Effects of indigenous Saccharomyces cerevisiae on intake, growth, gut histology, and serum biomarkers in pre-weaned Lohi lambs

The purpose of this study was to determine the comparative efficacy of locally-produced yeast to commercial yeast on growth performance, serum biomarkers, and gut histology. Thirty pre-weaned male Lohi lambs were divided into; 1) C (Control; starter diet with no supplement), 2) CY (starter diet with ewe milk supplemented with 1 g/animal/day commercial yeast), and 3) LY (starter diet supplemented with 1 g/animal/day local yeast). Animals were housed individually and fed a starter diet and orts were weighed daily. Dry matter intake (DMI) was determined daily while the body weights were determined on a weekly basis. At the end of the trial, blood samples were collected and analysed for blood urea nitrogen (BUN), glucose, β-Hydroxybutyrate (BHBA), and non-esterified fatty acids ( NEFA), while ruminal fluid and tissues were collected for rumen fermentation parameters and histomorphometry. The results revealed that the DMI and average daily weight gain (ADWG) were higher in CY and LY compared to C. The feed efficiency was also better in CY and LY lambs compared to C lambs. The BHBA, BUN, total proteins (TP), cholesterol, creatinine, blood glucose, and tissue histomorphometry were similar in LY and CY supplementation. In conclusion, supplementation with LY produced comparable results to the CY.


Introduction
Optimal growth and development of ruminants are imperative to meet the ever-increasing food demand of the growing population of the world.Sheep play a key role to cater for the nutritional demands of 220 million people in Pakistan and 31.6 million sheep serve as an important cohort for nutritional research.A number of previous studies have emphasised feed supplementation over the use of antimicrobials as feed additives in livestock to prevent the development of antimicrobial resistance in consumers and to improve digestibility and growth performance in ruminants (Ma et al., 2021).For instance, saponin-rich plants such as Sesbania sesban have shown strong anti-protozoal activity while leaving the rest of the rumen biomass unaltered (Wang et al., 2000).Similarly, supplementation of ruminant feed with Eucalyptus leaves (Manh et al., 2012) and essential oils has improved the physiological status of animals.
In Pakistan, most feed additives including S. cerevisiae are imported from technologically advanced countries.However, due to the recent challenges of the COVID-19 pandemic and consequent constraints in shipment, the reliance of the livestock industry on import items has become a point of concern for farmers and stakeholders.The positive impact of yeast supplementation in ruminants has been well documented but the effectiveness of locally-produced live yeast and its impact on pre-weaned lambs remains to be explored.It was hypothesised that the indigenous yeast would produce comparable outcomes when supplemented in comparison to the commercial yeast and therefore, the objective of this study was to compare the effect of the local strain of S. cerevisiae on anthropometric parameters, serum biomarkers, ruminal fermentation parameters, and tissue histomorphometry with that of commercial yeast in lambs.

Materials and Methods
A four-week research trial was conducted at the Small Ruminant Training Center, Pattoki, University of Veterinary and Animals Sciences, Lahore, Pakistan.All the animals were treated against endo-and ectoparasites using a sub-cutaneous injection of ivermectin (0.1 ml/5 kg) and were vaccinated against enterotoxaemia.This experiment was approved by the Animal Care and Use Committee (ACUC) UVAS, Lahore (DR/1136).
A total of 30 male Lohi lambs (body weight, BW, 5.0 ± 1.5 kg; age, 8 ± 2 days) were randomly allocated to three dietary treatments (n = 10/treatment), 1) control (C), without any supplementation; 2) commercial yeast (CY), commercial live yeast (Alltech Inc., Nicholasville, Kentucky, USA) @ 1 g/animal/day (2×10 9 CFU); and 3) local yeast (LY), local live yeast @ 1 g/animal/day (2×10 9 CFU).For the supplemented group, the yeast, either commercial or local, was added to ewe milk and hand-fed twice a day at 07,00 and 17,00 @ 10% of BW for 28 consecutive days.From the 20 th day of the trial, lambs were fed individually on a starter diet (Table 1) and orts were weighed daily.All the lambs were housed in individual pens of 1.5 × 1.5 m, bedded with sand, and had free access to fresh water during the entire experimental period.Multiple yeast strains were subjected to various biochemical tests, including wet mount examination, a germ tube test, a sugar utilization assay, and polymerase chain reaction (PCR) to facilitate their identification.Sabouraud dextrose broth was employed to support the growth of selected yeast cells (indigenous yeast).The culture was centrifuged at 6000 rpm for 10 min and the supernatant was carefully removed.The resulting cell pellets were collected and subsequently washed with phosphate-buffered saline (PBS).The washed cells were then suspended in PBS, and the concentration of the cell suspension was adjusted to an optical density (OD) of 2×10 9 colony-forming units per millilitre (CFU/mL).The resulting suspension was then transferred to a sterile container and saved for further supplementation The DMI was determined daily while the animals were weighed weekly.Body condition score was performed on a weekly basis using a scale of 0 to 5 (Phythian et al., 2012), where 0 was considered emaciated and 5 as obese.The faecal score was developed by visual scoring as described by Hu et al. (2012) and recorded daily on a scale from 1 to 4 where 1 was considered a solid pellet and 4 as watery.
At the end of the trial, 10 ml of blood sample was collected aseptically from the jugular vein, poured into non-EDTA vacutainers, and centrifuged for 15 min at 2200×g to harvest serum.The serum was stored at -20 °C for analysis of glucose, blood urea nitrogen (BUN; Biosystems, Spain), cholesterol (Human, Germany), β-hydroxybutyrate (BHBA, Randox, UK), and total protein (portable refractometer, ATAGO U.S., Inc., Bellevue, WA).All blood chemistry parameters were analysed using commercially available kits with an EPOCH microplate spectrophotometer (Biotek Instruments Inc., Winooski, USA).
At the end of the experimental period, a gastric tube was used to collect ruminal fluid as reported by Lodge-Ivey et al. (2009) 4h after feeding.The pH of the ruminal fluid was measured immediately after the collection of fluid using a digital pH meter (NeoMet model pH-200L, Korea).
At the completion of the trial, lambs (n = 5) from each treatment were slaughtered at the UVAS slaughtering facility to collect ventral rumen and jejunum tissue samples.Tissue samples were fixed in 10% formalin, dehydrated by graded alcohol in ascending order, and cleared in xylol.After tissue sectioning, samples were stained with a haematoxylin-eosin staining dye, and images were taken at 4X using an optical microscope (LX400, LABOMED, The Netherlands) fitted with a digital camera (DC, 1355-F050, CMEX Euromex, The Netherlands).To assess rumen developmental parameters; papillae length, papillae width, and surface areas were measured as described earlier by Malik & Rashid (2020).Jejunal histomorphometry was assessed in terms of villus height, villus width, and crypt depth by using a microscope and an image processing and analysis system, LABOMED Pixel Pro (Version 1, Leica Imaging Systems Ltd., Cambridge, United Kingdom).
The collected data were tested for a normal distribution using the Kolmogorov-Smirnov test and analysed using one-way analysis of variance (SPSS; version 20.0 IBM Corp., Armonk, New York, USA).Tukey's post-hoc test was employed to determine differences between treatments and the results were considered significant at P <0.05.

Results and Discussion
The results of the current study showed that the average daily weight gain (ADWG), dry matter intake (DMI), and feed efficiency were improved (P <0.05) in sheep supplemented with either LY or CY compared to C (Table 2).As expected, the supplementation of yeast substantially increased the DMI, which is in agreement with the previous findings (Abd El-Ghani, 2004;Stella et al., 2007), who reported that the DMI was higher in goats supplemented with yeast.An increase in DMI with yeast supplementation is generally attributed to its favourable capacity to create anaerobic conditions in the rumen, which aids ruminal microorganisms and fibre digestibility (Chaucheyras-Durand et al., 2008) Similar results were observed for the ADWG, in which CY and LY increased the ADWG by 8.5% and 11%, respectively, over the C group.These findings are in accordance with the previously published claims that yeast supplementation enhances DMI and increases fibre digestibility in the rumen as a result of cellulolytic bacteria (Newbold et al., 1996).
No effect (P >0.05) of yeast supplementation was observed for body condition score (BCS), which was also reported by Dann et al. (2000) in dairy cows.Despite the fact that all the animals were acclimatized and randomly selected, the faecal score in C (although within the normal range) was higher than CY and LY (P ˂0.05) and showed improvement with supplementation, probably as a result of better feed conversion (Ghazanfar et al., 2015) or an improved faecal microbiome (Huebner et al., 2019).Although previous studies have shown age-related improvements in faecal score rather than yeast supplementation (He et al., 2017), S. cerevisiae treatment does lower the diarrhoeal score in piglets (Trckova et al., 2014), primarily by improving the immune response.For the rumen fermentation parameters, rumen pH did not change (P >0.05) with supplementation.A slightly increased rumen pH in CY and LY could be due to an increase in the number of cellulolytic microbes or due to decreased VFA concentration, which negatively correlates with the rumen pH (Thrune et al., 2009).BHBA was substantially higher in the group supplemented with CY compared to the control group, while BUN was higher in LY than the control (Table 3).Interestingly, BHBA markedly increased in CY compared to the C but was similar to LY, despite a trend towards an increase.This could be due to strain differences that might have stimulated the ruminal milieu in different ways.However, both the yeast-supplemented groups were similar.An increasing trend of BHBA in LY compared to C (P =0.08) motivates further investigations and experiments with LY.There was also an increase in BUN in CY and LY compared to C, suggesting an increase in the microbial protein turnover and ammonia level in the rumen (Chuelong et al., 2011).For serum biochemical parameters, the total protein, cholesterol, and creatinine were not affected by yeast supplementation (Table 4).However, glucose decreased in CY, while uric acid decreased in LY, as compared to the C treatment.Serum total protein and cholesterol were similar among the treatments; these findings are in agreement with Wojcik (2010) and Elaref et al. (2020), respectively.The blood glucose showed a substantial decrease in CY compared to C with supplementation, which is contradictory to that previously reported by Sowińska et al. (2016).However, this trend could be due to an increase in fibre digestibility and less propionate production.During the process of digestion, ruminal microbes breakdown the fibres and the prime VFA produced from fibre fermentation is acetic acid, instead of propionic acid.Propionic acid is known as glucogenic VFA and high production leads to higher glucose in the blood and vice versa (Yost et al., 1977).Serum creatinine was similar among all the groups, which has been reported before (Elaref et al., 2020).Rumen papillae length and density were improved in both treatment groups compared to the control group (P ˂0.05); however, rumen papillae width was similar (Table 5).In the jejunum, villus height and the villus height to crypt ratio was substantially improved in supplemented groups compared to the control group; villus width and crypt depth were similar across treatments.Representative ruminal and jejunal histology samples from each treatment group are shown in Figure 1.These findings suggest that the yeast supplementation increased the digestibility of the fibre and, consequently, the availability of VFA in the gut milieu, thereby resulting in an adaptive response at the level of the epithelium (Steele et al., 2016).One of the proposed mechanisms for enhancing the growth of epithelium by VFA is through indirect effects of multiple hormones, including epidermal growth factor and IGF-1 (Cui et al., 2019).

Table 1
Ingredients and chemical composition of starter diets on a dry matter basis 2 DM = dry matter; CP = Crude protein; ME = Metabolizable energy; NDF = neutral detergent fibre; ADF = acid detergent fibre

Table 2
Growth performance and a faecal score of lambs supplemented with local yeast (LY) or commercial yeast (CY)

Table 3
Rumen fermentation parameters of lambs supplemented with local yeast (LY) and commercial yeast (CY) The data are presented as mean ± standard error of mean (SEM); values in the same row with different superscripts are significantly different at P <0.05; CY= Commercial yeast, LY= Local yeast

Table 4
Serum biochemical parameters of lambs supplemented with local yeast (LY) and commercial yeast (CY)

Table 5
Rumen and jejunum histomorphometry of lambs supplemented with local yeast (LY) and commercial yeast (CY) The data are presented as mean ± standard error of mean (SEM); values in the same row with different superscripts are significantly different at P <0.05; CY= Commercial yeast, LY= Local yeast