Effect of dietary oregano essential oil and milk replacer on physiological status and immunological responses of pre- and post-weaned Ghoongroo piglets

Abstract Ghoongroo pigs have good adaptability in a low input production system with high prolificacy. The present study was conducted on pre-and post-weaned Ghoongroo piglets from 2–3 days to 12 weeks of age to evaluate the effect of a milk replacer and oregano essential oil (EO) on growth, physiological and immunological responses. Thirty six piglets were randomly divided into three groups. The control group (n = 12) was allowed to suck mother’s milk. Second group piglets were provided milk replacer (MR) and piglets of the third group were provided milk replacer along with oregano EO at 500 mg/kg diet. After weaning, piglets were provided standard concentrate diets. The results showed that the body weight in MR and MR + EO groups were significantly higher compared with the control. The MR + EO group had better intestinal microbiota, greater nonspecific innate immunity with the phagocytosis efficacy of neutrophils, lower cortisol concentration and more stable thyroid hormones than the other groups. The better haematological status supported the rapid organ development and improved intestinal health status in both the experimental groups. In conclusion, milk replacer, especially with the inclusion of oregano EO, can lower weaning stress, enhance nonspecific immunity and improve growth and health status of piglets.


Introduction
Ghoongroo pig breed has been identified in the eastern Sub-Himalayan region of West Bengal, India, adjoining Nepal, 1 which has good adaptability in low input production system with high prolificacy. 2,3The litter size at birth (10.24 piglets/farrowing) is significantly larger compared to some exotic breeds, for example, Large Black (8.38 piglets/farrowing) pigs. 2 However, pre-weaned mortality of Ghoongroo pigs is high.Earlier research found that insufficient milk of the sows caused a higher pre-weaning mortality rate of Ghoongroo pig (2.64 pigs/litter) compared to exotic breeds such as Large Black pig (1.47 pigs/litter). 2arious antibiotic growth promoters are widely used in the diets of pigs for improvement of growth rate as well as health status and to reduce piglets' mortality rates.Antimicrobial growth promoters and immunomodulators with milk replacer resulted in a synergistic effect on various physiological and immunological performances, including the growth of the pre-weaned pigs. 4However, antibiotics uses in diets are growingly restricted due to emergence of antibiotic resistance against human and animal pathogenic microbes, and alternative approaches are investigated to tackle this issue. 5,6Various dietary phytogenic feed additives including essential oils (EO) and phenolic compounds due to their antimicrobial and antioxidant properties have been shown to improve growth rate, intestinal microbiota and immunity of weaned piglets. 5,6iglets experience physiological, social and environmental stresses after weaning, particularly at the first week of weaning, which predispose to intestinal and immune dysfunctions, consequently growth retardation and health. 7Supplementation of EO could be useful to ameliorate the weaning stress of piglets.][10] Other EO such as clove and cinnamon EO 10 has also been supplemented to the diets of weaned piglets to improve growth performance and health.Satisfactory growth performance and immune response were not evidenced in suckling piglets when oregano EO was directly added to the diet or supplemented to the lactating sows. 8Supplementation of oregano EO directly to the pre-weaned or suckling piglets is very limited and has not yet shown conclusive results on the performance of piglets.Satisfactory preweaned growth performance piglets had been observed by providing skim milk. 11Using bovine milk as a milk replacer in piglets reduced piglet mortality with increased weaning weight. 12Scanty literature is available on the improvement of health status, including growth, morphometry, haematological, endocrines and immunological performances of the pre-and postweaned Ghoongroo piglets using milk replacer and EO.Therefore, the present study was conducted to evaluate the effect of oregano EO, with or without a milk replacer on growth performance, haematobiochemical profile and immune responses in preweaned and post-weaned Ghoongroo piglets.

Experimental animals and site
A total of 36 Ghoongroo piglets with 18 males and 18 females were selected randomly for the study within 2-3 days after birth from different sows farrowing within 15 days.The herd had litter size of 8.86 ± 0.67 piglets/farrowing (mean ± standard deviation), litter weight of 9.30 ± 0.53 kg with mean body weight of 1.05 ± 0.25 kg at birth.The piglets were divided equally into three groups (n ¼ 12) viz.control (CON), feeding with a milk replacer (MR), and feeding with milk replacer and oregano essential oil (MR þ EO).Each group had same number of both sexes.Similar standard management practice with concentrate feeding was followed for all the animals rearing at the pig farm under the Indian Council of Agricultural Research -Indian Veterinary Research Institute, Eastern Research Station, Kalyani, Nadia, West Bengal, India (at 22 98 0 N and 88 44 0 E).The study was conducted from January to April 2021 with a temperature-humidity index (THI) of 70.4 ± 3.09.

Experimental design and treatments
The control group (CON) piglets were allowed to suck on mother's milk every hour for 15 min up to weaning (6th week of age).In the MR groups, the piglets were provided ad libitum milk replacer at every hour interval for 15 min for three weeks (Table S1-S4).In the MR þ EO group, diet schedule was same as MR group except oregano EO at 500 mg/kg dry matter (DM) was added with the milk replacer or concentrate feed in the as per the study of Pu et al. 10 The oregano EO was the extract of the plant Origanum vulgare (Oregano oil V R , purity 100%; Earth N Pure, New Delhi, India).Starter feed was gradually increased at 20 g/day from third week to 100 g/day at 6th week in all groups as a part of the early weaning standard operating procedure of the farm (Table S5-S7).Thereafter, the piglets were fed as per the standard requirement of the animals. 13An antibiotic (streptopenicillin; Strepticillin V R , Intracin Pharmaceuticals Pvt. Ltd., Nadiad, Gujrat, India) was added at 15 mL/L of water to the control group piglets throughout the study.A starter ration was offered from the 3rd week, followed by a grower ration from the 9th week onwards (Table 2).The milk replacer and concentrates feed were prepared to meet or exceed the minimum nutrient requirements recommended by the National Research Council. 13

Variables studied
Growth, morphometry and physiological variables All the physiological and morphometric variables were recorded at week 1, 3, 6, 9 and 12 at early morning (within 9:00 am).Rectal temperature was recorded by using digital thermometer, pulse rate was measured by placing finger over the femoral artery, and respiration rate was recorded by placing a hand on the piglet's flank.Body weights were measured with a pig weighing scale at week 1 and thereafter 3 weeks intervals up to 12 weeks.
The growth related morphometric features were measured using a measuring tape.Body length was measured from the base of the ear to the base of the tail, heart girth around the heart from the shoulder behind the forelegs, and height at fore limb from the front hoof to the top of front shoulder at the highest point.

Blood variables
Blood samples (2 mL) were collected from all piglets in vacuitaner tubes containing ethylenediamine tetraacetic acid at 1.5 mg/mL of blood on week 1, 3, 6, 9 and 12 and separated plasma was stored at À20 C for further analysis.The blood samples on week 6 were collected after two days of weaning to assess the weaning stress of piglets.Haematological parameters, i.e., hemoglobin (Hb) percentage by cyanmethemoglobin method as described by Molden, 14 packed cell volume (PCV) by Wintrobe Hematocrit tube method, 15 and total erythrocyte counts (TEC) and total leukocyte counts (TLC) using a hemocytometer following the method described by Jain 16 were analyzed.Differential leukocyte counts (DLC) were measured using Leishman stain as described by Jain. 16Blood indices were calculated from the obtained results using the specific formulas, viz.mean corpuscular volume (MCV, in femtolitres) ¼ PCV% Â 10/RBC number (millions/mm 3 ), mean corpuscular Hb (MCH, in picograms) ¼ Hb (gm/dL) Â 10/RBC number (millions/ mm 3 ), and mean corpuscular Hb concentration (MCHC, in gm/dL) ¼ Hb (gm/dL) Â 100/PCV (%).
Blood biochemical parameters were measured from plasma using commercially available kits following glucose oxidase-peroxidase method as described by Trinder 17 for determination of glucose, Biuret method (end point) test as described by Piotrowski 18 for total protein, end point method using bromocresol green dye as described by Doumas et al. 19 for albumin, and cholesterol oxidase phenol 4-aminoantipyrine peroxidase method modified by Roeschlau et al. 20 for total cholesterol.Plasma globulin was calculated by subtracting the value of albumin from total plasma protein and obtained the ratio of albumin-globulin thereof.Plasma was analyzed using the commercially available ELISA Kit (Calbiotech, USA) following the methods as described by Brock et al. 21for plasma cortisol, Rastogi and Sawhney 22 for triiodothyronine (T 3 ), and Ingbar et al. 23 for thyroxine (T 4 ) concentrations.

Immunology and fecal microbes
Phagocytic activity (PA) of neutrophils was determined by using nitroblue tetrazolium (NBT) as per the methods of Abuharfeil et al. 24 The lymphocyte proliferation response (LPR) was measured using the colorimetric MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5diphenyltetrazolium bromide) test according to the procedure described by Mosmann. 25Fecal bacteria (Escherichia coli, Salmonella spp.and Lactobacillus spp.) numbers were quantified by spread plate method using species-specific media. 26

Data analysis
Data were analyzed using PROC MIXED procedures of SAS, 27 and the model contained treatment, week and treatment x week interactions as main effects with repeated measures of week and animal as a random effect using the following statistical model: where Y ijk ¼ dependent variable, l ¼ overall mean, T i ¼ effect of treatment i, W j ¼ effect of week j, (T Â W) ij ¼ interaction effect of treatment i and week j, a k ¼ random effect of animal k, and e ijk ¼ overall residual error.Week was used as repeated measures with animal as subject in the model.The heterogeneous first-order autoregressive covariance matrix was used in the final model, which usually showed a better model fit.When an interaction effect was significant, the 'SLICE' option in the SAS model was used to find the significant (p < 0.05) difference among treatments in a week or among weeks within a treatment.Subsequently, significant differences (p < 0.05) among the treatments within a week or among the weeks within a treatment were detected using pair-wise comparisons specifying 'PDIFF' in the least-square means ('LSMEANS').Microbial data were log-transformed before statistical analysis.Statistical significance was set at p 0.05.

Growth, morphometry and physiological responses
The body weight of the piglets did not significantly alter among the groups up to weaning (Table 1).However, higher (p < 0.01) body weight was found in the MR and MR þ EO groups than in the control group during the post-weaning period.Persistent increased (p < 0.01) body weight was observed throughout the study in both experimental groups, whereas the control group had similar body weight at weaning (6 weeks) and immediately after the weaning at 9 weeks.The heart girth varied among the groups and age (Table 1).The height at withers and linear length of the piglets of all the groups were unaltered during the entire study period.Heart girth significantly (p < 0.05) increased up to the 12th week, with a higher value at the end of the study in the MR þ EO group than in the control and MR groups.Heart girth did not change in the latter groups immediately after weaning at the 9th week.
The rectal temperature was affected by group (p ¼ 0.05), week (p < 0.01), but not by group and week interaction.It was similar in different weeks in all groups from the 1st week to the 12th week.The mean rectal temperature of the control group was higher than the experimental groups.Pulse rate remained unchanged on week 1 and during the post-weaning period in all groups, but it was significantly (p < 0.01) higher in the control group during weaning (on week 6) and reverse was noticed on week 3. Respiratory rate was higher (p < 0.05) in the treatment groups at 3rd week, but was lower at weaning compared with the control.

Endocrine and biochemical profiles
The levels of T 3 and cortisol significantly (p < 0.01) differed among the groups throughout the study, whereas the T 4 level did not vary during and immediately after the weaning (Table 2).During weaning, the T 3 level increased but cortisol level decreased in both the experimental groups.A lower cortisol level was found in the MR þ EO group, which even did not change during weaning.Higher cortisol levels were found in the MR group before and after the weaning, and the similar pattern was noted in the control group.
The glucose was found to be highest (p < 0.01), and the total protein and albumin levels were found to be lowest (p < 0.01) in the MR þ EO group, followed by the MR group than the control group in the initial three weeks (Table 3).No biochemical profile variation was found during and after the weaning among the groups.The globulin and albumin-globulin ratio did not alter among the groups throughout the study.The cholesterol level was higher in both the experimental groups immediately before weaning, which was lowered during weaning.No such variation was observed among the groups during the remaining period of the study.The glucose and cholesterol levels were gradually decreased, albumin remained constant, and total protein and globulin inconsistently altered during the study in all the groups irrespective of groups.

Hematology
Among the various haematological and blood indices features, Hb concentration showed significant age effect (p < 0.01) with increased Hb concentration by age in all groups, and groups effect with higher (p < 0.01) mean concentrations in both the treatment groups compared with the control, but no group Â age interaction effect (Table 3).In the treatment groups, the higher TEC (p < 0.05) was noted only during weaning and at the beginning of the study, and the elevated PCV (p < 0.01) was found throughout the study, except in the 1st week.Both the TEC and the PCV gradually increased with age until weaning and remained constant after the weaning.Among the three blood indices, MCH lowered at the beginning of the study the MCV and MCHC lowered at first week and weaning in the treatment groups.The higher MCV was recorded in the treatment groups during weaning, which reversed from 1st week's feature and opposite to the character of MCHC.The elevated MCHC was also found in the MR þ EO and control groups than in the MR group at the end of the study.The MCH and MCV gradually increased (p < 0.01), and MCHC decreased (p < 0.01) with age.

Leukocyte and immunological features
Total leukocyte count (TLC) and the percentage of neutrophils, eosinophils and basophils did not alter among the groups, and all the granulocytes remained constant during the study period (Table 4).In the

Fecal microbial profile
Both the MR and MR þ EO groups had a higher (p < 0.01) mean count of Lactobacillus spp.throughout the study.During weaning, increased E. coli and Salmonella spp.counts were noticed in the experimental groups (Table 5).In the MR þ EO group, Salmonella spp.count reduced immediately after the weaning and remained constant during the entire study.However, Salmonella spp.count increased at the end of the study in the MR group, which always remained high in the control group, except during weaning.A similar pattern of E. coli counts was noticed in the control group, where its number gradually increased in both the experimental groups.

Growth, morphometry and physiological responses
The body weight in all the groups of piglets was in accordance with the earlier reports in Ghoongroo piglets at 1st week and lower during weaning, 28 but higher in MR and MR þ EO groups at weaning than the Landrace-Large white piglets 29 and at the age of 12th week compared with Ghoongroo piglets in the study of Hazorika et al. 30 The higher body weight in the experimental groups may be due to the anabolic effect caused by the higher levels of T 3 and T 4 , 31 lower cortisol levels during and after weaning, 32 and pre-weaning milk replacer feeding. 33Higher body weight in the experimental groups may also relate to higher number of Lactobacillus spp. in the gut of   1.36bn 1.15abmn 0.90am a Control: milk from dams; MR: milk replacer; MR þ EO: milk replacer added with oregano essential oil.b SEM: standard error of the mean for the treatment and week interaction.Means followed by different letters differ significantly within a specific parameter in the same row (a, b, c) among treatments within a week and in the same column (m, n, o, p) among weeks within a treatment.
Table 5. Fecal microbial profile of Ghoongroo piglets in pre-and post-weaning state.piglets, which compete with pathogenic bacteria for nutrients or absorption sites. 6The oregano EO provided to the piglets was also probably responsible for higher growth performance due to improvement of the immunity and antioxidant status. 34he height at withers was higher in all the groups of piglets; whereas the heart girth and linear length were similar to the report of Banik et al. 35 in Ghoongroo piglets, but lower than the Nigerian indigenous crossbred piglets 36 and crossbred (Hampshire Â Ghoongroo) piglets. 37Higher heart girth at the post-weaning stage was found only in the MR þ EO group, which was similar to Burmese Black piglets. 38This also reflected in higher body weight during the same time.Heart girth is considered as a most appropriate trait among the various biometric traits to predict body weight, which explained 89-96% variations in various breeds of pigs. 37,39he rectal temperature was static from 1st week to the 12th week in all groups of the piglets even after weaning, which was similar to the earlier findings of Vasdal et al. 40 The respiration rate and pulse rate decreased with the advancement of age.However, both the rates were higher immediately after the weaning in all the groups, which corroborated with the reports of Sipos et al. 41 Both the rates were also higher in the control group than other groups, probably because of higher cortisol levels caused by weaning stress 7 and consequently activation of the sympathetic nervous system and the hypothalamicpituitary-adrenal axis by the cortisol hormone.42 Lower respiration rate and pulse rate in both the experimental groups may be due to well physiological adaptation to a healthy state.43

Endocrine and biochemical profiles
The level of T 4 hormone in the present study was similar to the earlier finding in pigs. 44Levels of T 4 and T 3 were higher than the reports of Chakraborty et al. 45 Cortisol levels were higher than the earlier report in Ghoongroo piglets. 46The levels of T 3 and T 4 hormones decreased with age in piglets, mostly with age-related metabolism, and a little increase in T 3 during weaning and immediately after weaning due to stress. 44The higher levels of thyroid hormones were observed in the MR and MR þ EO groups, which influenced body growth. 47In the MR þ EO group, the T 3 and T 4 were more stable after weaning than the other two groups, likely due to their good health status with the homeostatic hypothalamic-pituitary-thyroid (HPT) axis.A similar observation was also noticed in the MR group of piglets for T 4 , indicating their better health position than the control group.
Reduced cortisol levels in MR and MR þ EO groups after weaning signified lesser stress than the control group, as a higher level of cortisol resulted in a negative impact on the growth in the post-weaning period in piglets. 32Cortisol concentration in the MR þ EO group remained static compared to the MR group, an indicative of better regulation of the hypothalamicpituitary-adrenocortical axis. 32,48This observation confirmed the supplementation of oregano EO to reduce weaning stress, which facilitated the increased body weight gain in the MR þ EO group.This result corroborated with the observations in Large White sows to reduce lactation stress and improvement in piglet performance, 49 in finishing pigs (Large White Â Landrace) to reduce transport stress and modulation of intestinal morphology using dietary oregano EO, 50 and to increase immune response in Suffolk hybrid preslaughter pigs. 51lucose levels decreased gradually in all the groups up to weaning. 52After weaning, the glucose level remained unaltered up to the 12th week in MR þ EO, followed by the other groups, which may be due to thyroid hormones depending on carbohydrate metabolism to maintain glucose homeostasis. 53Cholesterol levels altered in all the groups before weaning keeping parity with the thyroid hormones. 54Stable total protein and albumin concentrations may be due to a steady source of dietary protein from the supplemented milk in both the experimental groups. 55The plasma globulin content altered randomly in all the groups during the entire study period, which may be due to rapid alteration of liver function. 52

Hematology
The PCV and MCV levels increased in all the groups with the advancement of age, particularly during weaning, as they have significant associations keeping similarity with the RBC count. 56But, the Hb concentration increased significantly and MCV level decreased with age in all the groups.The MCHC also gradually decreased in all the groups up to the 12th week, which may be compensated with the increase of RBC numbers. 57But, the haematological indices indicated that the piglets were not affected by any anemia as the values of indices remained within the normal range for pigs. 56mproved intestinal health favors ferroprotein transportation in post-weaning piglets, which may cause a better haematological status, including Hb, TEC and PCV in the experimental groups than in the control group. 58,59The higher mean concentration of Hb in the experimental groups may be due to more weight gain in post-weaned piglets than in the control group, where a positive association between weight gain and Hb concentration was found. 59

Leukocyte and immunological responses
Total leukocyte counts and the percentage of neutrophils, eosinophils, basophils, lymphocytes and monocytes were in accordance with the earlier findings in pre-and post-weaned Mali piglets, 56,60 which remained unaltered in all the groups during the entire study period indicating the regulation of leukocyte homeostasis in the blood. 61Decreases in the percentages of lymphocytes were age-related and not due to weaning. 62The changes in the lymphocyte counts continue for about one week in the post-weaning period and might not indicate the piglets' immunological response to weaning stress. 63ncreased neutrophil phagocytosis, from weaning, in the control group compared with both the experimental groups may be due to higher cortisol levels. 64Higher phagocytic activity of neutrophils in both the experimental groups at the pre-and post-weaning (3-6 week and 12 week of age) may be caused by the elevation of circulating thyroid hormones. 65During weaning and immediately after weaning, no change in the lymphocyte proliferation response was found in all the groups, which may be due to the effect of T 4 hormones in maintaining immune system homeostasis as depicted in mice. 66The lymphocyte proliferation was not altered during the post-weaning period in the MR þ EO group.
Neutrophil phagocytotic activity decreased only at weaning in the MR group and up to the 9th week in the control group.The piglets of the MR þ EO group, followed by the MR group adjusted the short-term suppressive response of the immune system with the phagocytosis efficacy of neutrophils. 64It signified that MR þ EO group piglets were superior in overcoming weaning stress compared with the other two groups, followed by MR group than the control group.This may relate to their cortisol levels as explained by Reiske et al. 64 The present finding indicated that piglets fed with oregano EO along with milk replacer at the pre-weaning period might physiologically control the cortisol level and stimulate the nonspecific innate immune system during weaning stress.Better immune protection in the piglets of both the experimental groups than in control may also be due to better gut health. 67

Fecal microbial profile
Different groups were infected with E. coli and Salmonella spp. at the 1st week, likely due to maternal transfer, 68 which can persist for a long period 69 and its count increased in the post-weaning period. 7owever, the number of fecal E. coli was lower in all groups than in the crossbred (Duroc Â Landrace) piglets at the same age. 68The weaning stress as indicated by higher cortisol level may lead to exposure to microbes, 7 which may be reduced in the control group due to the use of antibiotics in this group.In MR þ EO group, no increase of Salmonella spp. was observed during and after weaning, which may be due to antimicrobial and antioxidative activities of oregano EO. 70 Fecal Lactobacillus spp. was higher in MR and MR þ EO groups than in the piglets of Moturi et al. 71 study.Higher Lactobacillus spp.may support the improved gut health with better intestinal morphology and antioxidative capacity in the MR þ EO group due to supplementation of oregano EO 9 with greater body weight. 68Higher Lactobacillus spp.may favor to reduce the number of E. coli and Salmonella spp. in MR þ EO group after the weaning. 72

Conclusion
Pre-weaning milk replacer feeding helped to maintain the piglets' health at weaning.Dietary supplementation of oregano EO at 500 mg/kg diet reduced weaning stress as indicated by lower cortisol concentration at weaning and also caused recovery of weaning stress early.Additionally, dietary oregano EO stimulated the nonspecific innate immune system with the increased phagocytosis efficacy of neutrophils, and modulated the intestinal microbiota and thyroid hormones favorably in Ghoongroo piglets, thus facilitating the increased body weight gain.The concentration of thyroid hormones was more stable after weaning in the oregano EO supplemented group.The better haematological status, including Hb, TEC and PCV, indicated the rapid organ development, formation and maturation of erythrocytes and improved intestinal health status in piglets fed milk replacer and combined with oregano EO Thus, milk replacer along with EO could be used in the diets of piglets to reduce weaning stress and better growth and health status.

Ethical approval
The study was performed with the approval of the Institutional Animal Ethics Committee of West Bengal University of Animal and Fishery Sciences, Kolkata, India b SEM: standard error of the mean for the treatment and week interaction.Means followed by different letters differ significantly within a specific parameter in the same row (a, b, c) among treatments within a week and in the same column (m, n, o, p) among weeks within a treatment.

Table 1 .
Growth, morphometry and physiological responses of pre-and post-weaned Ghoongroo piglets.
a Control: milk from dams; MR: milk replacer; MR þ EO: milk replacer added with oregano essential oil.b SEM: standard error of the mean for the treatment and week interaction.Means followed by different letters differ significantly within a specific parameter in the same row (a, b, c) among treatments within a week and in the same column (m, n, o, p) among weeks within a treatment.

Table 2 .
Endocrine and biochemical profiles of pre-and post-weaned Ghoongroo piglets fed milk replacer and oregano essential oil.
a Control: milk from dams; MR: milk replacer; MR þ EO: milk replacer added with oregano essential oil.b SEM: standard error of the mean for the treatment and week interaction.Means followed by different letters differ significantly within a specific parameter in the same row (a, b, c) among treatments within a week and in the same column (m, n, o, p) among weeks within a treatment.

Table 3 .
Haematological profile of pre-and post-weaned Ghoongroo piglets fed milk replacer and oregano essential oil.
a Control: milk from dams; MR: milk replacer; MR þ EO: milk replacer added with oregano essential oil.

Table 4 .
Leukocyte profile and immune responses of pre-and post-weaned Ghoongroo piglets fed milk replacer and oregano essential oil.