cottonseed protein is richer in methionine, arginine and proline but more deficient in leucine, lysine and isoleucine. 55Therefore, when utilising cottonseed meal as a protein source in animal feed, thoroughly considering the amino acid composition of cottonseed meal and adjusting the balanced formula accordingly is imperative to ensure that the feed meets the specific amino acid requirements of the animals.
Additionally, most of the crude fat in cottonseed meal is composed of unsaturated fatty acids, whose content is similar to that found in soybean meal.The effective energy value of cottonseed meal is primarily determined by its crude fibre content, which varies depending on the degree of dehulling during processing and typically ranges from 10% to 14%.Additionally, cottonseed meal contains a rich array of vitamins and minerals (Table S1).Given these nutritional characteristics, cottonseed meal is a high-quality source of edible and feed plant proteins.

Side effects of gossypol in cottonseed
Although cottonseed is highly nutritious, the presence of anti-nutritional factors, such as gossypol, restricts its efficient utilisation.Gossypol is an important secondary metabolite of the cotton plant that is produced in response to pathogen invasion and insect bites. 61Based on its existence form, gossypol can be classified into free and bound gossypol free and bound gossypol.Longterm consumption of cottonseed meal with high levels of gossypol can result in adverse effects for animals, including impaired weight gain, weakness, anaemia, respiratory distress, anorexia, apathy and heart failure. 62herefore, regulating the gossypol content when cottonseed meal is used as animal feed is crucial.

GOSSYPOL EXHIBITS A BROAD SPECTRUM OF MEDICINAL PROPERTIES
Despite the potential toxic side effects of the long-term consumption of cottonseed containing gossypol for animals, gossypol has great potential applications in medicine, agriculture, industry, and other fields.Gossypol is clinically used for the treatment of gynaecological diseases, such as uterine fibroids, endometriosis, and functional uterine bleeding.5][66] The combination of gossypol with paclitaxel and cisplatin has successfully passed phase III clinical trials for the treatment of non-small cell lung cancer, demonstrating its potential as an effective therapeutic agent in clinical medicine.
In addition, gossypol exhibits broad-spectrum antiviral activity and effectively inhibits plant viruses such as tobacco mosaic virus, 67 animal viruses such as avian influenza virus, 15 and zoonotic viruses including West Nile virus, 14 Zika virus, 14 and Hendra virus. 13urthermore, gossypol displays inhibitory effects on human viruses, such as HIV. 68Although gossypol has broad-spectrum antiviral effects, its mechanism of action needs to be analysed in depth.Recently, we discovered that gossypol targets the active pocket region of RNA-dependent RNA polymerase (RdRp) by blocking the binding sites of the RNA primers and templates, thereby effectively suppressing the activity of RdRp and inhibiting the replication of SARS-CoV-2 and its variants.Additionally, based on the highly conserved features of the RdRp in coronaviruses, our study also demonstrated that gossypol can broadly inhibit other livestock and poultry coronaviruses. 11esides, gossypol also exhibits broad-spectrum antibacterial activity by inhibiting various pathogenic bacteria in livestock and poultry, including Staphylococcus aureus, Streptococcus, and Edwardsiella tarda in vitro. 69,70In vivo studies have shown that gossypol increases the resistance of catfish to E. tarda. 71he aforementioned research indicates that, despite certain side effects, the broad-spectrum capacity of gossypol to inhibit pathogenic microorganisms in livestock and poultry may make it a valuable asset for animal husbandry.

DIALECTICAL DISCUSSION ON THE APPLICATION SCENARIOS OF COTTONSEED IN ANIMAL FEED
The insufficiency of protein resources and prevalence of infectious diseases are the two main challenges impeding the healthy development of animal husbandry.Addressing the issue of protein resource scarcity requires the identification of suitable protein source alternatives to soybean meal.Combating infectious diseases in animal husbandry often involves resorting to antibiotics or antiviral drugs, which can give rise to new problems, such as drug residues, heightened antibiotic resistance, and environmental pollution.Therefore, developing a two-pronged breeding strategy is necessary.
Gossypol in cotton poses the risk of inducing toxic diseases in livestock, thereby limiting the long-term use of cottonseed containing this substance in animal husbandry.However, gossypol exhibits broadspectrum inhibitory activity against pathogenic microorganisms and may emerge as a novel PBFA for nonantibiotic and -antiviral breeding purposes.Here, we discuss the potential applications of cottonseed and how to make good use of gossypol, a 'double-edged sword' for animal health.

Low-gossypol cottonseed meal can constitute high-quality daily feed for animal husbandry
Due to the presence of gossypol, the use of unprocessed cottonseed meal in large-scale livestock production is limited.However, with the development of MODERN AGRICULTURE -115 glandless cotton varieties and detoxification techniques, the restrictions on its use are decreasing and its use is increasing in the livestock industry.The utilization of low-gossypol cottonseed meal as a component of mixed diets in aquaculture, poultry, and swine production systems is advantageous because of its high nutritional value, favourable palatability, costeffectiveness, and normal growth production ability. 72elgado et al. investigated the functional properties of extruded shrimp feed containing ultra-low gossypol cottonseed meal and concluded that the use of cottonseed meal as a fishmeal substitute is a viable option for reducing the costs while maintaining balanced levels of minerals, amino acids, proteins, and essential fatty acids. 73In an independent study, the energy and nutrient digestibility of degossypolised cottonseed protein was evaluated as a supplementary feed source for nursery pigs. 74The results indicate that degossypolised cottonseed protein can be effectively utilised in nursery pig diets at proportions of 5% and 10% within 2 weeks and after 2 weeks of weaning, respectively.Moreover, the supplementation of cottonseed meal feed with iron and lysine can stimulate the growth of juvenile fish and enhance their immune system. 75Additionally, when the proportion of cottonseed meal in the feed reached 15%, the fattening performance of Holstein bulls was effectively improved. 76ow-gossypol cottonseed presents a promising opportunity as a plant-based animal protein feed with diverse applications.However, several key issues must be addressed before its widespread adoption.First, the absence of gossypol renders low-gossypol cotton vulnerable to pests and diseases, which results in less commonly cultivation. 62,77Therefore, breeding efforts should prioritise the selection of seeds with low gossypol levels and with a high resistance to pests and diseases.Second, although numerous methods such as physical, chemical, or biological methods are available for detoxifying cottonseed, [78][79][80][81][82] a unified standard should be implemented to ensure the consistency of the quality of the cottonseed meal products derived from different detoxification techniques.Furthermore, the functional effects and mechanisms of the various cottonseed meal detoxification treatments remain unclear and require further investigation.Finally, the optimisation of feed formulas utilising low-gossypol cottonseed meal as a protein source represents an important area for future research.Addressing these challenges is crucial to effectively harness the potential of cottonseed meal in animal nutrition.

Gossypol may act as a natural additive in cottonseed meal feed
As livestock and poultry farming continue to expand, the use of antiviral and antibiotic drugs during the growth process is increasing.Viruses, despite being simple noncellular microorganisms composed of nucleic acids and proteins, are responsible for a large number of diseases that spread rapidly owing to their high infectivity and pathogenicity.The excessive use of antiviral and antibiotic drugs in livestock and poultry farming not only results in drug residues, leading to the development of multidrug-resistant bacteria, but also poses a potential risk of the emergence of mutant strains.The overconsumption of animal products treated with these drugs can lead to drug accumulation in the human body, which may facilitate the development of antimicrobial resistance.In these cases, upon infection with viruses or bacteria, the efficacy of antiviral and antibiotic drugs on the human body may be limited.Therefore, implementing strict controls on the misuse of these medications can not only ensure food safety but also safeguard public health.
Nucleoside analogues are commonly used as antiviral drugs for viral prevention and control.Despite their efficacy in combating viral infections, these drugs carry inherent risks, such as gene mutation induction and high drug concentrations, which increase the likelihood of the development of drug residues.Plant-based nonnucleoside drugs have several advantages.Among these, gossypol, an RdRp protein inhibitor, exhibits broad-spectrum antiviral activity.The palm domain represents a key binding site for gossypol in coronavirus RdRp and is highly conserved across a variety of viral polymerases.This suggests that gossypol may effectively inhibit viral replication by targeting the polymerases of other genomes.We performed molecular docking simulations of gossypol molecules using polymerases from diverse viruses and observed that the binding energy between gossypol and the polymerases was less than −7.0 kcal/mol (data not shown), indicating its potential to inhibit the corresponding viruses, which warrants further investigation (Figure 1).
Additionally, gossypol exhibits broad-spectrum antibacterial activity.These findings suggest that undetoxified gossypol may serve as a natural feed additive for the prevention and control of pathogens in aquaculture, thereby having promising application prospects.
Furthermore, gossypol possesses contraceptive properties; however, its prolonged use is typically required to achieve the desired effect.Therefore, feed containing gossypol may be utilised as a short-term emergency 'medicine-feed homologous' feed and regularly administered for the prevention and control of viral or bacterial diseases in aquaculture.The likelihood of drug residue development caused by the short-term use of feed containing gossypol is relatively low.The treatment duration and dosage should be carefully considered.For example, in our previous study, when treated with a loading dose of 50 mg/kg on the first day, followed by a daily maintenance dose of 25 mg/kg for the next 4 days, gossypol significantly inhibited viral replication in mouse models infected with SARS-CoV-2.Moreover, the fertility of mice treated with 50 mg/kg gossypol for 20 days was not significantly affected, suggesting the feasibility of using gossypol as a shortterm treatment for animal diseases without causing animal sterility.However, few in vivo studies have used gossypol as an antiviral or antibacterial drug in animals, and its effective dose remains to be explored.As the current EU legislation on the use of gossypol-containing 116 -MODERN AGRICULTURE feed, the limit of free gossypol in complete feed for cattle (except for calves, 500 mg/kg), sheep (except for lambs, 300 mg/kg), goats (except for kids, 300 mg/kg), poultry (except for laying hens), and calves (100 mg/ kg), as well as rabbits, lambs, kids, and pigs (except for piglets, 60 mg/kg) may provide some reference information for the dose of gossypol to use to prevent disease in the future.

(þ)-Gossypol exhibits extensive potential for the application of cottonseed meal feed
Generally, natural gossypol is present as a racemic mixture of two enantiomers [(þ)-and (−)-enantiomers] with distinct optical properties owing to a restricted rotation around the binaphthyl ring.9][90][91] (−)-Gossypol, but not (þ)-gossypol, has male anti-fertility activity and is more toxic to animals than (þ)-gossypol. 83,89Wang et al. discovered that rats orally administered (−)-gossypol at a dose of 30 mg/kg for 1 week exhibited a significantly reduced body weight, whereas those treated with an equivalent amount of (þ)-gossypol showed no body weight reduce. 92In a feeding trial conducted by Bailey et al., broiler chickens fed a diet containing 5% cottonseed with a (þ)-to (−)-gossypol ratio of 83:17 exhibited weight gain rates equivalent to those observed in the control group receiving 100% soybean-based feed. 93egression analysis revealed that the cumulative weight gain of chickens decreased by approximately 126 g for every 100 mg increase in (−)-gossypol consumption, whereas no significant changes were observed in cumulative weight gain with a higher (þ)-gossypol consumption.In addition, Wang et al. discovered that (þ)-and (−)-gossypol exhibited comparable inhibitory effects on coronaviruses, indicating that the optical activity of gossypol does not affect its ability to inhibit coronaviruses.Yildirim et al. investigated the inhibitory effects of different gossypol enantiomers on Edwardsiella ictaluri and observed that (þ)gossypol exhibits greater inhibitory activity on this bacterium than (−)-gossypol. 70Yang et al. reported that (þ)-gossypol derivatives and their analogues exhibit greater activity against H5N1 than their corresponding (−)-gossypol counterparts. 15The aforementioned findings imply that cottonseed with high levels of (þ)gossypol possess promising potential for the application as natural 'substitute antibiotics (antivirals)' additives in animal feed.
CYP82D113, CYP71BE79, and 2-ODD-1; Finally, two hemigossypols are polymerised to form gossypol, in this process, dirigent proteins (DIR)-GhDIR3, GhDIR4, and GhDIR6 mediate the biogenic synthesis of (þ)-gossypol, while GhDIR5 mediates the biogenic synthesis of (−)gossypol. 61,97,98The knockdown of the GhDIR5 gene using CRISPR-Cas9-mediated gene editing technology can effectively prevent the formation of (−)-gossypol in cotton seeds but does not significantly alter the levels of (þ)-gossypol and other terpenoids, resulting in low or nontoxic cotton seeds.5][86] Besides, some key genes and transcription factors associated with the gossypol biosynthesis pathway have been identified, 99 which can be used to regulate gossypol content through genetic engineering techniques such as RNA interference and oligonucleotide-directed mutagenesis.
Therefore, creating a 'medical-feeding homologous' cottonseed feed for different purposes by utilising the nontoxic and broad-spectrum pathogenic microorganism inhibition characteristics of (þ)-gossypol in livestock and poultry is of great potential application and economic value.In addition to gossypol, many other active ingredients exist in cottonseed with many pharmacological activities (Table S2), which further endow cottonseed with feeding and medicinal value.Besides, reasonable reduction of gossypol content in cottonseed can not only improve the application of cottonseed meal in animal husbandry but also broaden the application field of other cottonseed products such as cottonseed oil and cottonseed shell.On the one hand, reducing the content of gossypol in cottonseed oil can improve the quality and safety of the oil; on the other hand, cottonseed oil with lower gossypol can be more suitable for use as a raw material for biofuels, providing more options for renewable energy.In addition, low-gossypol cottonseed shell may be more suitable for organic fertiliser, which helps to improve the quality of the agro-ecological environment.Therefore, regulating the content of gossypol and its enantiomers in cottonseed can not only enhance the feeding value of cottonseed meal but also increase the application value of other cottonseed products.

CONCLUSION
Protein deficiency, rampant infectious diseases, and antibiotic and antiviral abuse are major challenges faced by the animal husbandry industry.This article provides a comprehensive review of the viral diseases encountered during animal husbandry and their conventional prevention and control methods.In addition, we explored the use of cottonseed as an animal feed source and the obstacles that prevent its wide use.We dialectically propose the potential application of gossypol, particularly (þ)-gossypol, as a natural cottonseed feed additive.This highlights a new direction for farming and biological breeding using natural products as antibiotic substitutes.

F I G U R E 1
Application strategy of cottonseed in animal husbandry under different scenarios.(1) Glandless cottonseed with low-gossypol may provide high-quality daily feed for healthy animals; (2) Cottonseed containing gossypol may act as a short-term emergency 'medicinefeed homologous' feed for animals infected with viruses by targeting the viral polymerases.The viral polymerases RNA-dependent RNA polymerase (RdRp) is responsible for RNA (plus-sense [þ] ssRNA, minus-sense [−]ssRNA, and dsRNA) viruses, DNA-dependent DNA polymerases (DdDp, also called DNA polymerases) and DNA-dependent RNA polymerase (DdRp, also called RNA polymerases) are responsible for DNA (ssDNA and dsDNA) viruses, RNA-dependent DNA polymerase (RdDp, also called reverse transcriptase) is responsible for reverse-transcribing (ssRNA-reverse transcribing [rt] and dsDNA-rt) viruses.The targeted inhibition of gossypol on these viral polymerases was predicted by molecular docking (data not shown).114-MODERN AGRICULTURE