The research team, based at Ferdowsi University of Mashhad in Iran, worked with 390 male Ross 308 broiler chickens divided into six treatment groups, each with five replicates of 13 birds. The birds were fed either a basal control diet or diets supplemented with a six-strain probiotic containing Lactobacillus acidophilus, Lactobacillus rhamnosus, Pediococcus acidilactici, Bacillus subtilis, Bacillus megaterium and Bacillus amyloliquefaciens at a minimum concentration of one billion colony-forming units per gram, chelated zinc at 120 milligrams per kilogram of feed, chelated copper at 150 milligrams per kilogram, or all three additives combined. To reproduce the conditions under which necrotic enteritis strikes in commercial flocks, the researchers primed the birds with a twenty-fold dose of a coccidiosis vaccine on day 11 — coccidial damage being a classic predisposing factor for the disease — and then administered an aggressive culture of C. perfringens (strain ATCC 13124) at two times ten to the eighth CFU per millilitre on days 13, 14 and 15. The experiment ran to day 21, with growth, carcass traits, gut architecture, microbial populations, blood chemistry, cecal fatty acids, meat oxidation, lesion scores and mortality all measured along the way.

The challenge worked exactly as intended. Birds exposed to C. perfringens grew significantly less than their unchallenged counterparts, finishing the 21-day period at 541 grams of cumulative body weight gain against 584 grams for the clean control group, with feed conversion deteriorating from 1.58 to 1.78 grams of feed per gram of gain. The physiological explanation is well understood: the bacterium produces toxins that erode the intestinal epithelium, triggering inflammation that diverts energy and amino acids away from muscle growth toward immune defence, while the damaged gut lining absorbs nutrients less efficiently. The challenge also left its fingerprints everywhere else in the data — breast muscle yield dropped sharply, ileal lesion scores climbed to 2.80 on a four-point scale, mortality reached 6.15 percent, and meat lipid oxidation, measured as malondialdehyde, rose to 3.10 micrograms per gram.

Against this grim backdrop, the probiotic group emerged as the single most effective intervention. Birds receiving the strain mix on top of the challenge regained nearly all the lost ground, achieving 581 grams of weight gain and posting the best early feed conversion of any challenged group. Under the microscope, the difference was striking: duodenal villus height in the probiotic group reached almost 1600 micrometres, the highest of any treatment and far above the 1121 micrometres seen in challenged, unsupplemented birds, while crypt depth fell and the villus height-to-crypt depth ratio climbed to 8.22 — a classic signature of a healthy, absorptive intestine. The probiotic also reshaped the microbial landscape, cutting ileal C. perfringens counts from 14 million to 5.5 million CFU per gram and boosting resident Bacillus populations, the very organisms best known for producing antibacterial compounds such as lichenicidin that punch holes in pathogenic cell membranes.

Chelated zinc told a complementary story. Zinc-supplemented challenged birds matched the probiotic group in body weight, and their feed intake over the full rearing period was the highest of any treatment at 985 grams. The mineral’s benefits likely stem from its well-documented role in tightening the intestinal barrier and modulating inflammation: earlier work cited by the team shows chelated zinc upregulates the anti-inflammatory cytokine IL-10 while suppressing IL-8 and other pro-inflammatory signals, restoring intestinal cell membrane integrity during infection. Zinc’s superior performance relative to inorganic sources is usually attributed to the higher bioavailability of chelated forms, which are absorbed through different pathways and place less demand on plasma albumin for transport. Ileal lesion scores in the zinc group fell to 1.60, and lipid oxidation in thigh meat dropped to 2.65 micrograms per gram, reflecting zinc’s participation in the bird’s antioxidant machinery.

Copper, by contrast, was the weakest solo performer. Birds given chelated copper grew no better than the unsupplemented challenge group over the full period and actually showed the lowest early body weight of all treatments. Yet copper was not without value: challenged birds receiving it recovered breast muscle yield to 18.53 percent of live weight, essentially matching the unchallenged control, and mortality fell to 1.53 percent. The authors note that copper’s antibacterial reputation rests largely on pharmacological doses around 250 milligrams per kilogram, considerably above the 150 milligrams per kilogram used here, which may explain the muted growth response.

The triple combination produced a genuinely mixed verdict. On several fronts it excelled: birds receiving probiotics, zinc and copper together suffered zero mortality, scored the lowest ileal lesion severity of any challenged group at 1.40, restored breast yield to 18.92 percent, and preserved jejunum architecture so well that their villus height-to-crypt depth ratio of 7.68 rivalled the healthy control. Blood biochemistry also shifted, with the combination group showing the lowest serum glucose and HDL cholesterol, consistent with altered lipid and metal metabolism when both minerals arrive in organic complexes. However, the trio failed to replicate the probiotic’s standout gains in body weight and duodenal villus height, and it delivered only intermediate results for feed intake.

The cecal short-chain fatty acid data reveal why more is not always better. Probiotic supplementation raised acetate, propionate and butyrate concentrations toward or above control levels — butyrate being the preferred fuel of enterocytes and a critical reinforcement agent for tight junctions. Zinc and copper given alone, however, sharply suppressed the fatty acid profile, with acetate falling to 25.56 and 22.50 micrograms per gram respectively. This suppression is thought to reflect the sensitivity of Lactobacillus species, key fatty-acid producers, to elevated concentrations of both metals, with copper reported in prior studies to eliminate beneficial strains such as Lactobacillus johnsonii outright. The combination group, containing the probiotic, largely avoided this pitfall, posting acetate and butyrate values statistically comparable to the control — evidence that the live microbes could partially buffer the antibacterial pressure of the minerals on the fermentative community.

The study’s overall picture is nuanced rather than triumphalist. C. perfringens unquestionably devastates growth, gut morphology, meat quality and survival, and probiotic supplementation stands out as the most consistently protective single intervention, working through competitive exclusion, organic acid production, bacteriocin release and direct architectural repair of the villi. Zinc earns its place as a robust second pillar, particularly for feed intake and barrier integrity, while copper contributes to meat yield and survival but underperforms at the dose tested. The three-way combination shines where it matters most to producers — mortality, lesion severity and breast yield — but dilutes some of the probiotic’s growth-promoting power, possibly through mineral-driven suppression of the very bacteria that make probiotics work. For an industry searching for antibiotic alternatives, the message is that nutritional strategies must be designed with their interactions in mind: the goal is not simply to stack protective additives, but to balance their antimicrobial potency against their capacity to nurture the beneficial fermenters that keep the gut — and the flock — thriving.

Subject of Research: The effects of multi-strain probiotics, chelated zinc and chelated copper on broiler chickens challenged with Clostridium perfringens.

Article Title: The Effect of Multi‐Strain Probiotics, Zinc and Copper on the Performance of Broiler Chickens Challenged With Clostridium perfringens

Article References: Kazemi, A., Kermanshahi, H., Heravi, R. M., Jouzani, G. S., & Javadmanesh, A. (2026). The Effect of Multi‐Strain Probiotics, Zinc and Copper on the Performance of Broiler Chickens Challenged With Clostridium perfringens. Veterinary Medicine and Science, 12(5), Article e71176. https://doi.org/10.1002/vms3.71176

Image Credits: AI Generated

DOI: 10.1002/vms3.71176

Keywords: necrotic enteritis, Clostridium perfringens, broiler chickens, probiotics, zinc supplementation, copper supplementation, gut health, intestinal microbiota, short-chain fatty acids, poultry nutrition, antibiotic alternatives, feed conversion ratio