Performance responses of lactating Rahmani ewes fed diet supplemented with Enterococcus faecium NRC-3 or Lactobacillus rhamnosus

Abstract Production of new types of probiotics for animal nutrition mainly depends on the appropriate bacterial strain and efficient substrate. Therefore, this study aimed to evaluate the impact of two probiotic strains containing 1.2 × 108 (CFU/g), produced on permeate media on performance responses of Rahmani ewes. Thirty early lactating ewes (about 2–3 years old and weighting on average 43.2 ± 0.3 kg) were randomly divided into three groups of 10 animals each using a completely randomized design. The 1st group was fed the basal diet (60% concentrate feed mixture (CFM) + 30% Egyptian clover + 10% bean straw). While the ewes in 2nd and 3rd groups were fed the basal diet + 2 g of Enterococcus faecium NRC-3(EF) and Lactobacillus rhamnosus (LR), respectively for 9 weeks. Ewes’ diet supplementation with EF or LR increased (p < 0.05) dry matter, organic matter, crude protein, neutral detergent fiber, acid detergent fiber, and non-structural carbohydrates digestibility compared to ewes of the control group. Glucose, total protein, and albumin concentrations significantly increased in the blood of EF ewes than those of LR and control. Probiotics increased ewes’ milk yield as well as milk protein, fat, and lactose yields, but no differences were observed between treatments when milk components were expressed as percentage. Milk fatty acids profile not changed due to EF or LR supplementation. Probiotics (E. faecium and L. rhamnosus) produced on cheese industry waste (permeate) have proven their ability to improve the productive performance of the lactating Rahmani ewes.


Introduction
Livestock producers always aim to improve the health and productive performance of their herds in an economical manner.5][6] The global probiotics in the animal feed market were valued at USD 4.4 billion in 2020, with estimation to reach about USD 7.3 billion by 2026.The demand for probiotics in animal feed is expected to remain high due to the increasing awareness about their benefits on animal's health and production (Probiotics in animal feed market report 7 ).
In ruminants, supplementation with probiotics (living nonpathogenic microbes) aids to make favorable changes in the balance and activities of the microbiota in the rumen and lower gastrointestinal tract. 8,9It also offers a promising avenue to control the pathogens and harmful microorganisms to promote animal health and production. 10,11The most common direct microbial feed supplements in commercial formulas are yeast and lactic acid bacteria. 12Lactic acid bacteria (LAB) are gram-positive, either rod-shaped (bacilli) or spherical (cocci) bacteria that produce lactic acid as the major metabolic end product of carbohydrate fermentation. 13][14] Enterococci are facultative anaerobic bacteria found in the rumen, tolerant to acids and bile salts 15 and have the ability to produce vitamin B12, 16 and bacteriocin (enterocin) which enables it to inhibit the activity of harmful bacteria. 17Also, Lactobacilli are acid-tolerant bacteria found in ruminant's gastrointestinal tract (GIT), where they work on stimulate the growth of beneficial bacteria and limit the colonization of harmful bacteria. 18Like Enterococci, Lactobacilli have the ability to resistance of the antibiotics, produce antimicrobial agents, and confer good immune response for the host animal. 19,20he production of probiotics for livestock faces obstacles including the difficulty of selecting a specific bacterial strain with high biomass production and the high cost of nitrogen and carbon sources of the growth medium which represents about 40% of the production cost. 21Our research team succeeded in the production of a high biomass yield of Enterococcus faecium and Lactobacillus rhamnosus in economical way by using cheese industry waste (permeate) as a substrate. 22We hypothesize that utilization of the produced probiotics in the feeding of lactating ewes will improve ewes' productive performance, enrich their milk with healthy fatty acids (e.g., CLA); which would pave the way for large scale production of probiotics on agro-industrial wastes.Therefore, the aim of the current research was to test the effectiveness of new probiotics (E.faecium NRC-3 and L. rhamnosus locally produced using milk permeate) on nutrient digestibility, blood metabolites, milk production, milk composition, and milk fatty acid profile of Rahmani lactating ewes

Materials and methods
The experiment was carried out at the Agriculture Experimental and Research Station, Faculty of Agriculture, Al-Azhar University (Egypt) for 9 weeks (63 days).Management of the ewes was in accordance with the 3rd edition (2010) of the guide of Agricultural Research and Teaching of Federation of Animal Science Societies, Champaign, IL, USA, and approved by the technical committee of the Science, Technology & Innovation Funding Authority (STDF), Egypt (project STDF 33413).The microbiological and chemical analyses were performed at the laboratories of the National Research Center (Egypt).

Preparation of the bacterial probiotics
Two probiotic strains of lactic acid bacteria (Enterococcus faecium NRC-3 with NCBI accession number MW856656 (https://www.ncbi.nlm.nih.gov/nuccore/MW856656) and Lactobacillus rhamnosus) were used in this study.They were isolated from homemade yogurt and cheese samples and chosen depending on their ability to inhibit the activity of the pathogens especially E. coli and survival in good counts at GIT conditions.These bacteria were identified using MALDI-TOF MS technology and 16S rDNA sequencing.For economical production of the biomass, E. faecium NRC-3 (EF) and L. rhamnosus (LR) were grown on permeate (cheese industry waste) media.The used permeate media composed of (g/L permeate): yeast Extract; 5.0, Tryptophan; 0.3, Magnesium Sulfate; 0.5, Ammonium Chloride; 3 and Pantothenic acid; 0.004.Permeate media was inoculated individually with 5% (v/v) of EF or LR culture then incubated at 37 � C for 48 h.The cultured biomasses were separated by centrifugation at 6000 g for 15 min at 4 � C then were carried on corn starch and dried to be more suitable for animal feeding.Each gram of the produced probiotics EF or LR was containing 1.2 3 10 8 CFU.

Animals and management
After a week of parturition, thirty lactating Rahmani ewes (about 2-3 years old and weighting on average 43.2 ± 0.3 kg) were randomly divided into three groups of ten animals each using a completely randomized design.Each ewe's group was kept in a separate semiopened concrete floor pen (1.5 m 2 /ewe) with free access to fresh clean drinking water.All ewes received a basal (control) diet consisted of 60% pelleted concentrates feed mixture (CFM) which consisted of 55% yellow corn, 15% wheat bran, 15% soya bean meal, 10%cotton seed meal, 2% molasses, 1.5% limestone, 1% mineral and vitamin mixture and 0.5% NaCl, 30% fresh Egyptian clover, and 10% bean straw.The ewes of the first group were fed the basal (control) diet with no probiotic supplementation.While the ewes in the second and third groups received the basal diet þ 2 g of Enterococcus faecium NRC-3 (EF) and Lactobacillus rhamnosus (LR), respectively.The chemical composition of the feed ingredients and the basal diet is shown in Table 1.The ewes were fed dry matter according to 4% of their body weight in two equal amounts daily at 9.00 a.m. and 5:00 pm.The daily average dry matter intake (DMI) for control, EF, and LR group was 1.732, 1.728, and 1.720 kg, respectively.Ewes were first offered the allotted amounts of CFM, and then fresh Egyptian clover and bean straws were offered.

Nutrient digestibility trial
During the last week of each month of the experimental period, a nutrient digestibility trial was conducted using silica as an indigestible marker as described by Ferret et al. 23 Approximately 50 g of fecal grab samples were collected daily in a bag connected to the back of each animal for five successive days and then dried in an oven at 60 � C for 48 h.Approximately 250 g of each feed ingredient was collected each month of the experimental period, then dried and grinded.The dried feed and feces samples were grinded to pass a 1 mm screen and analyzed for dry matter (DM), ash, ether extract (EE), and crude protein (CP) contents according to AOAC 24 official methods.

Blood plasma sampling and analysis
Two blood samples were collected from each ewe at days 30 and 60 of the experimental period.After 4 h of the morning feeding, 5 mL of blood samples were taken from the animal jugular vein into heparinized tubes.The blood tubes were centrifuged at 5000 � g for 15 min at 15 � C. The separated plasma was transferred to 3 ml Eppendorf tubes and frozen at À 18 � C until analysis.For analysis of the blood plasma samples, Biodiagnostic specific kits (Biodiagnostic for diagnostic and research reagents, Dokki, Giza, Egypt) were used.The plasma glucose, total protein, albumin, cholesterol, and urea concentrations and glutamateoxaloacetate transaminase (GOT), glutamatepyruvate transaminase (GPT) units were determined as described in the manufacturer instructions.

Milk sampling and analysis
After 3 weeks of starting the experimental period, weekly milk samples were collected from each ewe up to the end of the experimental period.Ewes were milked by hand daily at 09:00 a.m. and the produced milk was recorded using 1000 ml graduated cylinder.The milk samples (100 ml of the recorded milk yield) were kept in clean plastic bottles for analysis.The fresh milk samples were analyzed for fat, lactose, protein, ash, solids not fat, and total solids using an ultrasonic milk analyzer (Milkotester master classic LM2, Belovo, Bulgaria).Fat corrected milk (4% fat percent) was calculated using formula of Gaines. 25at corrected milk (FCM) ¼ 0.4 M þ 15 F, Where: M ¼ milk yield and F ¼ fat yield.
The milk fatty acid profile was determined by the transmethylation of the fatty chains to fatty acid methyl esters (FAMEs) according to the modified method by Zahran and Tawfeuk. 26The FAMEs were separated with an HP 6890 plus gas chromatography (Hewlett Packard, USA), using a capillary column Supelco TM SP-2380 (60 m � 0.25 mm � 0.20 lm), (Sigma-Aldrich, USA), Detector (FID), and the injector and detector temperature was 250 � C. The column temperature was 140 � C (held for 5 min) and rose to 240 � C, at a rate of 4 � C/min, and held at 240 � C for 10 min.The carrier gas was helium at a flow rate of 1.2 mL min À 1 .The sample volume was 1 mL (in n-hexane) and injected through a split injector at a splitting ratio of 100:20.FAMEs were identified by comparing their relative and absolute retention times to those authentic standards of FAMEs (Supelco TM 37component FAME mix).The fatty acid composition was reported as a relative percentage of the total peak area.

Statistical analysis
Data were analyzed as a completely randomized design with repeated measures using the PROC MIXED procedure of SAS (SAS Institute, Cary, NC, USA), considering sampling time as repeated measures and individual ewes as the experimental unit.
The statistical model included the treatment effect, week effect, and the treatment � week interaction.Animal nested within treatment was considered the random effect, while treatment was the fixed effect.Two covariance structures were considered in the REPEATED statement in PROC MIXED: compound symmetry (cs) and auto-regressive (AR(1)).The error structure, with the lowest Akaike information criteria, that fits the statistics was selected for the model.When the F-test was significant at p < 0.05, means were

Effect of probiotics supplementation on nutrients digestibility
Data of Table 2 showed that supplementation of the lactating ewes diet with E. faecium NRC-3 (EF) and L. rhamnosus (LR) increased (p < 0.05) significantly dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and non-structural carbohydrates (NSC) digestibility compared to ewes of control.No impact of E. faecium NRC-3 or L. rhamnosus supplementation on EE digestibility was detected.

Effect of probiotics supplementation on ewes blood metabolites
The results showed that diet supplementation with EF increased ewes' blood glucose, total protein, and albumin concentrations significantly (p < 0.05) than those of LR and control ewes (Table 3).Ewes of LR group showed higher blood glucose concentration than those of control with no significant changes in total protein and albumin concentrations.No impact of EF or LR supplementation on ewes' blood concentration of globulin, albumin/globulin ratio, urea, cholesterol, glutamate-oxaloacetate transaminase (GOT), and glutamatepyruvate transaminase (GPT) when compared with control ewes.

Effect of probiotics supplementation on ewes' milk yield and composition
Milk yield and fat corrected milk yield increased significantly (p < 0.05) by probiotics (EF and LR) addition to ewes diets (   (p < 0.05) increased.The rate of increase in milk component yields was comparable to what was observed in milk and fat corrected milk yields.Despite the positive effect of adding probiotics to the diets of ewes on milk production and the yield of its components, it had no significant effect on the milk fatty acid profile (Table 5).The results showed an insignificant (p � 0.05) decrease in milk total saturated fatty acids (SFA) in supplemented ewes with probiotics (EF and LR) compared to control group.While the mono and poly unsaturated fatty acids (MUFA and PUFA) content of ewes' milk treated with probiotics (EF and LR) showed an insignificant (p � 0.05) increase compared to the control group.

Discussion
The effects of new two probiotic strains (E.faecium NRC-3 and L. rhamnosus) on ewes' nutrient digestibility, blood parameters, milk production, composition, and fatty acid profile have been evaluated in the current study.In the following lines, the most important results obtained from this study will be discussed.

Effect of probiotics supplementation on ewes' nutrients digestibility
Supplementation of ruminants' diets with direct-fed lactic acid bacteria has shown great impact in the manipulation of rumen fermentation through modulation of microflora composition, improved feed conversion rate, and enhancement of animals' immune system responses and productive performance. 4,12 the present study, the improvement of the nutrient digestibility by ewes fed diet supplemented with E. faecium NRC-3 or L. rhamnosus may be due to positive changes in the rumen fermentation led to more production of the volatile fatty acids (VFA) and flow of more nitrogen for microbial protein synthesis. 4hese positive changes include; enhancement of the ruminal cellulolytic fungi and bacteria in number and activity 14 as well as increase in microbial endogenous enzymes production and activity. 27Moreover, Azzaz et al., 28 stated that E. faecium as a probiotic supplementation increased significantly ruminal short chain fatty acids (SCFA) production (in vitro) and improved digestability of feed's DM, OM, CP, NDF, ADF, and NSC by lactating Holstein cows.Also, Mamuad et al. 14 found that E. faecium SROD supplementation at three different levels (0.1, 0.5, and 1%) significantly increased the production of ruminal acetate, propionate, butyrate, and total VFA when compared with the control.Furthermore, the dietary supplementation with E. faecium increased the number of cellulolytic fungi and F. succinogenes and R. flavefaciens ruminal bacteria, 14 which could explain the significant improvement of fiber and all nutrients digestibility in the current study.The effect of adding E. faecium NRC-3 to ruminant diets at (1, 2, and 3 g/kg DM) on ruminal diet degradability characteristics and fermentation pattern was previously studied (in vitro) by our research team. 22The results showed the positive impact of E. faecium NRC-3 on ruminal diet degradability characteristics and fermentation pattern.The maximum improvement of the diet's digestibility parameters, production of VFA, and ammonia concentration was at 2 g/kg DM of E. faecium NRC-3 addition level.Accordingly, we chose this level for application in the in vivo experiments in the current study.Moreover, Zhang et al. 27 reported that the addition of Lactobacillus rhamnosus GG to the diet of Holstein calves during the preweaning stage significantly increased the ruminal microbial enzymes (protease and amylase) activity, increased production of propionate, butyrate and total VFA in the rumen and improved feed nutrients digestion.In addition, Mani et al. 8 stated that ruminal fibrobacter (fibrolytic bacteria) was detected in the rumen after feeding sheep on two different strains of L. rhamnosus or their combination and it was not detected before L. rhamnosus inclusion, which support use of L. rhamnosus as a feed additive.

Effect of probiotics supplementation on ewes' blood metabolites
The higher blood glucose and protein concentrations in lactating ewes fed on probiotics (EF and LR) compared to the control group were expected due to the significant improvement in digestion of OM, CP, and NSC (Table 2).Specifically, increased ruminal fermentation for OM and NSC resulted in a numerical downward shift in the ratio of acetate to propionate which leads to increased propionate concentration in the rumen 14 and subsequently in the blood.The blood propionate encourages the liver to secrete certain enzymes (pyruvate carboxylase and phosphoenolpyruvate carboxykinase) which are involved in the gluconeogenesis pathway that converts propionate into glucose. 29,30It has been reported that ruminants receive little alimentary glucose because of rumen fermentation and limitation of glucose absorption from the intestine, and hence they depend largely on gluconeogenesis for their glucose supply. 31This scenario may give a simplified explanation for the reason for higher blood glucose concentration in ewes received E. faecium NRC-3 or L. rhamnosus in the current study.
In agreement with the current data, Nocek and Kautz 32 reported that Holstein cows consuming E. faecium had higher blood glucose than did animals of the control.Also, Azzaz et al. 28 found that the addition of E. faecium to the diet of lactating Holstein cows increased their blood glucose concentration significantly as a direct result of higher OM and NSC digestibility.In contrast, Azzaz et al. 28 did not observe any impact of E. faecium supplementation on cows' blood total protein and albumin concentration but observed a reduction of blood cholesterol and triglycerides concentrations.The discrepancy in some of the obtained results from the different studies may be attributed to the use of different animal breeds with different physiological conditions, different diets and management systems, different microbial strains used, and the dose of administration, among others … . 33In accordance with the results of the current study, Abu et al. 34 found that feeding Holstein heifers on rice straw treated with a mixture of probiotics containing L. rhamnosus led to a significant increase in blood glucose concentration without any effect on the level of albumin in the blood.Also, Zhang et al. 27 observed that the addition of L. rhamnosus GG to the diet of Holstein calves had no impact on the blood total protein and immunoglobulin.In this study, all the measured blood parameters were within the standard physiological ranges for healthy animals. 35The supplementation with the probiotics did not affect blood urea-N, GOT, and GPT concentrations, this indicating minimal effect of the treatments on ewes' muscle protein catabolism, and kidney and liver function. 28The higher blood glucose and total protein concentrations of the probiotics-supplemented ewes indicate that ewes have covered their protein and energy needs, 1 and suggest the efficacy of the probiotics in supporting their health and nutritional status.

Effect of probiotics supplementation on ewes' milk yield, composition, and fatty acid profile
Higher milk production by ewes fed diet supplemented with lactic acid bacteria (EF and LR) compared to ewes fed control diet can be expected because of enhancement of animal health and/or significant improvement in the efficiency of the digestion and metabolism. 32Consistent with our results, Azzaz et al. 28  (DSM 7061) produced 13.5% higher milk and 13.2% energy corrected milk than those of control.Similar to our findings, many previous studies revealed no significant effect of E. faecium or L. rhamnosus supplementation on percentages of milk components, but positively affects the milk component yields. 28,32,36he higher milk component yields in EF and LR supplemented ewes may be due to occurred improvement of digestibility and metabolism of feed nutrients leading to a greater level of aminogenic and glucogenic fuel provision for milk component production. 32Also, higher blood glucose concentration can provide the mammary gland with more glucogenic precursors. 37It has been reported that supplementation of lactating cows with E. faecium or L. rhamnosus tended to decrease CH 4 emission significantly. 28,38Suppression of CH 4 production in the rumen may lead to redistribution of energy toward improvement milk component production. 39In addition, lactic acid bacteria as feed supplements increase the release and activity of ruminal microbial enzymes, 14,27 produces bacteriocins to antagonism the harmful bacteria, 17 and provide the host animal with antioxidants and growth factors to support its immune system 20 which could also explain the higher milk and its component production in the current study.
It is worth noting that in all aforementioned studies, 28,32,37,38 there was no significant effect of adding E. faecium or L. rhamnosus to animals' diets on the daily dry matter intake (DMI).Therefore, it can be thought that the DMI has little effect on the feed efficiency/conversion (expressed as the amount of feed (kg) required for producing 1 kg of fat/energy corrected milk).In the present study, the offered feed amount was restricted (ewe fed DM according to 4% of its body weight) to avoid refusals and to neutralize the effect of the DMI on the productive performance of the tested ewes.However, the average DMI for control, EF, and LR ewes groups were 1.732, 1.728, and 1.720 (kg/h/d), respectively.The feed conversion/efficiency was found to be 4.34, 3.51, and 3.54 (kg feed/each kg of 4% fat corrected milk) for the control, EF, and LR ewes groups, respectively.Accordingly, the feed efficiency of lactating ewes was greatly improved by E. faecium NRC-3 or L. rhamnosus supplementation, resulting in increased milk production without additional feeding costs for the farmers.
In the current study, supplementation of ewes' diets with lactic acid bacteria (EF and LR) did not alter the milk fatty acids profile.Similarly, Philippeau et al. 38 found no effect of L. rhamnosus supplementation on the milk fatty acid profile of Holstein cows fed high-or low-starch diets.While Azzaz et al. 28 reported a positive effect of E. faecium supplementation on Holstein cow's milk fatty acid composition.They stated that E. faecium decreased the percentage of C23:0, but increased the percentage of C18:1 trans-9, C18:2 cis-9-12, and C18:2 trans-10 cis-12 in milk.They attributed this little change in the milk fatty acids profile to the biohydrogenation of polyunsaturated fatty acids of feed fat.Therefore, the effect of LAB on the ruminal microbes that mainly participate in dietary fat biohydrogenation should be specifically studied.

Conclusion
Production of two new types of probiotic preparations for feeding lactating ewes using permeate (a waste polluting the environment) as a substrate supports the concept of a sustainable farm.E. faecium NRC-3 and L. rhamnosus have been shown to improve the nutritional and productive status of Rahmani ewes at a level of 2 g/kg DM of feed.E. faecium NRC-3 was found to be superior to L. rhamnosus in improving the productive performance of the ewes.The effect of different LAB strains on the rumen's biohydrogenation of dietary fats and its relationship with the fatty acid content in milk requires further study.

Table 1 .
Chemical composition of feed ingredients and the basal diet (on DM basis).
compared by applying the probability of difference option of the least squares means statement.The probability of difference option of the least squares means statement was used for multiple comparisons of means.
Table 4).The milk yield increased in EF and LR ewes groups by 21.7 and 20.6%, respectively compared to control group.Also, fat corrected milk yield of EF and LR ewes increased significantly by 23.3 and 21.9%, respectively compared to control ewes.No impact of EF and LR supplementation on the percentage of milk component was noticed, but yields of milk components were significantly

Table 4 .
Effect of E. faecium NRC-3 and L. rhamnosus supplementation on ewe's milk yield and composition.
LR: Ewes fed the basal diet þ 2 g of L. rhamnosus.Means in the same row with different superscripts differ (p < 0.05).