WO2024044569A1 - Compositions and methods for improving hen welfare with beta-mannanase and probiotics - Google Patents
Compositions and methods for improving hen welfare with beta-mannanase and probiotics Download PDFInfo
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- WO2024044569A1 WO2024044569A1 PCT/US2023/072621 US2023072621W WO2024044569A1 WO 2024044569 A1 WO2024044569 A1 WO 2024044569A1 US 2023072621 W US2023072621 W US 2023072621W WO 2024044569 A1 WO2024044569 A1 WO 2024044569A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2494—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01078—Mannan endo-1,4-beta-mannosidase (3.2.1.78), i.e. endo-beta-mannanase
Definitions
- the present disclosure relates to supplements comprising beta-mannanase comprising beta-mannanase and/or a probiotic. Furthermore, methods of administering the supplements to avians such as chickens are provided to demonstrate beneficial effects on the avians.
- the compositions and methods described herein can benefit overall animal wellbeing and future productivity, especially in egg-laying chickens.
- Probiotics can reduce heat stress and abnormal behavior as well as improve health in broiler chickens. Such responses occur from the regulatory power of probiotics under the micro biota-gut-brain axis. Probiotics also alleviate the stress response along the hypothalamic-pituitary-adrenal axis, reducing plasma or brain levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone.
- Enzyme supplementation is another strategy that can benefit the gut health status by reducing the impacts of anti-nutritional components.
- the use of -mannanase can help nonruminant animals dealing with the non-starch polysaccharides, which can reduce nutrient digestibility.
- Such components are found in plant cell walls and are present in many ingredients largely used in animal feeding, such as soybeans.
- P-mannans Among the main hemicelluloses found in plant cell walls are P-mannans, which can also be found on the surface of microorganisms.
- the animal's innate immune system is activated when foods that contain P-mannans are ingested, which responds with the proliferation of monocytes, macrophages, dendritic cells, and increased production of cytokines.
- this enzyme can improve the digestibility of mannans, increasing the population of beneficial bacteria, improving immunity, digestion and absorption of nutrients, in addition to limiting the proliferation of potential pathogens in the intestine.
- the present disclosure provides supplements comprising beta-mannanase comprising beta-mannanase and/or a probiotic as well as associated methods.
- the compositions and methods of the present disclosure surprisingly provide a synergistic effect for improve avian behaviors and egg laying qualities.
- the present disclosure describes supplements comprising beta-mannanase (e.g., Hemicell HTTM) and a probiotic (e.g., ProtexinTM) that can be added to feed to improve animal health and animal welfare.
- Figure 1 shows examples of eggs with minor presence of feces.
- Figure 2 shows examples of eggs with major presence of feces.
- Figure 3 shows ultramicroscopy images collected in a single eggshell during the first phase.
- Figure 4 shows ultramicroscopy images collected in a single eggshell during the second phase.
- Figure 5 shows ultramicroscopy images collected in a single eggshell during the third phase.
- a method comprising a step of administering one or more supplements to a non-human animal.
- the animal is an avian.
- the avian is selected from the group consisting of a chicken, a turkey, and a duck.
- the avian is a chicken.
- the chicken is a hen.
- the hen is a laying hen.
- the avian is present in a cage system.
- the supplement comprises HemicellTM. In an embodiment, the supplement comprises Hemicell HTTM. In an embodiment, the supplement comprises beta- mannanase, also referred to herein as “0- mannanase”.
- the beta-mannanase is provided by fermentation of Paenibacillus lentus. In an embodiment, the beta-mannanase is administered at a dose between 100 g/ton and 500 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 100 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 200 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 300 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 400 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 500 g/ton.
- the supplement comprises ProtexinTM.
- the supplement comprises a probiotic supplement.
- the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles.
- the supplement comprises a probiotic supplement comprising Enterococcus faecium.
- the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181
- the probiotic supplement is administered at a dose between 10 g/ton and 100 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 10 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 20 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 30 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 40 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 50 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 60 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 70 g/ton.
- the probiotic supplement is administered at a dose of 80 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 90 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 100 g/ton.
- the supplements comprise i) beta-mannanase and ii) a probiotic.
- the beta-mannanase and the probiotic provide a synergistic effect in improvement in health of the animal.
- the supplement is present in a feed for the animal.
- the feed is a free choice feed.
- the administration provides an improvement in health of the animal.
- the improvement in health of the animal is an improvement in animal welfare.
- the improvement in health of the animal comprises an improvement in eating behavior.
- the improvement in health of the animal comprises an improvement in walking behavior.
- the improvement in health of the animal comprises an improvement in standing behavior.
- the improvement in health of the animal comprises an improvement in sitting behavior.
- the improvement in health of the animal comprises an improvement in liquid consumption behavior. In an embodiment, the improvement in health of the animal comprises an improvement in feather exploring behavior. In an embodiment, the improvement in health of the animal comprises an improvement in head scratching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in wing flapping behavior. In an embodiment, the improvement in health of the animal comprises an improvement in leg stretching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in stretching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in aggressive pecking behavior. In an embodiment, the improvement in health of the animal comprises an improvement in non- aggressive pecking behavior.
- the improvement in health of the animal comprises a decrease in lesions on the animal.
- the lesions are present on the neck.
- the lesions are present on the tail.
- the lesions are present on the cloaca.
- the lesions are present on the comb.
- the improvement in health of the animal comprises an improvement in intestinal health in the animal. In an embodiment, the improvement in health of the animal comprises prevention of intestinal dysbiosis in the animal.
- the improvement in health of the animal comprises a reduction in pro-inflammatory effect in the animal.
- the improvement in health of the animal comprises increased release of mannan-oiligosaccharides (MOS) in the animal.
- MOS mannan-oiligosaccharides
- the administration provides an improvement in egg quality of one of more eggs laid by the animal.
- the improvement in egg quality is indicated by a modification of a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh unit, shell weight, shell breaking strength, albumen pH, yolk pH, yolk color score, yolk lightness, yolk redness, yolk yellowness, chroma, and any combination thereof.
- the improvement in egg quality is indicated by a modification of weight.
- the improvement in egg quality is indicated by a modification of specific gravity.
- the improvement in egg quality is indicated by a modification of albumen height.
- the improvement in egg quality is indicated by a modification of albumen weight. In an embodiment, the improvement in egg quality is indicated by a modification of yolk height. In an embodiment, the improvement in egg quality is indicated by a modification of yolk length. In an embodiment, the improvement in egg quality is indicated by a modification of yolk index. In an embodiment, the improvement in egg quality is indicated by a modification of yolk weight. In an embodiment, the improvement in egg quality is indicated by a modification of Haugh unit. In an embodiment, the improvement in egg quality is indicated by a modification of shell weight. In an embodiment, the improvement in egg quality is indicated by a modification of shell breaking strength. In an embodiment, the improvement in egg quality is indicated by a modification of albumen pH.
- the improvement in egg quality is indicated by a modification of yolk pH. In an embodiment, the improvement in egg quality is indicated by a modification of yolk color score. In an embodiment, the improvement in egg quality is indicated by a modification of yolk lightness. In an embodiment, the improvement in egg quality is indicated by a modification of yolk redness. In an embodiment, the improvement in egg quality is indicated by a modification of yolk yellowness. In an embodiment, the improvement in egg quality is indicated by a modification of chroma. In an embodiment, the modification is an increase in measurement. In an embodiment, the modification is a decrease in measurement. [0029] In an embodiment, the administration provides an improvement in reactive oxygen species (ROS) in the animal. In an embodiment, the improvement in ROS is in the intestine of the animal. In an embodiment, the improvement in ROS is in the oviduct of the animal.
- ROS reactive oxygen species
- the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thios (TSH), and any combination thereof.
- the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS).
- the improvement in ROS is indicated by a modification of glutathione S-transferase (GST).
- the improvement in ROS is indicated by a modification of protein thios (TSH).
- the modification is an increase in measurement.
- the modification is a decrease in measurement.
- the administration provides an increase in egg laying rate of the animal. In an embodiment, the administration provides an increase in fresh egg weight for eggs laid by the animal. In an embodiment, the administration provides a reduction in coefficient of variability of egg weight for eggs laid by the animal. In an embodiment, the administration provides an increase in an egg component mass for eggs laid by the animal. In an embodiment, the egg component mass is egg mass. In an embodiment, the egg component mass is yolk mass. In an embodiment, the egg component mass is albumen mass. In an embodiment, the egg component mass is egg shell mass.
- the administration provides an increase in occurrence of clean eggs laid by the animal.
- the increase in occurrence of clean eggs is a reduction in the presence of feces on the eggs.
- the administration provides a modification in eggshell thickness in eggs laid by the animal.
- the modification in eggshell thickness is an increase in eggshell thickness.
- the modification in eggshell thickness is a decrease in eggshell thickness.
- the modification in eggshell thickness is observed during a first phase of about 40 weeks. In an embodiment, the modification in eggshell thickness is observed during a second phase of about 44 weeks. In an embodiment, the modification in eggshell thickness is observed during a third phase of about 48 weeks.
- a method comprising a step of administering one or more supplements to a nonhuman animal.
- probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles, or wherein the supplement comprises a probiotic supplement comprising Enterococcus faecium, optionally wherein the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181.
- NCIMB National Collection of Industrial, Food and Marine Bacteria
- control treatment a basal diet without supplementation with any other additive
- P-mannanase control diet supplemented with 300 g/ton of -mannanase
- probiotic control diet supplemented with 50 g/ton of a multi- cepa probiotic additive
- P-mannanase plus probiotic treatment control diet supplemented with 300 g/ton of P-mannanase and 50 g/ton of a multi-cepa probiotic additive.
- the P-mannanase (Hemicell HTTM, Elanco Animal Health) for the instant example was an exogenous enzyme from the fermentation of the Paenibacillus lentus bacteria.
- the probiotic additive (ProtexinTM Concentrate, Elanco Animal Health) for the instant example includes Lactobacillus acidophilus (2.06 x 10 8 UFC/g), Lactobacillus bulgaricus (2.06 x 10 8 UFC/g), Lactobacillus plantarum (1.26 x 10 8 UFC/g), Lactobacillus rhamnosus (2.06 x 10 8 UFC/g), Bifidobacterium bifdum (2.0 x 10 8 UFC/g), Enterococcus faecium (6.46 x 10 8 UFC/g), and Streptococcus thermophilus (4.10 x 10 8 UFC/g).
- the basal diet comprised a com-soybean meal-based feed formulated according to nutritional requirements and inert material (kaolin) was included in basal feed to replace - mannanase and/or probiotic additives. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.
- the birds were housed in conventional sheds, arranged in an east-west direction, with concrete floors and masonry walls complemented with wire mesh to the ceiling. The shed was equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. The average minimum and maximum temperature and air relative humidity values recorded were 18 and 36 °C, and 35.8 and 94.7%, respectively.
- the lighting regime was composed of 16 hours of light and 8 hours of dark per day.
- Behavioral assessments were performed via image capture combined with local feather scoping and comb abnormalities assessments.
- For the behavior assessment six birds per treatment (one per cage) were randomly selected for observation. The captured images were carried out for 7 consecutive days, in a period of 15 minutes in the morning (e.g., the highest peak of laying of the birds) and 30 minutes in the afternoon divided into two periods corresponding to the hottest and cooler times of the day. Images were recorded and analyzed by visual counting and frequency methods.
- the lesion scoring was performed through visual scoring attributed to three body regions (neck, tail, and vent) from 25 birds per treatment group that were randomly selected. Possible injuries and different degrees of severity were analyzed on a scale from 0 to 5 (best score being 0, indicating complete plumage and no damage, and the worst score being 5, indicating completely feathered areas with skin lesions). Comb abnormalities were observed in the same birds using a scale from 0 to 3, with the best score being 0 (no evidence of comb abnormalities) and the worst score being 3 (3 or more comb areas with evidence of abnormalities).
- the egg quality of birds in various treatment groups was evaluated.
- the egg quality was evaluated during storage periods.
- the experimental units were randomly selected among the hens housed in the commercial farm (about 28,000 light-weight laying hens, 36 weeks old, Hyline W 36 lineage) and assigned to the treatments, that were i) control (basal diet, without supplementation); ii) diet supplemented with 300 g/ton of the P-mannanase Hemicell®; iii) diet supplemented with 50 g/ton of the probiotic additive Protexin®; iv) diet supplemented with 50 g/ton of probiotic additive and 300 g/ton of P-mannanase (Hemicell + Protexin).
- the basal feed was formulated according to the nutritional requirements for the genetic (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the feed formulas. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.
- the albumen height was estimated by the average of three measurements taken at different points on the albumen at a distance of 10 mm from the yolk using a digital caliper (TMX PD - 150, China).
- TMX PD - 150 digital caliper
- Yolk color was determined in fifteen eggs per treatment at weekly intervals using the Roche colorimetric fan (DSM, Sao Paulo, Brazil), with a score ranging from 1 (light yellow) to 15 (reddish orange).
- DSM Roche colorimetric fan
- a spectrophotometer equipment (Delta Vista model 450G, Delta Color, Sao Leopoldo, Brazil) was also used for this evaluation, which determined colorimetric coordinates of luminosity (L*), red intensity (a*) and yellow intensity (b*). Chroma was estimated considering the following equation:
- the shell percentage was obtained after shell separation, washing, drying and weighing. Fifteen eggs from each treatment were evaluated on day 0, 21 and 42 for these variables.
- TBARS thiobarbituric acid
- Table 6 displays a summary of fresh egg quality analyses from laying hens fed P-mannanase and/or probiotics. The values represent averages from the three evaluated periods, considering only fresh eggs.
- Hemicell Hemicell
- Protexin probiotics
- Table 8 shows weight loss (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 9 shows weight loss (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 10 shows specific gravity (g/ml) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 11 shows specific gravity (g/ml) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 12 shows shows specific gravity (g/ml) of eggs from laying hens fedbeta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 13 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 14 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 15 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 16 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 17 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 18 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 19 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 20 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 21 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 22 shows yolk index of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 22 Standard error. Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. [0080] Table 23 shows yolk index of eggs from laying hens fed beta-mannanase
- Hemicell Hemicell
- Protexin probiotics
- Table 24 shows yolk index of eggs from laying hens fed beta-mannanase
- Hemicell Hemicell
- Protexin probiotics
- Table 25 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 25 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 26 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 27 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 28 shows Haugh unit of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 29 shows Haugh unit of eggs from laying hens fed beta-mannanase
- Hemicell Hemicell
- Protexin probiotics
- Table 30 shows Haugh unit of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 31 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 32 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 33 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 34 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 35 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 36 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 37 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 38 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- N shell breaking strength
- Table 39 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 40 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 41 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
- Table 42 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
- Table 43 shows yolk pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
- Table 43 Standard error. 2 Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
- Table 44 shows yolk pH of eggs from laying hens fed beta-mannanase
- Hemicell Hemicell
- Protexin probiotics
- Table 45 shows yolk pH of eggs from laying hens fed beta-mannanase
- Hemicell Hemicell
- Protexin probiotics
- Table 46 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
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Abstract
The present disclosure relates to compositions comprising capsicum, functional mineral compounds, and yeast cell wall. Furthermore, methods of feeding the composition to animals such as bovines are provided to demonstrate beneficial effects on the animals. The compositions and methods described herein can benefit overall animal wellbeing and future productivity, especially in younger animals.
Description
COMPOSITIONS
MANNANASE AND PROBIOTICS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Serial No. 63/400,098, filed on August 23, 2022, the entire disclosure of which is incorporated herein by reference.
HELD OF THE INVENTION
[0002] The present disclosure relates to supplements comprising beta-mannanase comprising beta-mannanase and/or a probiotic. Furthermore, methods of administering the supplements to avians such as chickens are provided to demonstrate beneficial effects on the avians. The compositions and methods described herein can benefit overall animal wellbeing and future productivity, especially in egg-laying chickens.
BACKGROUND
[0003] Improvement in animal health in avians is an important consideration for the agriculture and food production industries. In modern commercial operations, supplementation of avian feed can be undertaken in order to improve animal health and welfare.
[0004] For instance, the ability to maintain healthy avians that are able to improve their eating, walking, and consumption behavior is typically beneficial to overall avian wellbeing and future productivity. Furthermore, in commercial avian operations, improving egg laying productivity and the resulting quality of eggs is an important consideration. Thus, there exists a need for new compositions and methods to assist with management of avians in order to promote healthy behaviors and improve egg laying characteristics.
[0005] Probiotics can reduce heat stress and abnormal behavior as well as improve health in broiler chickens. Such responses occur from the regulatory power of probiotics under the micro biota-gut-brain axis. Probiotics also alleviate the stress response along the hypothalamic-pituitary-adrenal axis, reducing plasma or brain levels of corticotropin-releasing hormone, adrenocorticotropic hormone, and corticosterone.
[0006] Enzyme supplementation is another strategy that can benefit the gut health status by reducing the impacts of anti-nutritional components. The use of -mannanase can help nonruminant animals dealing with the non-starch polysaccharides, which can reduce nutrient digestibility. Such components are found in plant cell walls and are present in many ingredients largely used in animal feeding, such as soybeans. Among the main hemicelluloses found in plant cell walls are P-mannans, which can also be found on the surface of microorganisms.
Thus, the animal's innate immune system is activated when foods that contain P-mannans are ingested, which responds with the proliferation of monocytes, macrophages, dendritic cells, and increased production of cytokines. Such factors generate an unnecessary energy expenditure and an increase in inflammatory responses. By hydrolyzing the P-mannans, this enzyme can improve the digestibility of mannans, increasing the population of beneficial bacteria, improving immunity, digestion and absorption of nutrients, in addition to limiting the proliferation of potential pathogens in the intestine.
SUMMARY
[0007] The present disclosure provides supplements comprising beta-mannanase comprising beta-mannanase and/or a probiotic as well as associated methods. The compositions and methods of the present disclosure surprisingly provide a synergistic effect for improve avian behaviors and egg laying qualities. The present disclosure describes supplements comprising beta-mannanase (e.g., Hemicell HT™) and a probiotic (e.g., Protexin™) that can be added to feed to improve animal health and animal welfare.
[0008] Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of illustrative embodiments exemplifying the best mode of carrying out the disclosure as presently perceived.
BRIEF DESCRIPTIONS OF THE DRAWINGS
[0009] The detailed description particularly refers to the accompanying figures in which: [0010] Figure 1 shows examples of eggs with minor presence of feces.
[0011] Figure 2 shows examples of eggs with major presence of feces.
[0012] Figure 3 shows ultramicroscopy images collected in a single eggshell during the first phase.
[0013] Figure 4 shows ultramicroscopy images collected in a single eggshell during the second phase.
[0014] Figure 5 shows ultramicroscopy images collected in a single eggshell during the third phase.
DETAILED DESCRIPTION
[0015] In an illustrative aspect, a method comprising a step of administering one or more supplements to a non-human animal is provided. In an embodiment, the animal is an avian. In an embodiment, the avian is selected from the group consisting of a chicken, a turkey, and a
duck. In an embodiment, the avian is a chicken. In an embodiment, the chicken is a hen. In an embodiment, the hen is a laying hen. In an embodiment, the avian is present in a cage system.
[0016] In an embodiment, the supplement comprises Hemicell™. In an embodiment, the supplement comprises Hemicell HT™. In an embodiment, the supplement comprises beta- mannanase, also referred to herein as “0- mannanase”.
[0017] In an embodiment, the beta-mannanase is provided by fermentation of Paenibacillus lentus. In an embodiment, the beta-mannanase is administered at a dose between 100 g/ton and 500 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 100 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 200 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 300 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 400 g/ton. In an embodiment, the beta-mannanase is administered at a dose of 500 g/ton.
[0018] In an embodiment, the supplement comprises Protexin™. In an embodiment, the supplement comprises a probiotic supplement. In an embodiment, the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles. In an embodiment, the supplement comprises a probiotic supplement comprising Enterococcus faecium. In an embodiment, the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181
[0019] In an embodiment, the probiotic supplement is administered at a dose between 10 g/ton and 100 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 10 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 20 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 30 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 40 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 50 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 60 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 70 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 80 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 90 g/ton. In an embodiment, the probiotic supplement is administered at a dose of 100 g/ton.
[0020] In an embodiment, the supplements comprise i) beta-mannanase and ii) a probiotic. In an embodiment, the beta-mannanase and the probiotic provide a synergistic effect in improvement in health of the animal.
[0021] In an embodiment, the supplement is present in a feed for the animal. In an embodiment, the feed is a free choice feed.
[0022] In an embodiment, the administration provides an improvement in health of the animal. In an embodiment, the improvement in health of the animal is an improvement in animal welfare. In an embodiment, the improvement in health of the animal comprises an improvement in eating behavior. In an embodiment, the improvement in health of the animal comprises an improvement in walking behavior. In an embodiment, the improvement in health of the animal comprises an improvement in standing behavior. In an embodiment, the improvement in health of the animal comprises an improvement in sitting behavior.
[0023] In an embodiment, the improvement in health of the animal comprises an improvement in liquid consumption behavior. In an embodiment, the improvement in health of the animal comprises an improvement in feather exploring behavior. In an embodiment, the improvement in health of the animal comprises an improvement in head scratching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in wing flapping behavior. In an embodiment, the improvement in health of the animal comprises an improvement in leg stretching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in stretching behavior. In an embodiment, the improvement in health of the animal comprises an improvement in aggressive pecking behavior. In an embodiment, the improvement in health of the animal comprises an improvement in non- aggressive pecking behavior.
[0024] In an embodiment, the improvement in health of the animal comprises a decrease in lesions on the animal. In an embodiment, the lesions are present on the neck. In an embodiment, the lesions are present on the tail. In an embodiment, the lesions are present on the cloaca. In an embodiment, the lesions are present on the comb.
[0025] In an embodiment, the improvement in health of the animal comprises an improvement in intestinal health in the animal. In an embodiment, the improvement in health of the animal comprises prevention of intestinal dysbiosis in the animal.
[0026] In an embodiment, the improvement in health of the animal comprises a reduction in pro-inflammatory effect in the animal. In an embodiment, the improvement in health of the animal comprises increased release of mannan-oiligosaccharides (MOS) in the animal.
[0027] In an embodiment, the administration provides an improvement in egg quality of one of more eggs laid by the animal. In an embodiment, the improvement in egg quality is indicated by a modification of a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh unit, shell weight, shell breaking strength, albumen pH, yolk pH, yolk color score, yolk lightness, yolk redness, yolk yellowness, chroma, and any combination thereof.
[0028] In an embodiment, the improvement in egg quality is indicated by a modification of weight. In an embodiment, the improvement in egg quality is indicated by a modification of specific gravity. In an embodiment, the improvement in egg quality is indicated by a modification of albumen height. In an embodiment, the improvement in egg quality is indicated by a modification of albumen weight. In an embodiment, the improvement in egg quality is indicated by a modification of yolk height. In an embodiment, the improvement in egg quality is indicated by a modification of yolk length. In an embodiment, the improvement in egg quality is indicated by a modification of yolk index. In an embodiment, the improvement in egg quality is indicated by a modification of yolk weight. In an embodiment, the improvement in egg quality is indicated by a modification of Haugh unit. In an embodiment, the improvement in egg quality is indicated by a modification of shell weight. In an embodiment, the improvement in egg quality is indicated by a modification of shell breaking strength. In an embodiment, the improvement in egg quality is indicated by a modification of albumen pH. In an embodiment, the improvement in egg quality is indicated by a modification of yolk pH. In an embodiment, the improvement in egg quality is indicated by a modification of yolk color score. In an embodiment, the improvement in egg quality is indicated by a modification of yolk lightness. In an embodiment, the improvement in egg quality is indicated by a modification of yolk redness. In an embodiment, the improvement in egg quality is indicated by a modification of yolk yellowness. In an embodiment, the improvement in egg quality is indicated by a modification of chroma. In an embodiment, the modification is an increase in measurement. In an embodiment, the modification is a decrease in measurement. [0029] In an embodiment, the administration provides an improvement in reactive oxygen species (ROS) in the animal. In an embodiment, the improvement in ROS is in the intestine of the animal. In an embodiment, the improvement in ROS is in the oviduct of the animal.
[0030] In an embodiment, the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thios (TSH), and any combination thereof. In an embodiment, the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS). In an embodiment, the improvement in ROS is indicated by a modification of glutathione S-transferase (GST). In an embodiment, the improvement in ROS is indicated by a modification of protein thios (TSH). In an embodiment, the modification is an increase in measurement. In an embodiment, the modification is a decrease in measurement.
[0031] In an embodiment, the administration provides an increase in egg laying rate of the animal. In an embodiment, the administration provides an increase in fresh egg weight for
eggs laid by the animal. In an embodiment, the administration provides a reduction in coefficient of variability of egg weight for eggs laid by the animal. In an embodiment, the administration provides an increase in an egg component mass for eggs laid by the animal. In an embodiment, the egg component mass is egg mass. In an embodiment, the egg component mass is yolk mass. In an embodiment, the egg component mass is albumen mass. In an embodiment, the egg component mass is egg shell mass.
[0032] In an embodiment, the administration provides an increase in occurrence of clean eggs laid by the animal. In an embodiment, the increase in occurrence of clean eggs is a reduction in the presence of feces on the eggs.
[0033] In an embodiment, the administration provides a modification in eggshell thickness in eggs laid by the animal. In an embodiment, the modification in eggshell thickness is an increase in eggshell thickness. In an embodiment, the modification in eggshell thickness is a decrease in eggshell thickness.
[0034] In an embodiment, the modification in eggshell thickness is observed during a first phase of about 40 weeks. In an embodiment, the modification in eggshell thickness is observed during a second phase of about 44 weeks. In an embodiment, the modification in eggshell thickness is observed during a third phase of about 48 weeks.
[0035] The following numbered embodiments are contemplated and are non-limiting:
1. A method comprising a step of administering one or more supplements to a nonhuman animal.
2. The method of clause 1 , any other suitable clause, or any combination of suitable clauses, wherein the animal is an avian.
3. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the avian is selected from the group consisting of a chicken, a turkey, and a duck.
4. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the avian is a chicken.
5. The method of clause 4, any other suitable clause, or any combination of suitable clauses, wherein the chicken is a hen.
6. The method of clause 5, any other suitable clause, or any combination of suitable clauses, wherein the hen is a laying hen.
7. The method of clause 2, any other suitable clause, or any combination of suitable clauses, wherein the avian is present in a cage system.
8. The method of any one of clauses 1 to 7, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises Hemicell HT™.
9. The method of any one of clauses 1 to 8, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises beta-mannanase.
10. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is provided by fermentation of Paenibacillus lentus.
11. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose between 100 g/ton and 500 g/ton.
12. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 100 g/ton.
13. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 200 g/ton.
14. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 300 g/ton.
15. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 400 g/ton.
16. The method of clause 9, any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase is administered at a dose of 500 g/ton.
17. The method of any one of clauses 1 to 16, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises Protexin™.
18. The method of any one of clauses 1 to 17, any other suitable clause, or any combination of suitable clauses, wherein the supplement comprises a probiotic supplement.
19. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles, or wherein the supplement comprises a probiotic supplement comprising Enterococcus faecium, optionally wherein the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181.
20. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose between 10 g/ton and 100 g/ton.
21. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 10 g/ton.
22. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 20 g/ton.
23. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 30 g/ton.
24. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 40 g/ton.
25. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 50 g/ton.
26. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 60 g/ton.
27. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 70 g/ton.
28. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 80 g/ton.
29. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 90 g/ton.
30. The method of clause 18, any other suitable clause, or any combination of suitable clauses, wherein the probiotic supplement is administered at a dose of 100 g/ton.
31. The method of any one of clauses 1 to 30, any other suitable clause, or any combination of suitable clauses, wherein the supplements comprise i) beta-mannanase and ii) a probiotic.
32. The method of clause 31 , any other suitable clause, or any combination of suitable clauses, wherein the beta-mannanase and the probiotic provide a synergistic effect in improvement in health of the animal.
33. The method of any one of clauses 1 to 32, any other suitable clause, or any combination of suitable clauses, wherein the supplement is present in a feed for the animal.
34. The method of clause 33, any other suitable clause, or any combination of suitable clauses, wherein the feed is a free choice feed.
35. The method of any one of clauses 1 to 34, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an improvement in health of the animal.
36. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal is an improvement in animal welfare.
37. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in eating behavior.
38. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in walking behavior.
39. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in standing behavior.
40. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in sitting behavior.
41. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in liquid consumption behavior.
42. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in feather exploring behavior.
43. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in head scratching behavior.
44. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in wing flapping behavior.
45. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in leg stretching behavior.
46. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in stretching behavior.
47. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in aggressive pecking behavior.
48. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in non- aggressive pecking behavior.
49. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises a decrease in lesions on the animal.
50. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the neck.
51. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the tail.
52. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the cloaca.
53. The method of clause 49, any other suitable clause, or any combination of suitable clauses, wherein the lesions are present on the comb.
54. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises an improvement in intestinal health in the animal.
55. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises prevention of intestinal dysbiosis in the animal.
56. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises a reduction in pro- inflammatory effect in the animal.
57. The method of clause 35, any other suitable clause, or any combination of suitable clauses, wherein the improvement in health of the animal comprises increased release of mannan- oiligosaccharides (MOS) in the animal.
58. The method of any one of clauses 1 to 57, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an improvement in egg quality of one of more eggs laid by the animal.
59. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh unit, shell weight, shell breaking strength, albumen pH, yolk pH, yolk color score, yolk lightness, yolk redness, yolk yellowness, chroma, and any combination thereof.
60. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of weight.
61. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of specific gravity.
62. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of albumen height.
63. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of albumen weight.
64. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk height.
65. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk length.
66. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk index.
67. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk weight.
68. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of Haugh unit.
69. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of shell weight.
70. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of shell breaking strength.
71. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of albumen pH.
72. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk pH.
73. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk color score.
74. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk lightness.
75. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk redness.
76. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of yolk yellowness.
77. The method of clause 58, any other suitable clause, or any combination of suitable clauses, wherein the improvement in egg quality is indicated by a modification of chroma.
78. The method of any one of clauses 59 to 77, any other suitable clause, or any combination of suitable clauses, wherein the modification is an increase in measurement.
79. The method of any one of clauses 59 to 77, any other suitable clause, or any combination of suitable clauses, wherein the modification is a decrease in measurement.
80. The method of any one of clauses 1 to 79, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an improvement in reactive oxygen species (ROS) in the animal.
81. The method of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is in the intestine of the animal.
82. The method of clause 80, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is in the oviduct of the animal.
83. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thios (TSH), and any combination thereof.
84. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS).
85. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by a modification of glutathione S-transferase (GST).
86. The method of any one of clauses 80 to 82, any other suitable clause, or any combination of suitable clauses, wherein the improvement in ROS is indicated by a modification of protein thios (TSH).
87. The method of any one of clauses 83 to 86, any other suitable clause, or any combination of suitable clauses, wherein the modification is an increase in measurement.
88. The method of any one of clauses 83 to 86, any other suitable clause, or any combination of suitable clauses, wherein the modification is a decrease in measurement.
89. The method of any one of clauses 1 to 88, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in egg laying rate of the animal.
90. The method of any one of clauses 1 to 89, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in fresh egg weight for eggs laid by the animal.
91. The method of any one of clauses 1 to 90, any other suitable clause, or any combination of suitable clauses, wherein the administration provides a reduction in coefficient of variability of egg weight for eggs laid by the animal.
92. The method of any one of clauses 1 to 91, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in an egg component mass for eggs laid by the animal.
93. The method of clause 92, any other suitable clause, or any combination of suitable clauses, wherein the egg component mass is egg mass.
94. The method of clause 92 or clause 93, any other suitable clause, or any combination of suitable clauses, wherein the egg component mass is yolk mass.
95. The method of any one of clauses 92 to 94, any other suitable clause, or any combination of suitable clauses, wherein the egg component mass is albumen mass.
96. The method of any one of clauses 92 to 95, any other suitable clause, or any combination of suitable clauses, wherein the egg component mass is egg shell mass.
97. The method of any one of clauses 1 to 96, any other suitable clause, or any combination of suitable clauses, wherein the administration provides an increase in occurrence of clean eggs laid by the animal.
98. The method of clause 97, any other suitable clause, or any combination of suitable clauses, wherein the increase in occurrence of clean eggs is a reduction in the presence of feces on the eggs.
99. The method of any one of clauses 1 to 98, any other suitable clause, or any combination of suitable clauses, wherein the administration provides a modification in eggshell thickness in eggs laid by the animal.
100. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is an increase in eggshell thickness.
101. The method of clause 99, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is a decrease in eggshell thickness.
102. The method of any one of clauses 1 to 101, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is observed during a first phase of about 40 weeks.
103. The method of any one of clauses 1 to 101, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is observed during a second phase of about 44 weeks.
104. The method of any one of clauses 1 to 101, any other suitable clause, or any combination of suitable clauses, wherein the modification in eggshell thickness is observed during a third phase of about 48 weeks.
EXAMPLES
Example 1 Behavioral Assessments
[0036] For the instant example, birds were evaluated at a commercial farm in Salvador do Sul, state of Rio Grande do Sul, in southern Brazil. The experimental units were randomly selected among hens housed in a commercial farm with about 28 thousand lightweight laying hens (Hyline W 36 lineage, 36 weeks old). The replicates were assigned in a completely randomized design to the four treatments, that included i) control treatment (a basal diet without supplementation with any other additive), ii) ; P-mannanase (control diet supplemented with 300 g/ton of -mannanase), hi) probiotic (control diet supplemented with 50 g/ton of a multi- cepa probiotic additive), and iv) P-mannanase plus probiotic treatment (control diet supplemented with 300 g/ton of P-mannanase and 50 g/ton of a multi-cepa probiotic additive). [0037] The P-mannanase (Hemicell HT™, Elanco Animal Health) for the instant example was an exogenous enzyme from the fermentation of the Paenibacillus lentus bacteria. The probiotic additive (Protexin™ Concentrate, Elanco Animal Health) for the instant example includes Lactobacillus acidophilus (2.06 x 108 UFC/g), Lactobacillus bulgaricus (2.06 x 108 UFC/g), Lactobacillus plantarum (1.26 x 108 UFC/g), Lactobacillus rhamnosus (2.06 x 108 UFC/g), Bifidobacterium bifdum (2.0 x 108 UFC/g), Enterococcus faecium (6.46 x 108 UFC/g), and Streptococcus thermophilus (4.10 x 108 UFC/g).
[0038] The basal diet comprised a com-soybean meal-based feed formulated according to nutritional requirements and inert material (kaolin) was included in basal feed to replace - mannanase and/or probiotic additives. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.
[0039] The birds were housed in conventional sheds, arranged in an east-west direction, with concrete floors and masonry walls complemented with wire mesh to the ceiling. The shed was equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. The average minimum and maximum temperature and air relative humidity values recorded were 18 and 36 °C, and 35.8 and 94.7%, respectively. The lighting regime was composed of 16 hours of light and 8 hours of dark per day.
[0040] The birds remained in galvanized- wire cages (100-cm long x 40-cm wide x 45- cm high, resulting in a floor area of 500 cm2/hen) throughout the experimental period. Birds were allocated in each cage. Birds were supplemented for 84 days and the assessments were performed in the last week of the trial.
[0041] Behavioral assessments were performed via image capture combined with local feather scoping and comb abnormalities assessments. For the behavior assessment, six birds per treatment (one per cage) were randomly selected for observation. The captured images were carried out for 7 consecutive days, in a period of 15 minutes in the morning (e.g., the highest peak of laying of the birds) and 30 minutes in the afternoon divided into two periods corresponding to the hottest and cooler times of the day. Images were recorded and analyzed by visual counting and frequency methods.
[0042] The lesion scoring was performed through visual scoring attributed to three body regions (neck, tail, and vent) from 25 birds per treatment group that were randomly selected. Possible injuries and different degrees of severity were analyzed on a scale from 0 to 5 (best score being 0, indicating complete plumage and no damage, and the worst score being 5, indicating completely feathered areas with skin lesions). Comb abnormalities were observed in the same birds using a scale from 0 to 3, with the best score being 0 (no evidence of comb abnormalities) and the worst score being 3 (3 or more comb areas with evidence of abnormalities).
[0043] All behavioral tests were carried out in the last week of the experiment, allowing the birds to remain exposed to the treatments for a longer period. The same animal was used only in one of the tests, thus preventing one test from interfering with the result of the other.
[0044] The frequency of the main behaviors observed in laying hens fed P-mannanase and/or probiotics are shown in Table 1.
Table 1.
Treatments1 P-value2
. T..rai .t.s C „ont .ro -l He ZmIIi,cell Protexin H .. Avg . . . • .. ,
_ (H) _ (P) _ + P time _ Treat Time T x T Eating
times were polled together (indicated at ‘Avg treat’. The averages obtained when the fourth treatments were polled together in each observation time are also presented (indicated as ‘Avg time’) and compared within the columnXYZ.
2Probability of treatment effect (treat), time of observation (time), and interaction (T x T). [0045] The times expended (minutes/bird) in each of the main behaviors by laying hens fed P-mannanase and/or probiotics are shown in Table 2.
Table 2.
Walking
^eans followed b different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. Comparisons were performed among treatments - line A.,B,C,D each observation time and also for averages obtained when the three observation times were polled together (indicated at ‘Avg treat’. The averages obtained when the fourth treatments were polled together in each observation time are also presented (indicated as ‘Avg time’) and compared within the columnXYZ.
2Probability of treatment effect (treat), time of observation (time), and interaction (T x T).
[0046] The times expended (minutes/bird) in other behaviors by laying hens fed p- mannanase and/or probiotics are shown in Table 3.
Table 3.
Avg treat 0.33 0.09 0.01 0.09
^eans followed b different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. Comparisons were performed among treatments - line A B,C,D wjp]jn each observation time and also for averages obtained when the three observation times were polled together (indicated at ‘Avg treat’. The averages obtained when the fourt treatments were polled together in each observation time are also presented (indicated as ‘Avg time’) and compared within the columnXYZ.
2Probability of treatment effect (treat), time of observation (time), and interaction (T x T).
[0047] The frequency (%) of birds with lesions (disregarding the score) observed in groups of laying hens fed P-mannanase and/or probiotics are shown in Table 4.
Table 4.
[0048] The lesion score observed in groups of laying hens fed 3-mannanase and/or probiotics are shown in Table 5. Scores from 0 (no lesion) to 5 (severe lesion) were attributed to the birds individually, except for crest in which the scores ranged from 0 (no lesion) to 2
(severe lesion). Means (LS/means) followed by different uppercase letters differ statistically at
5%.
Table 5.
Example 2 Egg Quality Assessments
[0049] For the instant example, the egg quality of birds in various treatment groups was evaluated. The egg quality was evaluated during storage periods.
[0050] The experimental units were randomly selected among the hens housed in the commercial farm (about 28,000 light-weight laying hens, 36 weeks old, Hyline W 36 lineage) and assigned to the treatments, that were i) control (basal diet, without supplementation); ii) diet supplemented with 300 g/ton of the P-mannanase Hemicell®; iii) diet supplemented with 50 g/ton of the probiotic additive Protexin®; iv) diet supplemented with 50 g/ton of probiotic additive and 300 g/ton of P-mannanase (Hemicell + Protexin).
[0051] The basal feed was formulated according to the nutritional requirements for the genetic (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the feed formulas. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.
[0052] Three 28-day production cycles were carried out and 480 eggs (120 from each treatment group) were randomly collected on the last day of each cycle for evaluation of egg quality. Cracked eggs were excluded from the experiment.
[0053] Part of the eggs were immediately subjected to quality analysis to represent the characteristics of fresh eggs (zero days of storage). The other eggs were stored at room temperature and randomly separated for quality assessment at each storage interval (7, 14, 21, 28, 35 and 42 days).
[0054] Fifteen eggs from each treatment were randomly chosen and weighed individually at weekly intervals during the storage period. The weight loss (%) of eggs during storage was calculated using the following equation:
^
[0055] The albumen height was estimated by the average of three measurements taken at different points on the albumen at a distance of 10 mm from the yolk using a digital caliper (TMX PD - 150, China). Thus, the Haugh Unit (HU) was obtained through the equation proposed by Haugh (1937), where h= thickness of albumen (mm); W= mass of the entire egg (g):
[0056] Yolk width and height (mm) were measured with a digital caliper (TMX PD -
150, China) on 15 eggs from each treatment at weekly intervals. After, the yolk index was calculated as:
[0057] Yolk color was determined in fifteen eggs per treatment at weekly intervals using the Roche colorimetric fan (DSM, Sao Paulo, Brazil), with a score ranging from 1 (light yellow) to 15 (reddish orange). Complementarily, a spectrophotometer equipment (Delta Vista model 450G, Delta Color, Sao Leopoldo, Brazil) was also used for this evaluation, which determined colorimetric coordinates of luminosity (L*), red intensity (a*) and yellow intensity (b*). Chroma was estimated considering the following equation:
[0058] After yolk and albumen separation, the dense and the fluid albumen were homogenized for 20 seconds and then the pH was determined using a digital pH meter (Kasvi model k39-2014B, Parana, Brazil) previously calibrated with buffer solutions of pH 4, pH 7 and pH 10. The pH of the yolk was determined using the same pH meter.
[0059] The value of specific gravity was based on Archimedes’ principle, using the equation:
[0060] Total solid content was determined separately in albumen and yolk. Five grams of albumen and yolk were weighed separately in previously dried porcelain crucibles. The albumen and yolk samples were kept in an oven at 60 °C for 12 hours and weighed. After weighing, the samples were kept at 105 °C for 12 hours, and weighed again. Seven eggs from each treatment were evaluated to determine the total solids.
[0061] The shell percentage was obtained after shell separation, washing, drying and weighing. Fifteen eggs from each treatment were evaluated on day 0, 21 and 42 for these variables.
[0062] The substances that react to thiobarbituric acid (TBARS) were used to access the lipid oxidation in a pool of three yolks per treatment collected in three storage periods (0, 21, and 42 days). Only treatments with Hemicell and Protexin isolated were tested due to the previous results obtained in egg quality analysis.
[0063] Table 6 displays a summary of fresh egg quality analyses from laying hens fed P-mannanase and/or probiotics. The values represent averages from the three evaluated periods, considering only fresh eggs.
Table 6.
Treatments
, Hemicell Protexin ,, _ ,, , ,
Storage time Control . H + P SE H) (r) 1 P-value
( 2
Weight 62.74 63.11 62.51 62.71 0.290 0.904
Specific gravity (g/ml) 1.006B 1.007A 1.006B 1.006B 0.001 <0.001
Albumen height (mm) 8.04 8.06 8.18 8.17 0.104 0.129
Albumen weight (g) 36.82 37.39 36.30 36.59 0.239 0.424
Yolk height (mm) 17.98B 18.15A 18.27A 18.18A 0.063 0.037
Yolk length (mm) 40.67B 41.62A 41.25AB 41.82A 0.118 0.002
Yolk index 0.443 0.435 0.443 0.435 0.017 0.184
Yolk weight (g) 15.33B 15.70AB 15.45B 16.08A 0.096 0.004
Haugh unit 89.40 90.10 89.88 89.55 0.558 0.132
Shell weight (g) 5.81B 6.15A 5.96AB 6.11A 0.041 0.09
Shell breaking strength (N) 2.47 2.39 2.17 2.25 0.106 0.563
Albumen pH 8.41 8.40 8.38 8.44 0.028 0.178
Yolk pH 6.04B 5.96A 5.99A 6.00A 0.013 0.002
Yolk color score (palette) 5.60B 5.98A 5.77 AB 5.87AB 0.052 0.032
Yolk lightness (L* color) 50.86B 50.66B 51.33AB 52.16A 0.161 0.002
Yolk redness (a* color) 7.12B 7.66A 7.67A 7.66A 0.100 <0.001
Yolk yellowness (b* color) 57.41 58.88 58.93 58.75 0.354 0.122
Chroma 57.85B 59.67A 59.67A 59.25A 0.357 0.003
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at f0%.
[0064] Table 7 shows weight loss (g) of eggs from laying hens fed beta-mannanase
(Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 7.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0065] Table 8 shows weight loss (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 8
(H) (P) value2
^Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0066] Table 9 shows weight loss (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 9.
Only 3 treatments 0.062 0.300
Only Hemicell 0.085 0.152
Only Protexin 0.055 0.670
Day 21 1.523 1.562 1.436 1.719
All treatments AB AB A B 0.051 0.022
Only 3 treatments 0.062 0.424
Only Hemicell 0.058 0.708
Only Protexin 0.061 0.402
Day 28 2.137 2.300 2.018 2.243
All treatments 0.053 0.169
Only 3 treatments 0.065 0.170
Only Hemicell 0.074 0.241
Only Protexin 0.080 0.476
Day 35 2.439 2.121 2.539 2.592
All treatments 0.052 0.182
Only 3 treatments 0.058 0.218
Only Hemicell 0.077 0.291
Only Protexin 0.057 0.358
Day 42 3.675 3.263 3.132 3.382
All treatments B B A AB 0.049 0.007
Only 3 treatments B A A 0.057 0.003
Only Hemicell B A 0.069 0.020
Only Protexin B A 0.076 <0.001
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0067] Table 10 shows specific gravity (g/ml) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 10.
Treatments
.. . Hemicell Protexin „ „ P-
Storage time Control (H) (p) H + P SE1
Day l 1.066 1.059 1.064 1.057
All treatments A B B B 0.007 <0.001
Only 3 treatments A B A 0.007 <0.001
Only Hemicell A B 0.009 <0.001
Only Protexin 0.008 0372
Day 7 1.051 1.057 1.041 1.054
All treatments 0.259 0.997
Only 3 treatments 0.345 0.983
Only Hemicell 0.520 0.959
Only Protexin 0.503 0.922
Day 14 1.054 1.048 1.050 1.049
All treatments 0.016 0.653
Only 3 treatments 0.016 0.348
Only Hemicell 0.025 0.242
Only Protexin 0.025 0.381
Day 21 1.062 1.050 1.051 1.060
All treatments 0.028 0.293
Only 3 treatments 0.036 0.345
Only Hemicell 0.008 0.226
Only Protexin 0.008 0.353
Day 28 1.042 1.042 1.039 1.041
All treatments 0.018 0.954
Only 3 treatments 0.021 0.845
Only Hemicell 0.022 0.976
Only Protexin 0.026 0.522
Day 35 1.042 1.042 1.055 1.057
All treatments 0.018 0.954
Only 3 treatments 0.021 0.845
Only Hemicell 0.022 0.976
Only Protexin 0.026 0.622
Day 42 1.047 1.056 1.055 1.057
All treatments B A A A 0.006 <0.001
Only 3 treatments B A A 0.007 <0.001
Only Hemicell B A 0.009 <0.001
Only Protexin B A 0.009 <0.001
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0068] Table 11 shows specific gravity (g/ml) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 11.
Treatments
.. . i Hemicell Protexin ,, .. P-
Storage time Control H + P SE1 yalue2
Day l 1.075 1.083 1.073 1.074
All treatments B A B B 0.008 <0.001
Only 3 treatments B A B 0.010 <0.001
Only Hemicell B A 0.011 <0.001
Only Protexin 0.010 0.246
Day ? 1.069 1.066 1.071 1.066
All treatments AB AB A B 0.007 0.026
Only 3 treatments AB B A 0.008 0.031
Only Hemicell a b 0.008 0.065
Only Protexin 0.010 0.415
Day 14 1.059 1.059 1.053 1.062
All treatments A A B A 0.0065 <0.001
Only 3 treatments A A B 0.0071 <0.001
Only Hemicell 0.0067 0.526
Day 28 1.059 1.063 1.061 1.071
All treatments B B B A 0.007 <0.001
Only 3 treatments B A AB 0.006 0.040
Only Hemicell B A 0.007 0.011
Only Protexin 0.007 0.251
Day 35 1.036 1.039 1.038 1.038
All treatments B A AB AB 0.004 0.022
Only 3 treatments B A A 0 0.004 0.016
Only Hemicell B A 0.006 0.020
Only Protexin B A 0.005 0.011
Day 42 1.048 1.049 1.050 1.049
All treatments 0.004 0.301
Only 3 treatments 0.005 0.143
Only Hemicell 0.006 0.657
Only Protexin b a 0.006 0.054
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0069] Table 12 shows shows specific gravity (g/ml) of eggs from laying hens fedbeta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 12.
Treatments
, Hemicell Protexin _T _ P-
Storag se time Control H + P SB (H) (P) 1 , value 7 2
Day l 1.059 1.065 1.063 1.160
A B A A B 0.004 <0.001
Only 3 treatments B A A 0.005 <0.001
Only Hemicell B A 0.007 <0.001
Only Protexin B A 0.006 0.001
Day 7 1.064 1.061 1.068 1.064
All treatments BC C A B 0.004 <0.001
Only 3 treatments B C A 0.005 <0.001
Only Hemicell A B 0.004 0.001
Only Protexin B A 0.005 <0.001
Day 14 1.052 1.053 1.053 1.056
All treatments B AB AB A 0.004 0.036
Only 3 treatments 0.005 0.607
Only Hemicell 0.006 0.347
Only Protexin 0.007 0.742
Day 21 1.038 1.046 1.042 1.047
All treatments C A B A 0.005 <0.001
Only 3 treatments C A B 0.005 <0.001
Only Hemicell B A 0.008 <0.001
Only Protexin B A 0.005 <0.001
Day 28 1.031 1.033 1.031 1.032
All treatments 0.003 0.223
Only 3 treatments 0.004 0.104
Only Hemicell b a 0.005 0.079
Only Protexin 0.004 0.780
Day 35 1.038 1038 1.040 1.039
All treatments 0.004 0.954
Only 3 treatments 0.004 0.319
Only Hemicell 0.004 0.862
Only Protexin 0.005 0.240
Day 42 1.032 1.037 1.035 1.037
All treatments B A A A 0.005 <0.001
Only 3 treatments B A A 0.005 <0.001
Only Hemicell B A 0.007 <0.001
Only Protexin B A 0.005 0.003
1 Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0070] Table 13 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 13.
Treatments e. .. , Hemicell Protexin ,, „ P-
Storag 6e time Control (H) (P) H + P SE1 va .lue 2 2
Day l 9.34 9.69 9.73 8.55
All treatments AB A A B 0.14 0.009
Only 3 treatments 0.15 0.524
Only Hemicell 0.18 0.347
Only Protexin 0.17 0.250
Day 7 6.03 5.90 6.33 5.18
All treatments A A A B 0.08 <0.001
Only 3 treatments 0.08 0.102
Only Hemicell 0.08 0.461
Only Protexin 0.10 0.161
Day 14 4.74 4.30 4.79 4.39
All treatments AB B A AB 0.07 0.035
Only 3 treatments AB B A 0.09 0.048
Only Hemicell B A 0.11 0.044
Only Protexin 0.11 0.827
Day 21 3.97 3.67 3.86 3.56
All treatments A AB AB B 0.05 0.041
Only 3 treatments 0.06 0.186
Only Hemicell a b 0.08 0.096
Only Protexin 0.06 0.433
Day 28 3.24 3.16 3.31 3.09
All treatments 0.04 0.397
Only 3 treatments 0.05 0.511
Only Hemicell 0.06 0.538
Only Protexin 0.07 0.666
Day 35 2.98 2.88 2.77 2.77
All treatments 0.04 0.343
Only 3 treatments 0.05 0.339
Only Hemicell 0.08 0.534
Only Protexin 0.06 0.144
Day 42 2.70 2.76 2.58 2.71
All treatments 0.04 0.545
Only 3 treatments 0.04 0.285
Only Hemicell 0.05 0.644
Only Protexin 0.05 0.298
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0071] Table 14 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 14.
Treatments e. .. , Hemicell Protexin ,, „ P-
St r tim C ntr l H + P SE1
Day l 6.83 6.65 7.05 6.52
All treatments 0.11 0.384
Only 3 treatments 0.14 0.519
Only Hemicell 0.13 0.504
Only Protexin 0.20 0.595
Day 7 5.33 4.91 5.26 5.08
All treatments A B AB B 0.06 0.064
Only 3 treatments A B AB 0.06 0.006
Only Hemicell A B 0.08 0.007
Only Protexin 0.08 0.710
Day 14 3.92 3.58 3.73 3.34
All treatments A AB AB B 0.06 0.006
Only 3 treatments 0.07 0.163
Only Hemicell a b 0.08 0.058
Only Protexin 0.09 0.336
Day 21 3.23 2.89 3.06 2.75
All treatments A AB AB B 0.05 0.007
Only 3 treatments A B AB 0.05 0.061
Only Hemicell A B 0.07 0.016
Only Protexin 0.06 0.219
Day 28 2.74 2.58 2.72 2.75
All treatments 0.04 0.470
Only 3 treatments 0.05 0.406
Only Hemicell 0.06 0.242
Only Protexin 0.06 0.893
Day 35 2.75 2.57 2.71 3.06
All treatments AB B B A 0.05 0.004
Only 3 treatments 0.04 0.325
Only Hemicell 0.06 0.173
Only Protexin 0.06 0.762
Day 42 2.62 2.67 2.74 2.51
All treatments AB AB A B 0.03 0.069
Only 3 treatments 0.03 0.404
Only Hemicell 0.0o4 0.531
Only Protexin 0.04 0.160
1 Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0072] Table 15 shows albumen height (mm) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 15.
Treatments
.. „ . , Hemicell Protexin .. P-
Storag
&e time Control ,T„ H + P SE ) (P) 1 . (H value 2 2
Day l 7.97 7.83 7.56 7.64
All treatments 0.07 0.209
Only 3 treatments 0.09 0.201
Only Hemicell 0.12 0.576
Only Protexin 0.11 0.119
Day 7 5.49 5.28 5.50 5.25
All treatments 0.06 0.303
Only 3 treatments 0.07 0.394
Only Hemicell 0.09 0.249
Only Protexin 0.10 0.975
Day 14 4.74 4.30 4.79 4.39
All treatments A AB A B 0.05 0.011
Only 3 treatments a b a 0.07 0.085
Only Hemicell a b 0.08 0.051
Only Protexin 0.08 0.925
Day 21 3.67 3.13 3.51 3.31
All treatments A B AB AB 0.06 0.033
Only 3 treatments A B AB 0.07 0.012
Only Hemicell A B 0.01 0.016
Only Protexin 0.06 0.214
Day 28 2.96 2.82 2.93 2.73
All treatments a a a b 0.03 0.098
Only 3 treatments 0.04 0.424
Only Hemicell 0.05 0.245
Only Protexin 0.05 0.791
Day 35 2.66 2.48 2.52 2.77
All treatments AB B B A 0.03 0.018
Only 3 treatments 0.04 0.303
Only Hemicell a b 0.04 0.057
Only Protexin 0.06 0.295
Day 42 2.88 2.47 2.48 2.36
All treatments A AB AB B 0.04 0.050
Only 3 treatments 0.06 0.176
Only Hemicell 0.06 0.106
Only Protexin 0.08 0.130
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0073] Table 16 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 16.
Treatments
, Hemicell Protexin ....| P-
Storage time Control H + P SE1 value2
Day l 40.63 41.25 41.32 41.50
All treatments 0.25 0.565
Only 3 treatments 0.24 0.467
Only Hemicell 0.30 0.319
Only Protexin 0.29 0.261
Day 7 43.49 44.38 43.50 44.38
All treatments 0.23 0.319
Only 3 treatments 0.27 0.323
Only Hemicell 0.39 0.262
Only Protexin 0.27 0.986
Day 14 45.02 45.58 44.48 44.52
All treatments 0.23 0.285
Only 3 treatments 0.24 0.188
Only Hemicell 0.31 0.391
Only Protexin 0.27 0.328
Day 21 49.84 46.85 45.71 47.91
All treatments A B B AB 0.42 0.00
Only 3 treatments A B B 0.52 0.001
Only Hemicell A B 0.73 0.005
Only Protexin A B 0.73 0.005
Day 28 47.91 48.00 46.71 46.00
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0074] Table 17 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 17.
Only Protexin A B 0.50 0.003
Day 35 47.90 48.50 48.47 47.96
All treatments 0.28 0.816
Only 3 treatments 0.34 0.733
Only Hemicell 0.35 0.416
Only Protexin 0.45 0.536
Day 42 51.59 51.91 51.02 49.76
All treatments 0.39 0.238
Only 3 treatments 0.48 0.739
Only Hemicell 0.56 0.782
Only Protexin 0.66 0.680
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0075] Table 18 shows yolk length (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 18.
Treatments o. , Hemicell Protexin ,, „ P-
Storage time Control H + P SE1 yalue2
Day l 39.94 41.10 41.40 41.96
All treatments B AB AB A 0.19 0.002
Only 3 treatments b a a 0.22 0.051
Only Hemicell B A 0.26 0.024
Only Protexin B A 0.27 0.032
Day 7 42.03 42.63 42.76 44.01
All treatments B AB AB A 0.23 0.017
Only 3 treatments 0.23 0.400
Only Hemicell 0.28 0.301
Only Protexin 0.28 0.208
Day 14 46.63 45.40 45.93 46.21
All treatments 0.24 0.355
Only 3 treatments 0.31 0.248
Only Hemicell a b 0.37 0.099
Only Protexin 0.39 0.378
Day 21 46.08 46.10 46.18 47.66
All treatments 0.25 0.107
Only 3 treatments 0.27 0.989
Only Hemicell 0.37 0.977
Only Protexin 0.29 0.872
Day 28 46.08 45.47 47.16 48.99
All treatments B B AB A 0.31 <0.001
Only 3 treatments 0.33 0.108
Only Hemicell 0.30 0.328
Only Protexin 0.41 0.217
Day 35 48.06 47.96 48.39 47.44
All treatments 0.35 0.809
Only 3 treatments 0.42 0.913
Only Hemicell 0.54 0.928
Only Protexin 0.60 0.790
Day 42 50.00 49.04 48.80 49.71
All treatments 0.35 0.611
Only 3 treatments 0.43 0.502
Only Hemicell 0.58 0.418
Only Protexin _ 0.47 _ 0.206
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0076] Table 19 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 19.
Treatments
.. , Hemicell Protexin ,, „ P-
Storag
6e time Control H + P SE (H) (P) 1 . value 2 2
Day l 18.66 18.44 19.13 18.34
All treatments AB AB A B 0.10 0.035
Only 3 treatments b b a 0.12 0.060
Only Hemicell 0.12 0.367
Only Protexin 0.16 0.147
Day 7 15.77 15.22 15.36 14.98
All treatments A AB AB B 0.10 0.57
Only 3 treatments 0.11 0.154
Only Hemicell 0.13 0.132
Only Protexin 0.15 0.184
Day 14 13.15 13.09 13.10 13.05
All treatments 0.10 0.987
Only 3 treatments 0.12 0.974
Only Hemicell 0.14 0.820
Only Protexin 0.18 0.873
Day 21 11.82 12.08 11.94 11.96
All treatments 0.07 0.717
Only 3 treatments 0.08 0.520
Only Hemicell 0.11 0.295
Only Protexin 0.11 0.633
Day 28 10.51 10.48 11.21 11.40
All treatments B B A A 0.09 <0.001
Only 3 treatments B B A 0.10 0.003
Only Hemicell 0.11 0.875
Only Protexin B A 0.12 0.002
Day 35 10.07 10.03 11.01 9.81
All treatments B B A B 0.11 <0.001
Only 3 treatments B B A 0.13 0.001
Only Hemicell 0.14 0.894
Only Protexin B A 0.14 <0.001
Day 42 9.96 9.51 10.07 10.26
All treatments b b a ab 0.11 0.088
Only 3 treatments 0.13 0.193
Only Hemicell 0.14 0.119
Only Protexin 0.17 0.776
^Standard error. 2Prohability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0077] Table 20 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 20.
Treatments
Day l 17.88 17.79 17.94 18.11
All treatments 0.08 0.273
Only 3 treatments 0.09 0.551
Only Hemicell 0.11 0.633
Only Protexin 0.10 0.215
Day 7 15.50 15.77 16.27 16.01
All treatments B AB A AB 0.09 0.018
Only 3 treatments B AB A 0.11 0.020
Only Hemicell 0.13 0.327
Only Protexin B A 0.15 0.013
Day 14 13.47 13.42 13.78 13.10
All treatments 0.10 0.122
Only 3 treatments 0.11 0.400
Only Hemicell 0.13 0.880
Only Protexin 0.15 0.306
Day 21 12.33 11.83 12.23 12.17
All treatments 0.08 0.180
Only 3 treatments a b a 0.09 0.067
Only Hemicell 0.13 0.127
Only Protexin 0.09 0.510
Day 28 10.86 10.46 10.83 10.88
All treatments 0.09 0.431
Only 3 treatments 0.09 0.205
Only Hemicell 0.11 0.103
Only Protexin 0.11 0.900
Day 35 10.88 11.00 11.39 10.96
All treatments 0.09 0.266
Only 3 treatments 0.12 0.206
Only Hemicell 0.15 0.700
Only Protexin A B 0.12 0.040
Day 42 9.64 9.59 10.49 10.07
All treatments B B A AB 0.10 0.002
Only 3 treatments B B A 0.12 0.001
Only Hemicell 0.14 0.860
Only Protexin _ B _ A _ 0.16 _ 0.007
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0078] Table 21 shows yolk height (mm) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 21.
Treatments o. .. „ . , Hemicell Protexin ,, „ P-
Stora e time Control H + P SE1
Day l 17.59 17.92 17.75 18.11
All treatments 0.10 0.333
Only 3 treatments 0.12 0.542
Only Hemicell 0.15 0.304
Only Protexin 0.16 0.636
Day 7 15.97 15.70 15.81 16.27
All treatments 0.09 0. 194
Only 3 treatments 0.10 0.592
Only Hemicell 0.13 0.324
Only Protexin 0.13 0.564
Day 14 14.05 13.61 13.65 13.81
All treatments 0.08 0.247
Only 3 treatments 0.09 0.131
Only Hemicell 0.12 0.102
Only Protexin 0.12 0.103
Day 21 13.07 12.96 12.67 12.87
All treatments 0.10 0.569
Only 3 treatments 0.12 0.392
Only Hemicell 0.12 0.756
Only Protexin 0.13 0.131
Day 28 12.39 12.28 12.38 11.95
All treatments 0.08 0.221
Only 3 treatments 0.08 0.863
Only Hemicell 0.08 0.525
Only Protexin 0.12 0.961
Day 35 11.48 11.28 11.23 11.36
All treatments 0.08 0.802
Only 3 treatments 0.09 0.557
Only Hemicell 0.10 0.373
Only Protexin 0.11 0.315
Day 42 10.74 10.41 10.79 10.39
All treatments 0.10 0.385
Only 3 treatments 0.12 0.432
Only Hemicell 0.17 0.351
Only Protexin 0.14 0.872
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0079] Table 22 shows yolk index of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 22.
Standard error. Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0080] Table 23 shows yolk index of eggs from laying hens fed beta-mannanase
(Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 23.
Treatments
. - Hemicell Protexin rT „ P-
Storage time Control ( /THT). ( ZTPV)l H + P SE1 va ,lue 2 2
Day l 0.429 0.418 0.433 0.435
All treatments ab a b b 0.002 0.083
Only 3 treatments 0.003 0.142
Only Hemicell 0.003 0.113
Only Protexin 0.003 0.600
Day 7 0.347 0.369 0.383 0.369
All treatments B A A A 0.002 <0.001
Only 3 treatments B A A 0.003 <0.001
Only Hemicell B A 0.003 0.002
Only Protexin B A 0.004 <0.001
Day 14 0.293 0.294 0.312 0.289
All treatments B B A B 0.002 0.002
Only 3 treatments B B A 0.003 0.012
Only Hemicell 0.002 0.947
Only Protexin B A 0.004 0.020
Day 21 0.260819 0.248324 0.270605 0.258164
All treatments AB B B AB 0.002 0.032
Only 3 treatments AB B B 0.003 0.013
Only Hemicell 0.003 0.101
Only Protexin 0.003 0.190
Day 28 0.214 0.218 0.228 0.225
All treatments 0.002 0.286
Only 3 treatments 0.003 0.156
Only Hemicell 0.003 0.602
Only Protexin 0.003 0.074
Day 35 0.228 0.232 0.236 0.229
All treatments 0.002 0.730
Only 3 treatments 0.003 0.631
Only Hemicell 0.004 0.622
Only Protexin 0.003 0.240
Day 42 0.186 0.185 0.205 0.202
All treatments B B A A 0.003 0.023
Only 3 treatments AB B A 0.003 0.033
Only Hemicell 0.003 0.907
Only Protexin b a 0.005 0.064
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0081] Table 24 shows yolk index of eggs from laying hens fed beta-mannanase
(Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 24.
Treatments
, Hemicell Protexin .. , P-
Storag 6e time Control (H) (P) H + P SE1 va .lue 2 2
Day l 0.441 0.436 0.432 0.432
All treatments 0.002 0.623
Only 3 treatments 0.003 0.571
Only Hemicell 0.004 0.618
Only Protexin 0.004 0.340
Day 7 0.380 0.369 0.366 0.370
All treatments 0.003 0.407
Only 3 treatments 0.003 0.266
Only Hemicell 0.003 0.148
Only Protexin 0.004 0.155
Day 14 0.314 0.299 0.301 0.299
All treatments 0.002 0.156
Only 3 treatments 0.003 0.146
Only Hemicell 0.004 0.108
Only Protexin 0.004 0.147
Day 21 0.284 0.271 0.275 0.270
All treatments 0.003 0.486
Only 3 treatments 0.004 0.435
Only Hemicell 0.003 0.291
Only Protexin 0.003 0.197
Day 28 0.271 0.271 0.263 0.243
All treatments A A AB B 0.003 0.003
Only 3 treatments 0.003 0.589
Only Hemicell 0.002 0.947
Only Protexin 0.004 0.399
Day 35 0.239 0.235 0.229 0.240
All treatments 0.002 0.434
Only 3 treatments 0.003 0.416
Only Hemicell 0.003 0.598
Only Protexin 0.004 0.27
Day 42 0.220 0.214 0.222 0.210
All treatments 0.003 0.483
Only 3 treatments 0.003 0.682
Only Hemicell 0.005 0.575
Only Protexin 0.003 0.848
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0082] Table 25 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 25.
y
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0083] Table 26 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 26.
' Slandard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0084] Table 27 shows yolk weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 27.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0085] Table 28 shows Haugh unit of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 28.
Only Hemicell b a 1.06 0.080
Only Protexin 0.82 0.921
Day 7 76.76 75.55 78.39 70.26
All treatments A A A B 0.74 <0.001
Only 3 treatments 0.73 0.292
Only Hemicell 0.84 0.484
Only Protexin 0.38 0.369
Day 14 85.55 60.84 67.17 61.36
All treatments AB B A 0.89 0.126
Only 3 treatments 1.08 0.044
Only Hemicell 1.44 0.120
Only Protexin 1.16 0.496
Day 21 58.18 53.70 53.91 51.21
All treatments A AB AB B 0.94 0.075
Only 3 treatments 1.09 0.176
Only Hemicell 1.48 0.132
Only Protexin 1.16 0.163
Day 28 48.49 44.55 48.51 48.09
All treatments 1.02 0.458
Only 3 treatments 1.17 0.294
Only Hemicell 1.52 0.200
Only Protexin 1.49 0.994
Day 35 44.22 42.70 42.03 42.41
All treatments 1.05 0.309
Only 3 treatments 1.24 0.185
Only Hemicell 1.56 0.152
Only Protexin 1.63 0.112
Day 42 39.62 41.97 39.88 42.16
All treatments 0.93 0.582
Only 3 treatments 109 0.857
Only Hemicell 1.56 0.466
Only Protexin 1.22 0.117
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0086] Table 29 shows Haugh unit of eggs from laying hens fed beta-mannanase
(Hemicell) and/or probiotics (Protexin), according to various storage Limes in a second trial.
Table 29.
Treatments
.. z . . Hemicell Protexin .. ,, P-
Storage time Control H + P SE1
Day l 84.42 82.53 85.31 79.85
All treatments A AB A B 0.58 0.003
Only 3 treatments 0.62 0.181
Only Hemicell 0.76 0.221
Only Protexin 0.76 0.566
Day 7 70.58 69.01 69.00 66.96
All treatments 0.69 0.343
Only 3 treatments 0.81 0.195
Only Hemicell 0.90 0.140
Only Protexin 1.00 0.443
Day 14 58.24 53.89 57.42 48.30
All treatments A AB A B 1.01 0.001
Only 3 treatments 1.07 0.213
Only Hemicell 1.34 0.106
Only Protexin 1.23 0.746
Day 21 47.09 43.03 46.60 41.53
All treatments A A A B 0.92 0.094
Only 3 treatments 1.12 0.287
Only Hemicell 1.41 0.154
Only Protexin 1.40 0.863
Day 28 41.03 39.52 41.68 38.71
All treatments 0.77 0.543
Only 3 treatments 0.87 0.587
Only Hemicell 1.18 0.533
Only Protexin 1.04 0.759
Day 35 43.40 41.17 39.60 46.44
All treatments ABA B B A 0.88 0.030
Only 3 treatments 0.96 0.292
Only Hemicell 1.12 0.331
Only Protexin 1.35 0.163
Day 42 38.10 41.18 39.96 38.45
All treatments 0.85 0.593
Only 3 treatments 1.09 0.557
Only Hemicell 1.39 0.280
Only Protexin 1.31 0.497
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0087] Table 30 shows Haugh unit of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 30.
Treatments
. Hemicell Protexin P-
Storag
6e time Control H + P SE1 . (H) (P) value 2 2
Day l 86.93 87.22 87.38 87.14
All treatments 0.42 0.990
Only 3 treatments 0.55 0.58
Only Hemicell 0.76 0.858
Only Protexin 0.66 0.770
Day 7 71.65 68.71 70.85 67.55
All treatments 0.88 0.134
Only 3 treatments 0.78 0.297
Only Hemicell 0.98 0.139
Only Protexin 0 96 0 577
1 Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0088] Table 31 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 31.
Only Protexin 0.55 0.200
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0089] Table 32 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 32.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0090] Table 33 shows albumen weight (g) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 33.
Only Hemicell 0.62 0.153
Only Protexin 0.61 0.966
Day 21 38.56 38.43 36.77 37.07
All treatments 0.43 0.342
Only 3 treatments 0.52 0.294
Only Hemicell 0.66 0.926
Only Protexin 0.58 0.132
Day 28 36.07 37.92 37.18 35.89
All treatments 0.46 0.369
Only 3 treatments 0.56 0.411
Only Hemicell 065 0.162
Only Protexin 0.71 0.445
Day 35 34.49 35.36 34.86 34.55
All treatments 0.43 0.900
Only 3 treatments 0.49 0.92
Only Hemicell 0.68 0.536
Only Protexin 0.61 0.775
Day 42 33.99 33.36 33.12 33.80
All treatments 0.43 0.891
Only 3 treatments 0.49 0.761
Only Hemicell 0.57 0.589
Only Protexin 0.65 0.518
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0091] Table 34 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 34.
Treatments
... .. , Hemicell Protexin ,, „ P-
Storage time Control H + P SE1 yalue2
Day l 5.78 6.07 6.05 6.03
All treatments 0.07 0.482
Only 3 treatments 0.09 0.371
Only Hemicell 0.12 0.262
Only Protexin 0.07 0.071
Day 7 6.17 6.10 6.15 6.19
All treatments 0.04 0.919
Only 3 treatments 0.05 0.873
Only Hemicell 0.07 0.635
Only Protexin 0.06 0.901
Day 14 6.01 6.02 6.18 6.07
All treatments 0.05 0.636
Only 3 treatments 0.06 0.489
Only Hemicell 0.06 0.931
Only Protexin 0.08 0.300
Day 21 5.81 6.08 5.93 6.11
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0092] Table 35 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 35.
Day 35 5.95 5.90 5.75 6.13
All treatments 0.05 0.112
Only 3 treatments 0.05 0.278
Only Hemicell 0.06 0.737
Only Protexin 0.06 0.130
Day 42 6.22 6.26 6.01 6.39
All treatments 0.06 0.245
Only 3 treatments 0.07 0.394
Only Hemicell 0.09 0.859
Only Protexin 0.10 0.307
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0093] Table 36 shows shell weight (g) of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 36.
Treatments
.. „ . , Hemicell Protexin .. _ P-
Storag se time Control (H) (P) H + P SE1 val .ue2 ,
Day l 5.40 6.18 5.90 6.13
All treatments B A AB A 0.07 <0.001
Only 3 treatments B A A 0.09 0.002
Only Hemicell B A 0.12 0.001
Only Protexin B A 0.10 0.016
Day 7 5.32 6.26 5.84 4.97
All treatments C A B AB 0.06 <0.001
Only 3 treatments C A B 0.08 <0.001
Only Hemicell B A 0.11 <0.001
Only Protexin B A 0.09 0.003
Day 14 5.42 6.17 6.03 6.09
All treatments B A A A 0.07 <0.001
Only 3 treatments B A A 0.09 0.001
Only Hemicell B A 0.11 0.001
Only Protexin B A 0.10 0.002
Day 21 5.70 6.03 5.83 6.21
All treatments B AB B A 0.05 0.003
Only 3 treatments B A B 0.05 0.089
Only Hemicell b a 0.08 0.051
Only Protexin 0.06 0.320
Day 28 5.76 6.18 6.09 6.17
All treatments 0.06 0.101
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0094] Table 37 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 37.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0095] Table 38 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 38.
y
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0096] Table 39 shows shell breaking strength (N) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 39.
' Slandard error. Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0097] Table 40 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 40.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0098] Table 41 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 41.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[0099] Table 42 shows albumen pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 42.
Treatments
, Hemicell Protexin , P-
Storag 6e time Control (H) (P) H + P SE1 va .lue 2 2
Day l 8.13 8.05 8.09 8.10
All treatments 0.01 0.178
Only 3 treatments 0.01 0.163
Only Hemicell 0.02 0.101
Only Protexin 0.02 0.481
Day 7 8.30 8.35 8.35 8.37
All treatments 0.01 0.127
Only 3 treatments 0.01 0.239
Only Hemicell 0.01 0.171
Only Protexin 0.01 0.155
Day 14 8.46 8.48 8.49 8.50
All treatments 0.01 0.175
Only 3 treatments 0.01 0.552
Only Hemicell 0.01 0.545
Only Protexin 0.01 0.361
Day 21 8.57 8.59 8.60 8.58
All treatments 0.01 0.417
Only 3 treatments 0.01 0.274
Only Hemicell 0.01 0.437
Only Protexin 0.01 0,125
Day 28
All treatments 0.01
Only 3 treatments 0.01
Only Hemicell 0.01
Only Protexin 0.01
Day 35 8.75 8.77 8.78 8.76
All treatments 0.01 0.490
Only 3 treatments 0.01 0.336
Only Hemicell 0.01 0.403
Only Protexin 0.01 0.192
Day 42 9.08 9.09 9.09 9.09
All treatments 0.01 0.946
Only 3 treatments 0.01 0.879
Only Hemicell 0.01 0.669
Only Protexin 0.01 0.704
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00100] Table 43 shows yolk pH of eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 43.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00101] Table 44 shows yolk pH of eggs from laying hens fed beta-mannanase
(Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 44.
' Slandard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00102] Table 45 shows yolk pH of eggs from laying hens fed beta-mannanase
(Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 45.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00103] Table 46 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 46.
All treatments 0.10 0.100
Only 3 treatments 0.13 0.254
Only Hemicell b a 0.13 0.075
Only Protexin 0.17 0.999
Day 7 4.87 4.87 4.80 5.21
All treatments 0.07 0.221
Only 3 treatments 0.08 0.935
Only Hemicell 0.10 0.999
Only Protexin 0.10 0.764
Day 14 5.42 5.13 6.00 5.27
All treatments B B A B 0.08 <0.001
Only 3 treatments B B A 0.10 0.001
Only Hemicell 0.08 0.102
Only Protexin B A 0.13 0.031
Day 21 6.36 6.07 6.20 6.07
All treatments 0.08 0.574
Only 3 treatments 0.08 0.430
Only Hemicell 0.10 0.166
Only Protexin 0.12 0.525
Day 28 5.53 6.64 6.60 6.62
All treatments B A A A 0.07 0.001
Only 3 treatments B A A 0.08 <0.001
Only Hemicell B A 0.11 <0.001
Only Protexin B A 0.10 0.001
Day 35 6.38 6.67 6.64 6.29
All treatments 0.08 0.289
Only 3 treatments 0.09 0.446
Only Hemicell 0.10 0.202
Only Protexin 0.13 0.348
Day 42 6.18 6.91 6.82 6.38
All treatments B A A AB 0.08 <0.001
Only 3 treatments B A A 0.09 0.001
Only Hemicell B A 0.12 0.002
Only Protexin B A 0.10 0.001
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00104] Table 47 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 47.
Treatments
... , Hemicell Protexin .. „ P-
S to rag
6e time Control , (T_ H + P SE H) (P) 1 , value 7 2
Day l 5.33 6.00 5.93 5.60
All treatments B A A AB 0.07 0.004
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00105] Table 48 shows yolk color score (palette) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 48.
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00106] Table 49 shows yolk lightness (L* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 49.
Only Protexin 0.45 0.849
Day 7 58.18 58.72 57.51 57.20
All treatments a a ab b 0.21 0.051
Only 3 treatments 0.24 0.125
Only Hemicell 0.27 0.336
Only Protexin 0.32 0.300
Day 14 57.60 58.70 58.27 61.36
All treatments 0.61 0.133
Only 3 treatments 0.61 0.772
Only Hemicell 0.89 0.548
Only Protexin 0.39 0.408
Day 21 57.13 58.45 57.48 58.16
All treatments B B AB AB 0.18 0.036
Only 3 treatments B A AB 0.21 0.032
Only Hemicell B A 0.23 0.003
Only Protexin 0.26 0.512
Day 28 55.27 58.72 57.86 58.54
All treatments B A A A 0.313 <0.001
Only 3 treatments B A A 0.377 <0.001
Only Hemicell B A 0.505 <0.001
Only Protexin B A 0.491 0.006
Day 35 55.38 58.60 57.53 56.25
All treatments C A AB BC 0.31 0.001
Only 3 treatments B A A 0.35 <0.001
Only Hemicell B A 0.49 <0.001
Only Protexin B A 0.43 0.010
Day 42 57.77 59.14 58.96 58.53
All treatments 0.22 0.121
Only 3 treatments b a ab 0.26 0.071
Only Hemicell B A 0.32 0.029
Only Protexin b a 0.35 0.092
1 Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00107] Table 50 shows yolk lightness (L* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 50.
Treatments
.. z , . Hemicell Protexin .. ,, P-
Storage time Control H + P SE1
Day l 50.99 51.65 52.06 53.73
All treatments B B AB A 0.27 0.001
Only 3 treatments 0.27 0.300
Only Hemicell 0.32 0.311
Only Protexin a 0.30 0.079
Day 7 55.77 56.35 56.45 56.07
All treatments 0.23 0.741
Only 3 treatments 0.26 0.544
Only Hemicell 0.26 0.276
Only Protexin 0.35 0.343
Day 14 57.87 57.87 58.54 59.04
All treatments 0.19 0.101
Only 3 treatments 0.22 0.377
Only Hemicell 0.29 0.995
Only Protexin 0.30 0.284
Day 21 56.62 58.01 58.93 58.32
All treatments B AB A AB 0.25 0.008
Only 3 treatments B AB A 0.32 0.008
Only Hemicell b a 0.41 0.099
Only Protexin B A 0.43 0.005
Day 28 58.25 58.11 58.31 59.82
All treatments B B B A 0.18 0.003
Only 3 treatments 0.16 0.885
Only Hemicell 0.21 0.757
Only Protexin 0.18 0.870
Day 35 58.73 58.82 58.89 58.97
All treatments 0.17 0.970
Only 3 treatments 0.21 0.954
Only Hemicell 0.28 0.878
Only Protexin 0.24 0.750
Day 42 59.07 58.84 59.10 59.88
All treatments 0.22 0.385
Only 3 treatments 0.24 0.899
Only Hemicell 0.37 0.762
Only Protexin 0.26 0.948
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00108] Table 51 shows yolk lightness (L* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 51.
Treatments
, Hemiccll Protexin ¥¥ „ P-
Storage time Control H + P SE1
Day l 50.26 50.01 50.45 50.88
All treatments 0.23 0.777
Only 3 treatments 0.25 0.776
Only Hemicell 0.29 0.677
Only Protexin 0.32 0.768
Day 7 53.37 56.34 54.60 55.78
All treatments B A AB 0.32 0.003
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00109] Table 52 shows yolk redness (a* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 52.
Only Hemicell b a 0.19 0.081
Only Protexin 0.18 0.572
Day 14 7.21 7.62 7.28 6.69
All treatments 0.12 0.067
Only 3 treatments 0.14 0.494
Only Hemicell 0.17 0.265
Only Protexin 0.10 0.860
Day 21 7.02 7.41 6.71 6.36
All treatments 0.15 0.112
Only 3 treatments 0.18 0.298
Only Hemicell 0.17 0.266
Only Protexin 0.12 0.534
Day 28 6.57 7.46 7.15 6.20
All treatments AB A A B 0.1 0.003
Only 3 treatments B A AB 0.14 0.034
Only Hemicell B A 0.17 0.007
Only Protexin 0.16 0.201
Day 35 5.80 6.79 7.28 6.27
All treatments B AB A AB 0.15 0.005
Only 3 treatments B AB A 0.18 0.005
Only Hemicell B A 0.24 0.040
Only Protexin B A 0.17 0.003
Day 42 7.01 6.95 7.45 7.24
All treatments 0.17 0.757
Only 3 treatments 0.13 0.245
Only Hemicell 0.16 0.862
Only Protexin 0.14 0.203
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00110] Table 53 shows yolk redness (a* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 53.
Treatments
.. , Hemicell Protexin ,, „ P-
Storage time Control H + P SE1
Day l 6.98 7.20 7.97 6.02
All treatments AB A A B 0.16 <0.001
Only 3 treatments B B A 0.13 0.003
Only Hemicell 0.14 0.460
Only Protexin B A 0.10 0.002
Day 7 6.17 6.98 7.16 6.87
All treatments B A A AB 0.11 0.017
Only 3 treatments B A A 0.14 0.008
Only Hemicell B A 0.18 0.025
Only Protexin B A 0.12 0.006
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00111] Table 54 shows yolk redness (a* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 54.
All treatments 0.14 0.370
Only 3 treatments 0.17 0.900
Only Hemicell 0.20 0.920
Only Protexin 0.17 0.751
Day 21 6.09 6.04 6.38 5.09
All treatments AB AB A B 0.18 0.058
Only 3 treatments 0.21 0.777
Only Hemicell 0.28 0.937
Only Protexin 0.10 0.512
Day 28 5.49 6.05 6.11 5.93
All treatments 0.14 0.420
Only 3 treatments 0.18 0.332
Only Hemicell 0.22 0.233
Only Protexin 0.12 0.235
Day 35 5.58 6.33 6.22 5.29
All treatments AB AB A B 0.14 0.024
Only 3 treatments b a a 0.14 0.088
Only Hemicell b a 0.20 0.069
Only Protexin 0.10 0.125
Day 42 5.39 6.38 5.89 5.48
All treatments B A AB B 0.12 0.011
Only 3 treatments B A AB 0.12 0.002
Only Hemicell B A 0.16 0.001
Only Protexin b a 0.11 0.097
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00112] Table 55 shows yolk yellowness (b* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a first trial.
Table 55.
Treatments
.. , , . , Hemicell Protexin .. P-
Storag
6e time Control ,T„ H + P SE (H) (P) 1 , , value2
Day l 6.05 6.16 5.96 6.12
All treatments 0.33 0.167
Only 3 treatments AB A B 0.34 0.060
Only Hemicell 0.43 0.224
Only Protexin 0.33 0.178
Day 7 5.98 6.12 6.00 5.98
All treatments 0.35 0.445
Only 3 treatments 0.40 0.302
Only Hemicell 0.45 0.118
Only Protexin 0.53 0.880
Day 14 6.02 6.40 5.98 5.98
All treatments B A B B 0.53 0.013
Only 3 treatments B A B 0.67 0.018
Only Hemicell B A 0.89 0.030
Only Protexin 0.64 0.799
Day 21 5.96 6.23 5.96 5.85
All treatments AB A AB B 0.43 0.011
Only 3 treatments B A B 0.48 0.025
Only Hemicell B A 0.50 0.005
Only Protexin 0.55 0.997
Day 28 5.63 6.16 5.98 5.87
All treatments C A AB BC 0.45 <0.001
Only 3 treatments B A A 0.55 <0.001
Only Hemicell B A 0.78 <0.001
Only Protexin B A 0.85 0.006
Day 35 5.53 5.83 5.61 5.36
All treatments ab a ab b 0.66 0.072
Only 3 treatments 0.73 0.204
Only Hemicell b a 0.90 0.089
Only Protexin 0.97 0.688
Day 42 5.55 5.67 5.77 5.69
All treatments 0.60 0.666
Only 3 treatments 0.72 0.487
Only Hemicell 0.88 0.507
Only Protexin 0.92 0.252
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00113] Table 56 shows yolk yellowness (b* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a second trial.
Table 56.
Treatments o. , Hemicell Protexin ,, „ ,1T,. P-
Storage time Control H + P SE1
Day l 5.70 5.68 6.07 5.66
All treatments b b a b 0.62 0.055
Only 3 treatments B B A 0.70 0.034
Only Hemicell 0.86 0.895
Only Protexin B A 0.90 0.036
Day 7 5.25 5.45 6.02 5.70
All treatments B B A AB 0.68 0.012
Only 3 treatments B B A 0.82 0.006
Only Hemicell 1.03 0.752
Only Protexin B A 1.07 0.015
Day 14 5.62 5.80 5.80 5.53
All treatments 0.55 0.210
Only 3 treatments 0.63 0.422
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00114] Table 57 shows yolk yellowness (b* color) of eggs from laying hens fed beta- mannanase (Hemicell) and/or probiotics (Protexin), according to various storage times in a third trial.
Table 57.
Day 28 5.21 5.82 5.68 5.54
All treatments B A A AB 0.64 0.003
Only 3 treatments B A A 0.80 0.003
Only Hemicell B A 1.16 0.006
Only Protexin B A 1.08 0.026
Day 35 5.55 5.80 5.74 5.50
All treatments 0.51 0.102
Only 3 treatments 0.54 0.156
Only Hemicell b a 0.74 0.091
Only Protexin 0.74 0.219
Day 42 5.41 5.78 5.65 5.36
All treatments B A AB B 0.51 0.007
Only 3 treatments B A AB 0.53 0.011
Only Hemicell B A 0.73 0.009
Only Protexin B A 0.59 0.036
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
Example 3
Oxidant and Antioxidant Assessments
[00115] For the instant example, assessment of oxidant and antioxidant levels birds was evaluated. The status in intestines and oviducts from laying hens in one of the four treatment groups described in Example 2 was evaluated.
[00116] To determine the levels of reactive oxygen species (ROS), the methodology described by Halliwell and Gutteridge (2007) was followed. The levels of non-homogeneous muscle lipid peroxidation were determined as levels of antibodies reactive to thiobarbituric acid (TBARS), measured by the absorbance of the red product at 532 nm, according to the method described by Ohkawa et al. (1978) and expressed as nmol MDA/mg of protein.
[00117] GST activity was measured using spectrophotometry at 340 nm, according to Habig et al. (1974). The mixture contained muscle homogenate supernatant as a test, 0.1 M potassium phosphate buffer (pH 7.4), 100 mM GSH and 100 mM CDNB, used as substrate. Enzyme activity was expressed as pmol/CDNB/mg protein. Protein thiols (PSH) were determined by the method described by Sedlak and Lindsay (1968), a technique that uses DTNB (5,5-dithiobis- 2- nitrobenzoic acid; Sigma). Protein thiols were measured by the sediment formed by the precipitated protein that material was resuspended, using a
homogenization buffer to determine the PSH content. The readings absorbances (405 nm) were performed using a spectrofluorometer (Biotek, Synergy HT).
[00118] Table 58 shows oxidant and antioxidant status in intestines and oviducts from laying hens fed beta-mannanase (Hemicell), and/or probiotics (Protexin). The variables evaluated included TBARS (thiobarbituric acid reactive substances; mmol MDA/mg proteins); GST (glutathione S-transferase; pmolCDNB/min/mg protein); and TSH (protein thios; pmol TSH/mg protein).
Table 58.
Treatments
Variables Control P H H + P SE1 P- value2
Intestine
TBARS 1.84A 1.79A 1.39AB 1.06B 0.12 0.018
GST 975. IB 912.6B 914.9B 1251A 10.1 0.006
TSH 1.49G 2.81A 1.78B 1.70B 0.04 <0.001
Oviduct
TBARS 0.70 0.907 0.77 0.81 0.08 0.440
GST 861.2a 794.9a 741.7b 832.9a 15.3 0.061
TSH 1.25 1.30 1.05 1.22 0.14 0.420
Standard error. 2Probability of treatment effect (model did not included the effect of storage time). Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
Example 4
Performance Assessments
[00119] For the instant example, various performance were undertaken. The experimental units were randomly selected among the hens housed in the commercial farm (about 28,000 light-weight laying hens, 36 weeks old, Hyline W 36 lineage) and assigned to the treatments, that were i) control (basal diet, without supplementation); ii) diet supplemented with 300 g/ton of the P-mannanase Hemicell®; iii) diet supplemented with 50 g/ton of the probiotic additive Protexin®; iv) diet supplemented with 50 g/ton of probiotic additive and 300 g/ton of -mannanase (Hemicell+Protexin).
[00120] The basal feed was formulated according to the nutritional requirements for the genetic (Hy-Line, 2020). Corn and soybean meal were the main ingredients in the feed formulas. Feed and water were both provided ad libitum throughout the experimental period using nipple drinkers and gutter feeders.
[00121] The birds were housed in conventional sheds, arranged in an east-west direction, with concrete floors and masonry walls complemented with wire mesh to the ceiling. The shed
was equipped with side curtains, which were managed according to weather conditions to provide thermal comfort. Average maximum and minimum temperature and air relative humidity values recorded were 36 and 18.1 °C, and 94.7 and 35.8%, respectively. Only natural light illumination was used, allowing a lighting program of 16 hours and 30 minutes of light per day.
[00122] The instant example started when birds were 36 weeks old and lasted 144 days. Egg production was individually recorded at weeks 4, 8, and 12 in a subgroup of 120 cages with 4 birds each, corresponding to 30 replicates per treatment. All eggs produced in these subgroups were individually weighed and analyzed for the presence of feces in the shells, which was classified by the same observer through visual analysis as: absent (clean eggs), minor presence (e.g., presence of small spots; see Figure 1), or major presence (e.g., presence of large spots; see Figure 2).
[00123] Laying rate and egg mass were calculated considering all eggs (including non- marketable eggs) for each replicate (cage). The coefficient of variability was calculated for each cage considering the individual weight of all the eggs produced in each week. A similar procedure was adopted for evaluation of egg masses.
[00124] Blood samples were collected at the end of the project from 8 birds per treatment (random selection). Samples were processed and analyzed (Bio-Plus®, Biochemical Analyzer, Bioplus, Sao Paulo, Brazil) for total protein, albumin, uric acid, total cholesterol, triglycerides, glucose, alkaline phosphatase, alanine aminotransferase, and aspartate aminotransferase using commercial kits (Wiener Lab Group, Sao Paulo, Brazil) and semiautomatic analyzer.
[00125] Table 59 shows performance of laying hens fed diets supplemented with beta- mannanase (Hemicell) and/or probiotics (Protexin).
Table 59.
Treatments
.. „ . , Hemicell Protexin .. „ P-
Responses Control H + P SE1 2
Laying rate (%)
36 - 40 wk 85.31 C 93.71 B 83.25 C 97.66 A 0.11 <0.001
41 - 44 wk 78.34 B 96.69 A 96.36 A 90.55 A 0.11 <0.001
45 - 48 wk 91.05 B 92.59 AB 92.60 AB 95.90 A 0.09 <0.001
36 - 48 wk 84.90 C 94.33 A 90.74 B 94.70 A 0.59 <0.001
Weight of fresh eggs (g)
36 - 40 wk 61.95 B 62.36 B 61.38 B 63.18 A 0.14 <0.001
41 - 44 wk 61.28 C 63.09 A 61.98 B 62.79 A 0.15 <0.001
45 - 48 wk 64.13 B 65.21 A 63.76 B 65.52 A 0.16 <0.001
36 - 48 wk 62.47 B 63.55 A 62.37 B 63.83 A 0.09 <0.001
62 wk (no treat.)3 62.53 B 64.40 A 64.10 A 64.28 A 0.13 0.013
Coefficient of variability in egg weight (%)
36 - 40 wk 5.944b 5.897 b 5.193 a 5.292 ab 0.014 0.072
41 - 44 wk 7.152 B 5.625 A 5.750 A 5.734 A 0.018 0.007
45 - 48 wk 7.088 B 5.397 A 5.608 A 5.405 A 0.019 0.004
36 - 48 wk 6.728 B 5.640 Q 5.517 A 5.477 A 0.010 <0.001
62 wk (no treat.)3 7.940 8.285 8.272 7.891 0.124 0.564
'Slandard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. treatments were not provided from wk 48 to 62. Thus, the last evaluation was performed after 14 wk without supplementation.
[00126] Table 60 shows egg component masses produced by hens fed diets supplemented with beta-mannanase (Hemicell) and/or probiotics (Protexin).
Table 60.
Treatments
Hemicell Protexin ¥¥ P-
Responses Control H + P SE1 2
Egg mass (g/hen/day)
36 - 40 wk 5B2.85 B 58.44 A 51.10 B 61.70 A 0.67 <0.001
41 - 44 wk 48.01 C 61.00 A 59.72 AB 56.86 B 0.39 <0.001
45 - 48 wk 58.30 B 60.38 A 59.04 AB 62.83 A 0.63 <0.001
36 - 48 wk 53.08 C 59.94 A 56.62 B 60.46 A 0.39 <0.001
Yolk mass (g/hen/day)
36 - 40 wk 15.89 B 17.36 A 14.57 B 18.46 A 0.19 <0.001
41 - 44 wk 13.45 B 16.69 A 16.71 A 16.24 A 0.11 <0.001
45 - 48 wk 14.81 C 15.58 B 15.67 B 16.70 A 0.18 <0.001
36 - 48 wk 14.77 V 16.56 A 15.66 B 17.14 A 0.11 <0.001
Albumen mass (g/hen/day
36 - 40 wk 31.81 B 35.22 A 31.35 B 36.98 A 0.42 <0.001
41 - 44 wk 29.54 B 38.05 A 36.95 A 34.72 A 0.23 <0.001
45 - 48 wk 38.06 B 38.54 A 37.30 B 39.69 A 0.49 <0.001
36 - 48 wk 33.09 C 37.26 A 35.18 B 37.12 A 0.24 <0.001
Egg shell mass (g/hen/day)
36 - 40 wk 5.14 B 5.78 A 5.21 B 6.26 A 0.07 <0.001
41 - 44 Wk 5.02 B 6.26 A 6.06 A 5.90 A 0.04 <0.001
45 - 48 wk 5.42 C 6.26 A 6.06 B 6.44 A 0.06 <0.001
36 - 48 wk 5.22 C 6.13 A 5.78 B 6.20 A 0.04 <0.001
1 Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00127] Table 61 shows occurrence of clean eggs or minor/major presence of feces (%) in eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin).
Table 61.
' Si andard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%. treatments were not provided from wk 48 to 62. Thus, the last evaluation was performed after 14 wk without supplementation.
[00128] Table 62 shows serum biochemistry and intestinal morphometry of laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin).
Table 62.
Villi area, pm2 367039 288838 343272 450579 1798 0.670 Crypt depth, pm 224.9 186.4 227.6 202.1 4.96 0.397 Villi height : Crypt depth 6.839 B 7.245 AB 6.304 B 8.295 A 0.171 0.007
Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
Example 5
Egg Microstructure Assessments
[00129] For the instant example, microstructure of the eggs was evaluated. The eggshells obtained from eggs of laying hens in one of the four treatment groups described in Example 2 were evaluated.
[00130] Scanning electron microscopy analyzes were performed to assess the microstructure of the eggshells. For the evaluations, 20 shells from de last trial were used, in which fragments of approximately 5 cm2 were removed and stored in falcon tubes. After this period, the samples were mounted on a stubs, coated with gold-palladium of 35 nm for 3 minutes (Sputter Coater - SCD 050 Balzers, Germany) and analyzed through a scanning electron microscope (JEOL 6060, Japan) at magnifications from 100 to 2000x. In the images, the thickness of mammillary layer, palisade layer, membrane layer, and vertical crystal layer were measured in three locations aleatorily chosen in each eggshell image. The size of column or mammillary bodies and the distance between mammillary knobs were also accessed, but five measurements were made in each image due to the larger variability among measurements.
[00131] Table 63 summarizes eggshell traits accessed through ultramicroscopy in eggs from laying hens fed beta-mannanase (Hemicell) and/or probiotics (Protexin) depending on storage times. Evaluations were obtained at the first phase (40 weeks), second phase (44 weeks), and third phase (48 weeks).
Table 63.
Treatments
First Phase - 40 wk
Mammillary layer, pm 73.29 71.81 59.94 69.26 24.17 0.155
Palisade layer, pm 238.6B 260.2AB 262.7A 254.0AB 22.50 0.035
Mammill. -rPalisade, pm 317.8 332.0 315.4 315.7 28.10 0.319
Membrane layer, pm 57.39 29.84 68.88 68.20 20.01 0.294
Crystal layer, pm 28.39B 26.89B 26.80B 30.92A 19.32 0.429
Mammillary bodies, pm 68.18B 65.82B 62.42B 71.34A 6.928 0.009
Mam. Knobs distance, pm 83.89 82.34 68.13 78.30 21.41 0.148
Second Phase - 44 wk
Mammillary layer, pm 50.86B 66.81A 65.56A 59.88A 12.03 0.003
Palisade layer, pm 259.7B 284.3A 266.7 A 270.7 A 9.953 0.013
1 Standard error. 2Probability of treatment effect. Means followed by different uppercase letters differ statistically at 5%, while lowercase letters were used to indicate differences at 10%.
[00132] Figure 3 shows ultramicroscopy images collected in a single eggshell at the first phase. Figure 4 shows ultramicroscopy images collected in a single eggshell at the second phase. Figure 5 shows ultramicroscopy images collected in a single eggshell at the third phase.
Claims
1. A method comprising a step of administering one or more supplements to a non-human animal, wherein the supplements comprise i) beta-mannanase and ii) a probiotic.
2. The method of claim 1, wherein the animal is an avian.
3. The method of claim 2, wherein the avian is selected from the group consisting of a chicken, a turkey, and a duck.
4. The method of claim 2, wherein the avian is a chicken.
5. The method of claim 3, wherein the chicken is a hen.
6. The method of claim 4, wherein the hen is a laying hen.
7. The method of claim 1, wherein the beta-mannanase is administered at a dose of 300 g/ton and wherein the probiotic is administered at a dose of 50 g/ton.
8. The method of claim 1, wherein the beta-mannanase and the probiotic provide a synergistic effect in improvement in health of the animal.
9. The method of claim 1, wherein the beta-mannanase is administered at a dose between 100 g/ton and 500 g/ton.
10. The method of claim 1, wherein the beta-mannanase is administered at a dose of 300 g/ton.
11. The method of any one of claims 1 to 5, wherein the supplement comprises a probiotic supplement comprising Enterococcus faecium.
12. The method of claim 11, wherein the Enterococcus faecium is National Collection of Industrial, Food and Marine Bacteria (NCIMB) culture number 11181.
13. The method of any one of claims 1 to 5, wherein the probiotic supplement comprises Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus plantarum, Lactobacillus rhamnosus, Bifidobacterium bifidum, Enterococcus faecium, and Streptococcus thermophiles.
14. The method of claim 12, wherein the probiotic supplement is administered at a dose of 50 g/ton.
15. The method of any one of claims 1 to 8, wherein the supplement is present in a feed for the animal.
16. The method of claim 8, wherein the feed is a free choice feed.
17. The method of any one of claims 1 to 8, wherein the administration provides an improvement in health of the animal.
18. The method of claim 17, wherein the improvement in health of the animal is an improvement in animal welfare.
19. The method of claim 17, wherein the improvement in health of the animal comprises a decrease in lesions on the animal.
20. The method of claim 17, wherein the improvement in health of the animal comprises an improvement in intestinal health in the animal.
21. The method of claim 17, wherein the improvement in health of the animal comprises prevention of intestinal dysbiosis in the animal.
22. The method of claim 17, wherein the improvement in health of the animal comprises a reduction in pro-inflammatory effect in the animal.
23. The method of claim 17, wherein the improvement in health of the animal comprises increased release of mannan-oiligosaccharides (MOS) in the animal.
24. The method of any one of claims 1 to 8, wherein the administration provides an improvement in egg quality of one of more eggs laid by the animal.
25. The method of claim 24, wherein the improvement in egg quality is indicated by a modification of a quality selected from the group consisting of weight, specific gravity, albumen height, albumen weight, yolk height, yolk length, yolk index, yolk weight, Haugh unit, shell weight, shell breaking strength, albumen pH, yolk pH, yolk color score, yolk lightness, yolk redness, yolk yellowness, chroma, and any combination thereof.
26. The method of any one of claims 1 to 8, wherein the administration provides an improvement in reactive oxygen species (ROS) in the animal.
27. The method of claim 26, wherein the improvement in ROS is indicated by a modification of thiobarbituric acid reactive substances (TBARS), glutathione S-transferase (GST), protein thios (TSH), and any combination thereof.
28. The method of any one of claims 1 to 8, wherein the administration provides an increase in egg laying rate of the animal.
29. The method of any one of claims 1 to 8, wherein the administration provides an increase in fresh egg weight for eggs laid by the animal.
30. The method of any one of claims 1 to 8, wherein the administration provides a reduction in coefficient of variability of egg weight for eggs laid by the animal.
31. The method of any one of claims 1 to 8, wherein the administration provides an increase in an egg component mass for eggs laid by the animal.
32. The method of any one of claims 1 to 8, wherein the administration provides an increase in occurrence of clean eggs laid by the animal.
33. The method of any one of claims 1 to 8, wherein the administration provides a modification in eggshell thickness in eggs laid by the animal.
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| CN101715879A (en) * | 2009-12-25 | 2010-06-02 | 北京联合盛邦生物技术有限公司 | Compound beta-mannase and lactobacillus feed additive |
| CN112167475A (en) * | 2020-11-17 | 2021-01-05 | 广东顺德鼎一生物科技有限公司 | Eel feed and immunity-improving additive thereof |
| WO2022067052A1 (en) * | 2020-09-25 | 2022-03-31 | Elanco Us Inc. | Probiotic bacillus compositions and methods of use |
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