Effects of protease enzyme supplementation in weanling pigs’ diet with different crude protein levels on growth performance and nutrient digestibility

Abstract

The purpose of this study is to determine the impact of different levels of crude protein (CP) diets supplemented with dietary protease on the growth performance and nutrient digestibility of the weanling pigs. In a 5-week study, 100 crossbred ([Landrace × Yorkshire] × Duroc) of weaner pigs that have an average initial body weight (BW) of 7.17±1.06 kg were assigned to one of four dietary treatments with 5 replications and 5 pigs (3 gilts and 2 castrated male pigs) per pen in a randomized complete block design. The dietary treatments were as follows: Phase 1: CON: basal diets (20.60% CP); low protein (LP): CON − 0.30% CP; PLP1: (CON − 0.30% CP) + 0.05% protease; PLP2: (CON − 0.50% CP) + 0.05% protease. Phase 2: CON: basal diets (18.88% CP); LP: CON − 0.30% CP; PLP1: (CON − 0.30% CP) + 0.05% protease; PLP2: (CON − 0.50% CP) + 0.05% protease. The addition of protease to low CP diets significantly increased the feed conversion ratio (FCR) (p = 0.039), BW (p = 0.046), average daily gain (ADG) (p = 0.049), and average daily feed intake (ADFI) tended to increase (p = 0.053) in the young pigs during phase 1. However, FCR tended to increase throughout the experiment but did not change during phase 2, whereas BW, ADG, and ADFI stayed unchanged throughout phase 2 and overall. There was no significant difference in dry matter, nitrogen (N), and gross energy of nutrient digestibility in all phases and overall in weaned pigs with low CP when protease was fed. In contrast, adding protease to the low CP diets increased the tendency of N digestibility (p = 0.059) during phase 1. It is concluded that dietary protease supplementation tended to increase N retention during the first phase of the weaning period, hence increasing piglet performance.

INTRODUCTION

The pig’s weaning period is possibly the most stressful time of its productive life and is the foundation of a thriving lifetime of growth performance [1]. During this critical period, weaned piglets face a variety of problems, including post-weaning diarrhea and reduced feed intake due to an immature immune system and limited enzyme secretory capacity [2], both of which are economically significant in today’s pig industry. Therefore, it is critical to improve the weanling pigs’ ability to digest nutrients and establish a healthy and strong foundation for long-term growth and profitability. The addition of exogenous enzymes to the pig diets with reduced crude protein (CP) can improve the growth performance. This is due to improved protein digestibility and amino acid levels, allowing weanling pigs to thrive and continue to benefit later in life. As a result, excessive nutrient excretion is prevented, allowing for lower production costs. Exogenous enzymes supplemented as pig diet additives hydrolyze non-starch polysaccharides (NSP), break the cell wall that encapsulates them, degrade anti-nutritional factors (protease inhibitors, antigenic proteins, non-protein amino acids), and break glycolytic bonds that are not hydrolyzed by endogenous enzymatic activity [3–5]. Exogenous enzymes that can be introduced into pig diets include phytases, carbohydrases, proteases, and lipases [6]. Pigs can produce digestive proteases such as pepsin, trypsin, and chymotrypsin to break down proteins [7]. However, the secretion of endogenous proteases (trypsin, chymotrypsin) decreases dramatically during the weaning stage [8]. In a study by Lindemann et al. [2], weanling piglets experienced a severe and rapid drop in endogenous protease (trypsin and chymotrypsin) secretion after weaning. This drop was due to post-weaning stress or a lack of solid feed intake after weaning and it required more than two weeks to return to normal secretion levels. Because of the decrease in endogenous protease production, there is a possibility of an increase in the flow of undigested protein to the hindgut, leading to the production of ammonia, which is harmful to gut health and digestive capacity. It has been suggested that adding exogenous enzymes (such as protease) to post-weaning diets can aid in controlling post-weaning challenges by compensating for underdeveloped endogenous enzyme secretory capacity and enhancing nutrient digestibility in weaned pigs [1]. Proteases are enzymes that can be produced in the digestive tract [9]. Based on their catalytic mechanisms, proteases can be divided into six groups: Metalloproteases, cysteine, serine, and threonine proteases, aspartic, and glutamic [10]. Several studies have shown that protease improves the apparent ileal digestibility of gross energy (GE), dry matter (DM), CP, starch, NSP, phytate, isoleucine, valine, and aspartic acid during the weaning stage. Apparent total tract digestibility of DM, GE, CP, starch, NSP, phytate, and utilization of phosphorus; nutrient digestibility and modification of microbial communities in the posterior intestine; concentrations of acetic, propionic, and butyric acids in the cecum and colon; fermentation capacity in the intestine and a longer transit time are improved by protease [5,11–13]. Many studies have shown that exogenous protease supplementation in diets improves growth performance and nutrient digestibility, particularly for CP and amino acids [14,15]. Even though the objective of exogenous enzyme supplementation in the diet is to promote growth performance and nutrient digestibility, a review of the literature revealed that the protease results published to date are inconsistent [7]. The numerous functions of proteases may be affected by dietary formulation and ingredients [16]. Nevertheless, dietary protease supplementation can compensate for decreased endogenous protease secretion and boost digestive capacity in weanling pigs. Therefore, the focus of this research was to assess the effect of protease supplementation on growth performance and nutrient digestibility of the weanling pigs.

MATERIALS AND METHODS

This experiment was conducted at Dankook University’s Swine Research Unit in Jeonui (Sejong, Korea). Dankook University’s Institutional Animal Care and Use Committee in Cheonan, Korea, examined and approved the outlined protocols (Protocol number DK-1-2106) and weanling pigs used in this study.

Protease enzyme

The protease enzyme used in this study was a commercial product purchased from (Ronozyme ProAct^®, Novozymes A/S, Bagavaerd, Denmark) synthesized from Bacillus licheniformis submerged fermentation.

Experimental design, animal housing, and diets

In the 5-weeks of the experiment, a total of a hundred crossbred [(Landrace × Yorkshire) × Duroc] weanling pigs were assigned to one of four dietary treatments based on the average initial body weight (BW) of (7.17 ± 1.06 kg) and sex. They were then divided into 5 replications, with each pen containing 5 pigs (3 gilts and 2 castrated barrows) in a randomized complete block design. The nutritional diets were provided in two distinct phases, phase 1 (0 to 14 days) and phase 2 (15 to 35 days). The dietary treatments were as follows:

Phase 1: CON: basal diets (20.60% CP); low protein (LP): CON - 0.30% CP; PLP1: (CON − 0.30% CP) + 0.05% protease; PLP2: (CON − 0.50% CP) + 0.05% protease.

Phase 2: CON: basal diets (18.88% CP); LP: CON − 0.30% CP; PLP1: (CON − 0.30% CP) + 0.05% protease; PLP2: (CON − 0.50% CP) + 0.05% protease.

The basal diets were prepared following National Research Council (NRC) standards and were designed to meet or exceed the nutrient requirements of pigs [17]. The dietary composition of weanling pigs utilized in this experiment is illustrated in Table 1. The room temperature was kept at 28°C, and each pen was outfitted with a self-feeder and nipple drinker, allowing the weanling pigs’ free access to feed and water throughout the research.

Table 1. Diets composition (as-fed basis).

	Phase 1 (0–14 days)				Phase 2 (15–35 days)
	CON1)	LP	PLP1	PLP2	CON	LP	PLP1	PLP2
Corn	36.17	36.87	36.78	37.28	62.56	63..30	63.20	63.68
Expeller-pressed corn	15.00	15.00	15.00	15.00	5.00	5.00	5.00	5.00
Lactose	10.00	10.00	10.00	10.00	3.00	3.00	3.00	3.00
Dehulled soybean meal	12.14	11.49	11.50	11.03	10.36	9.66	9.68	924
Hamlet protein	10.00	10.00	10.00	10.00	10.00	10.00	10.00	10.00
Plasma protein	6.00	6.00	6.00	6.00	4.00	4.00	4.00	4.00
Whey protein	5.00	5.00	5.00	5.00	-	-	-	-
Soybean oil	1.26	1.26	1.29	1.28	0.70	0.69	0.72	0.72
Limestone powder	1.20	120	120	120	0..99	0..99	0..99	0..99
Mono-calcium phosphate	1.23	123	123	123	133	133	133	133
Methionine	0.15	0.14	0.14	0.14	0.12	0.11	0.11	0.10
Lysine	0.32	028	028	026	0.41	0.39	0.39	036
Choline chloride	0.10	0.10	0.10	0.10	0.10	0.10	0.10	0.10
Zinc oxide	0.23	0.23	0.23	0.23	0.23	0.23	0.23	0.23
Vitamin-mineral mix2)	0.50	0.50	0.50	0.50	0.50	0.50	0.50	0.50
Saline	0.40	0.40	0.40	0.40	0.40	0.40	0.40	0.40
Chromium oxide	0.30	030	030	030	030	030	030	030
Protease	-	-	0.05	0.05	-	-	0.05	0.05
Total	100.00	100.00	100.00	100.00	100.00	100.00	100.00	100.00
Calculated value
Moisture	9.85	9.87	9.86	9.87	11.43	11.45	11.44	11.45
Crude protein (%)	20.60	20.30	20.30	20.10	18.88	18.58	18.58	18.38
Crude fat (%)	3.30	3.32	3.35	3.35	324	325	328	329
Crude fiber (%)	1.66	1.66	1.65	1.65	1.82	1.81	1.81	1.80
Crude ash (%)	6.48	6.45	6.45	6.43	5.83	5.80	5.80	5.78
Calcium (%)	0.79	0.79	0.79	0.79	0.69	0.69	0.69	0.69
Phosphorus (%)	0.62	0.62	0.62	0.62	0.61	0.60	0.60	0.60
Lysine (%)	1.53	1.48	1.48	1.45	1.40	136	136	133
Methionine (%)	0.44	0. 43	0.43	0.42	0.40	0.39	0.39	0.38
Metabolizable energy (kcal)	3.400	3.400	3.400	3.400	3.350	3.350	3.350	3.350

¹⁾CON, basal diet; LP, CON - 0.3% CP; PLP1, LP + 0.05% protease; PLP2, (CON - 0.50% CP) + 0.05% protease.

²⁾Provided per kilogram of complete diet: vitamin A, 10,000 IU; vitamin D₃, 2,000 IU; vitamin E,48 IU; vitamin K₃, 1.5 mg; riboflavin, 6 mg; niacin, 40 mg; d-pantothenic, 17 mg; biotin, 0.2 mg; folic acid, 2 mg; choline, 166 mg; vitamin B₆, 2 mg; and vitamin B₁₂, 28 mg, Fe (as FeSO₄∙7H₂O), 90 mg; Cu (as CuSO₄∙5H₂O), 15 mg; Zn (as ZnSO₄), 50 mg; Mn (as MnO₂), 54 mg; I (as KI), 0.99 mg; and Se(as Na₂SeO₃∙5H₂O), 0.25 mg.

LP, low protein; CP, crude protein.

Sampling, measurements, and laboratory procedures

To compute the growth performance, the individual piglet’s BW was measured using a GL-6000S balance machine (G-Tech International, Uijiengbu, Korea) on days 14 and 35 to determine the average daily gain (ADG). Meanwhile, the amount of feed ingested and feed residues (on a per-pen basis) were recorded to calculate the average daily feed intake (ADFI) and feed conversion ratio (FCR).

Seven days before fecal sample collection, 2 g/kg of indigestible marker chromium oxide (Cr₂O₃) was added to weanling pig diets to determine nutrient digestibility of nitrogen (N), DM, and GE. Fresh fecal samples were collected by qualified personnel at the end of week 5 from two pigs per pen (1 gilt and 1 barrow) using a rectal massage technique, immediately placed in an ice box, delivered to the laboratory, and maintained at −20°C until examined. Then the samples were dried for 72 hours in a forced-air oven at 70°C before being finely powdered to pass through a 1-mm screen sieve. The AOAC recommended procedures were used to analyze the DM. UV absorption spectrophotometry was used to determine the amount of chromium in the sample (UV-1201, Shimadzu, Kyoto, Japan) and the findings were recorded for statistical analysis. 2 grams of feed and fecal sample were measured and assigned in an oxygen bomb calorimeter Parr 6400 to calculate GE (Parr Instrument Company, Moline, Il, USA). A Kjeltec 8600 analyzer was used to determine the N content (Foss Tecator AB, Hoeganaes, Sweden). The following formula was used to calculate the nutrient digestibility: N digestibility = 1 − [(Nf × Cd) / (Nd × Cf)]; N – nitrogen, Nf – nutrient concentration in feces, Nd – nutrient concentration in diet, Cd – chromium concentration in diet, and Cf – chromium concentration in feces.

Statistical analysis

The data from the experiments were cleaned and exported to SAS software (SAS Institute, Cary, NC, USA) for analysis. They were analyzed using a completely randomized block design by using SAS’s Generalized Linear Model procedure. The pen was regarded as the experimental unit. The GLM approach was used to assess significant differences in mean values among dietary treatments using repeated measurements and Duncan’s tests. The standard error means were used to express data variability. Probabilities less than 0.05 were deemed significant.

RESULTS

Growth performance

Table 2 displays the growth performance data. The findings indicated that the inclusion of protease in low CP diets enhanced the weanling pigs’ BWG (p = 0.046), ADG (p = 0.049), FCR (p = 0.039), and ADFI tended to increase (p = 0.053) during phase 1. However, FCR tended to increase (p = 0.076) throughout the experiment but was not affected during phase 2, and also BW, ADG, and ADFI remained unaltered throughout phase 2 and overall.

Table 2. Effect of supplementing dietary protease to low CP diet on growth performance in weanling pigs.

Items	CON1)	LP	PLP1	PLP2	SEM	p-value
Body weight (kg)
Initial	7.17	7.17	7.17	7.17	0.003	0.887
Phase 1	11.74a	11.38b	11.61ab	11.34b	0.100	0.046
Phase 2	21.66	21.17	21.54	21.39	0.220	0.463
Phase 1 (0–14 days)
ADG (g)	326a	300b	317ab	297b	7.0	0.049
ADFI (g)	413	385	405	385	8.0	0.053
FCR	1.266b	1.284ab	1.279ab	1.296a	0.006	0.039
Phase 2 (15–35 days)
ADG (g)	472	466	473	479	11.0	0.867
ADFI (g)	718	724	724	742	15.0	0.717
FCR	1.521	1.553	1.530	1.550	0.014	0.312
Overall
ADG (g)	414	400	410	406	6.0	0.476
ADFI (g)	596	588	596	599	6.0	0.858
FCR	1.441	1.472	1.453	1.475	0.010	0.076

¹⁾CON, basal diet; LP, CON − 0.3% CP; PLP1, LP + 0.05% protease; PLP2, (CON − 0.50% CP) + 0.05% protease.

^a,bMeans groups that have different superscripts in the same row are distinct (p < 0.05).

CP, crude protein; LP, low protein; ADG, average daily gain; ADFI, average daily feed intake; FCR, feed conversion ratio.

Nutrient digestibility

The nutrient digestibility of N, DM, and GE is illustrated in Table 3. Dietary protease supplementation in weaned pigs with low CP had no effect on N, DM, and GE on nutrient digestibility in all phases and overall. However, the addition of protease to the low CP diets increased tendency of N digestibility (p=0.059) during phase 1.

Table 3. Effect of dietary protease supplementation on nutrient digestibility in weanling pigs fed a low-CP diet.

Items	CON1)	LP	PLP1	PLP2	SEM	p-value
Phase 1 (0–14 days)
Dry matter	77.28	76.71	78.11	78.17	0.725	0.373
Nitrogen	69.26	70.98	73.26	72.17	1.008	0.059
Gross energy	74.89	75.02	75.79	75.42	0.670	0.814
Phase 2 (15–35 days)
Dry matter	74.87	74.89	75.26	76.81	1.187	0.625
Nitrogen	67.85	68.66	68.56	69.88	1.838	0.888
Gross energy	72.97	72.40	72.62	74.38	1.340	0.727

¹⁾CON, basal diet; LP, CON − 0.3% CP; PLP1, LP + 0.05% protease; PLP2, (CON − 0.50% CP) + 0.05% protease.

^a,bMeans groups that have different superscripts in the same row are distinct (p < 0.05).

CP, crude protein; LP, low protein.

DISCUSSION

Growth performance

Exogenous protease enzyme supplementation to the diets to promote protein usage has attracted the pig industry’s interest. Generally, dietary protease has been utilized as a mono-component or as part of an enzyme combination in poultry and swine diets [13,18]. Protein is required for the metabolism and physiology of animals, and changing CP levels in isoenergetic diets, on the other hand, is linked to changes in energy-to-protein ratios, which can have a big impact on body composition such as body fat content [19]. Exogenous enzymes should be studied because they have been found to assist lower the amount of nutrients in animal diets, especially CP. Various researchers have reported that the inclusion of protease in the diet of swine can enhance their growth performance. Protease supplementation increased ADG and G: F ratio (p < 0.05) in finishing pigs fed a protease-supplemented diet during weeks 6–12 [20]. Moreover, Lei et al. [21] reported that supplementing protease to a low CP diet increased growth performance. In the current study, weanling pigs fed low CP diets and treated with protease had better BW, ADG, and FCR during phase 1, and also the ADFI tended to increase when protease was added to the weaned pigs’ diets. Contrary to expectations, this study found no significant differences in growth performance between the four groups during phase 2 or throughout the trial. The release of endogenous enzymes is lower in younger animals due to post-weaning stress or a lack of solid feed intake after weaning [22]. As a result, young animals are more responsive to exogenous enzyme supplementation, which is likely why phase 1exhibited such a great positive result. Although these findings differ from those of some previously published studies [9,23], they are consistent with a study conducted on broiler chickens by Yan et al. [24] who found that supplementing protease to broiler chickens’ diet during the starter phase had a stronger effect than during other growth phases. Moreover, the current study’s findings are consistent with those of Lei et al. [21], who discovered improved growth performance in finishing pigs fed protease-supplemented diets. Further research on the effect of protease on pig growth performance is needed to resolve the inconsistencies in available data.

Nutrient digestibility

Protease is recognized as a dietary enzyme that aims to increase protein availability by targeting tight protein binding. Protease improves protein digestibility by hydrolyzing proteins into free amino acids and peptides that can be absorbed in the small intestine [25]. Protease supplementation has been shown to improve performance in both normal and reduced protein diets [26]. In the current experiment, we discovered that protease had no effect on N, DM, and GE on the nutrient digestibility in either each phase or overall. The lack of effect of the supplemented protease in the second phase could be attributed to the post-weaning phase, where pancreatic protease secretions were in an exponential phase [18] and the endogenous enzyme metabolism could have reduced the activity of the supplemented protease. However, adding protease to the low CP diets, increased the tendency of N digestibility during phase 1. This negative nutrient digestibility response in the current study could be attributed to the substrate released by the enzyme, which may have been used by commensal bacteria rather than the pig’s metabolism, as improvements in nutrient digestibility do not always translate to significant increases in metabolic energy. Although many prior studies claimed considerable improvements in nutrient digestibility [11,27–29]. For example, Tactacan et al. [11] discovered that pigs fed the protease diet had higher apparent total tract digestibility of DM and N (p < 0.05) than pigs fed the CON diet. In addition, Guggenbuhl et al. [18] found that dietary serine protease generated by B. licheniformis fermentation increased apparent ileal amino acid digestibility in weaned piglets. However, other research studies produces the results that are consistent with our current findings. For instance, Mohammadigheisar and Kim [9] discovered that the addition of dietary protease did not affect N digestibility. It is possible that young animals are more sensitive to supplemental protease, in agreement with the findings of this study.

CONCLUSIONS

By incorporating protease into the feed of weaned pigs, we were able to reduce dietary CP levels while having no negative effect on piglets’ performance. The introduction of exogenous protease to the diets of weanling pigs improved their BW, ADG, and FCR during phase 1, and also the ADFI tended to increase when protease was added to the weaned pigs’ diets. Furthermore, the findings revealed that adding exogenous protease to weaned pig feed tended to improve N digestibility during phase 1. Therefore, supplementing dietary protease into piglets’ diets should be considered, as the results of the current study show that it can improve piglet performance.