Using lactoferrin and N-acetylcysteine to augment the growth rate and hemato-biochemical parameters of Egyptian Baladi goats kids

Abstract

This study investigated the influence of lactoferrin (LF) and N-acetylcysteine (NAC) on the growth rate and immune response of Egyptian Baladi goats. Thirty-five goat kids (2.765 ± 0.135) were comprised seven groups, each with five individuals. The control group received neither LF nor NAC. The subsequent groups received daily doses at 50 mg, 100 mg, or 200 mg NAC. Similarly, the other groups were administered LF at 50 mg, 100 mg, or 200 mg per day. Biweekly assessments were used to measure the weight and daily weight gain (DWG) of the offspring. Additionally, blood samples were collected every 20, 40, and 60 days post-treatment for hemoglobin, total leukocyte counts, and subtypes. Additionally, blood serum was analyzed for total protein, albumin, globulin, creatinine, and alanine aminotransferase levels. The findings showed significant enhancements in the DWG, hemoglobin concentration, total leukocyte count, and various white blood cell types in the groups treated with LF and NAC compared to those in the control group. Notably, the group receiving LF at 200 mg/ml exhibited the most significant improvements with control and other level of LF. LF at 50 mg/ml/day resulted in higher total protein, albumin, and creatinine levels than did both the control and the other treatments with similar dose of 50 mg/mg/day of NAC. Administration of either LF or NAC notably enhanced the growth rate and blood parameters compared to those of the control group. In conclusion, our findings underscore the potential of daily LF and NAC administration during the weaning phase for Egyptian Baladi goat kids to enhance their immune response and accelerate their growth rate.

Keywords:

• Antioxidants
• Baladi goat
• hematology
• immunity
• liver functions
• welfare

REVIEWING EDITOR:

• Pedro Gonzalez-Redondo, Universidad de Sevilla, Spain

SUBJECTS:

• Agriculture & Environmental Sciences
• Plant & Animal Ecology
• Zoology
• Immunology
• Biotechnology
• Nutrition
• Food Additives & Ingredients

1. Introduction

Lactoferrin (LF) is a crucial component of innate defense mechanisms found in both humans and animals. Lactoferrin (LF) is an iron-binding glycoprotein (molecular weight 3125.8 g/mol), this multifunctional protein is present in various body secretions, such as milk, saliva, bile, tears, and sperm, and was initially discovered in milk whey in 1939 (Saeed et al., 2023). Extensive research since the 1960s has explored its structure, functions, and potential applications in healthcare, including therapeutic management and as a dietary supplement (Cao et al., 2022). The bovine LF is a glycoprotein consisting of 708 amino acids that features two spherical lobes with iron-binding sites. It is also present in external secretions and in the secondary granules of neutrophils and epithelial cells (El-Fakharany et al., 2022; Zarzosa-Moreno et al., 2020). Iron binds simultaneously with bicarbonate anions, which is crucial for its structural function (Kell et al., 2020).

Eminently, the antibacterial activity of LF is linked with two different mechanisms. Firstly, LF has the ability to bind iron, an essential element in the replication of microorganisms, that way indirectly acting as a bacteriostatic agent (Niaz et al., 2019). Secondly, LF is able to enhance the generation of peroxides, which in turn affect bacterial membrane permeability, acting directly for the sake of bacterial cell lysis (Braun & Braun, 2002). LF has broad antimicrobial effects on various pathogens, such as E. coli, S. aureus, and C. albicans, while also playing essential roles in granulopoiesis, cytokine production, antibody synthesis, and the regulation of immune cells, such as natural killer cells and lymphocytes. It activates complement and mediates the production of essential cytokines (Bukowska-Ośko et al., 2022; Karlsson, 2021). Despite its efficacy against many pathogens, the impact of LF on several pathogens, such as Streptococcus uberis and Streptococcus agalactiae, has remained limited. Studies suggest that the virulence of Streptococcus uberis might increase its pathogenicity by binding to LF, potentially mediating interactions between bacteria and mammary epithelial cells (Kabelitz et al., 2021; Xu et al., 2022).

The chemical compound N-acetyl cysteine (NAC), derived from L-cysteine, is an exceptional source of sulfhydryl groups. This compound, known as NAC, has a longstanding reputation as a mucolytic drug used in infertility management (Ezzat et al., 2023). Its versatility extends to robust antioxidant properties influencing insulin receptors, insulin secretion, and vascular integrity, and it also has immunological advantages (Zhou et al., 2019). Recent studies have highlighted its role in enhancing insulin sensitivity in type 2 diabetes patients and improving ovulation induction outcomes (Abu El-Ella et al., 2023).

In vitro, Lactoferrin and N-acetyl cysteine, stimulates the growth of lymphocytes (Clark et al., 1998), natural killer activity (Cooper et al., 2001), and the release of interleukin-8 (IL-8) from neutrophils. Further, Lactoferrin stimulates the release of IL 1, IL 2, and tumor necrosis factor (TNF) from leukocytes or complement activation (Cornish et al., 2004). LF and its derivatives have pleiotropic functions, including broad-spectrum antimicrobial activity, regulation of cell growth and differentiation, and intonation of inflammatory as well as humoral and cellular immune responses (Dealtry et al., 2000).

Furthermore, NAC is a recognized pharmaceutical agent with anti-inflammatory, antioxidant, and organ-protective effects because it functions as a precursor to glutathione (Comino-Sanz et al., 2021). Its ability to preserve mitochondrial integrity has emerged as a promising intervention against bisphenol A-induced renal injury due to oxidative stress (Abdelrazik et al., 2022). Clinical studies have demonstrated its efficacy in reducing recurrent pre-term labor in patients with a history of bacterial vaginosis and in positively impacting maternal inflammatory cytokines to provide neuroprotection against maternal chorioamnionitis (Liu et al., 2022; Schoeps et al., 2022).

Therapeutically, NAC is widely used as an expectorant and has shown antibacterial effects against Mycobacterium tuberculosis (potential antimycobacterial property), suggesting that NAC has immunomodulatory and antioxidant effects (Shiozawa et al., 2020). Its antioxidant functions include countering free radicals and restoring intracellular defenses by facilitating glutathione synthesis, which results in protection against various types of tissue damage (Kim et al., 2023; Kong et al., 2023).

However, limited studies have investigated the use of LF or NAC as supplementary tools for enhancing newborn goat kids’ growth performance, hematological parameters and immune status. Therefore, the impact of LF and NAC on the immunological response and growth rate of Egyptian Baladi goat progeny was investigated in this study.

2. Materials and methods

The experimental protocol was approved by the Institutional Animal Care and Use Committee, Faculty of Veterinary Medicine, Cairo University. Approval number: Vet CU 25122023860.

This study compared blood parameters (hematological and biochemical) among several kids over six months (December 2022 to May 2023) using a randomized prospective observational and cohort design. A total of 35 healthy Egyptian Baladi goat kids were housed at the El-Qarada Animal Production Research Center, a part of the Animal Production Research Institute (APRI), Ministry of Agriculture, Egypt. This is where the study was carried out. Although they were kept indoors with their mothers, the kids were allowed to spend time outdoors and experienced seasonal changes in temperature, relative humidity, and photoperiod. In the interior, extreme weather was managed by using heaters, fans, windows, and shade. The kids were left with their mothers to nurse them until they were ready to wean.

2.1. Study design

A total of 35 healthy Baladi meat kids were included in the study; the participants were isolated in separate pens under controlled environmental conditions and were distributed across seven groups. Straw bedding, thermostats, distinct ventilation systems, and manually controlled feed dispensers were provided in each enclosure. The following parameters were observed and used to categorize and compare kids. (1) Date of birth: All the kids in the groups were approximately the same age. (2) Weight: The kids’ weights were recorded as soon as they were born and were measured every 15 days until they were weaned. (3) Treatment: Three groups were given daily oral doses of three different concentrations of LF (Sigma), and another three groups were given daily oral doses of five ml of NAC (Sigma).

Seven groups were enrolled in this study during the study period of six months, during which all kids who delivered during this period were enrolled directly and observed for 60 days. Group 1 was the control group and received no treatment; groups 2, 3, and 4 were kids who received LF at dose rates of 50, 100, and 200 mg/ml, respectively; and groups 5, 6, and 7 were kids who received NAC at dose rates of 50, 100 and 200 mg/ml, respectively. All the treated groups received the treatment as a single oral dose which was determined according to Ripani et al. (2022), and the treatment was administered every day after the morning breastfeeding session until weaning.

2.2. Animal management and environment

The facility managed all the ewes in the same way, controlling the environment to prevent heat and cold stress. The intensity of heat stress relies primarily on the dry bulb temperature and RH%, which can be amalgamated into a THI or temperature–humidity index (Goma & Phillips, 2022). Marai et al. adapted the THI equation (in °C) for sheep and goats as follows: THI = db°C − {(0.31 – 0.31 RH) (db°C − 14.4)}, where db°C represents the dry bulb temperature (°C) and RH signifies the relative humidity (RH%)/100. Accordingly, the ideal THI for sheep and goats under Egyptian conditions was <22.2 = no heat stress, 22.2 to <23.3 = mild heat stress, 23.3 to <25.6 = significant heat stress, and above 25.6 indicates extremely severe heat stress (Table 1) (Marai et al., 2001). Kids with does were housed in shaded, semi-open quarters and fed a well-balanced ration according to the NRC as a concentrate (cottonseed cake, corn, wheat bran, soya bean meal) and green fodder (green herbage, grass, berseem, and darawa). Each doe was provided with a high-quality concentrate ration containing 14% protein. The animals were fed twice daily, with an initial quantity of 100 g during the first month, 150 g during the second month, and 200 g during the third month (El-Sayed et al., 2020; Nrc, 1985), while water was always available ad libitum. Does were provided with veterinary care according to standard management practices to ensure their welfare and health.

Table 1. Descriptive statistics (Max.–Min.) of temperature (T°C), RH%, air velocity (AV, m/s), and THI during the study 6 months in the farm outdoor and indoor climates.

December			January		February		March		April		May
	Outdoor	Indoor	Outdoor	Indoor	Outdoor	Indoor	Outdoor	Indoor	Outdoor	Indoor	Outdoor	Indoor
T°C	22.5–16.8	24.1–18.5	20.5–14.0	24.0–17.8	17.9–13.3	22.9–18.4	22.4–17.1	23.3–21.7	24.8–19.0	21.6–20.0	26.8–20.1	23.6–21.1
RH%	75.6–50.4	72.6–47.5	79.7–49.4	74.1–45.1	74.4–45.5	65.1–35.9	70.1–48.2	65.9–43.5	67.1–41.8	80.1–55.0	70.4–44.9	83.3–58.1
AV	16.2–8.1	7.3–4.1	19.2–8.7	7.5–5.4	18.3–9.8	8.3–2.8	21.3–12.3	10.7–4.5	20.1–10.9	7.7–3.8	21.4–12.2	9.0–4.7
THI	21.2–16.6	22.5–18.1	19.5–14.0	22.4–17.5	17.3–13.3	21.2–17.9	21.0–16.8	21.7–20.9	22.9–18.5	20.6–19.7	24.7–19.6	22.4–20.8

2.3. Blood parameters

Blood samples were collected via jugular venipuncture (5 ml) and transferred into standard 10 ml EDTA vacuum tubes (Vacutainer® System Europe; Becton Dickinson, France) and another 5 ml into a standard 10 ml plain tube from each animal using aseptic techniques. Samples were collected at specific time points during the early morning hours every 20 days of age (1^st sample at the 20^th day of age, then at the 40^th day of age, and, finally, at the 60^th day of age) (Pesántez-Pacheco et al., 2019).

2.4. Hematological parameters

EDTAed blood samples were collected to evaluate complete blood count (CBC) using an automated blood cell counter or hematology analyzer and a CELL-DYN 3700SL system (Abbott Diagnostika GmbH, Wiesbaden, Germany). A number of factors, such as the hemoglobin concentration (Hb) and total and differential leukocyte counts, were included in the CBC (Jain, 1993).

2.5. Biochemical parameters

The serum was separated from the plain blood samples by centrifugation at 3000 rpm for 15 minutes and then kept at -20 °C for subsequent biochemical analysis, during which the serum biochemical parameters, such as total protein, albumin, and globulin in mg/dl, were measured spectrophotometrically in accordance with the protocol outlined by Yin et al. (2022). Creatinine and alanine aminotransferase (ALT) levels in mg/dl were measured spectrophotometrically according to Lippi et al. (2019) and Wiles et al. (2019), respectively.

2.6. Statistical analysis

The data and results were collected and analyzed using the Statistical Package for Social Sciences software, version 25.0 (SPSS Inc., Chicago, IL). We computed descriptive statistics, including means and standard errors, for each group and parameter. One-way analysis of variance (ANOVA) was used to compare daily weight gain and blood parameters among the various groups. Post hoc tests, such as Tukey’s or Dunn’s test, were performed for pairwise comparisons if significant differences were detected (Campbell, 2021).

3. Results

During the six-month study period, all quantifiable and observed parameters were noted and tallied, where temperature (T°C), relative humidity (RH%), air velocity (AV, m/s), and temperature-humidity index (THI) were recorded for both outdoor and indoor farm environments. During the period of investigation (December to May), the outdoor T°C fluctuated between 26.8 °C and 13.3 °C, while the indoor T°C ranged from 24.1 °C to 17.8 °C. RH% values varied between 79.7% and 41.8% outdoors and between 74.1% and 35.9% indoors during the same period. The air velocity ranged from 21.4 m/s to 8.1 m/s outdoors and 10.7 m/s to 2.8 m/s indoors. Similarly, the THI did not significantly change from month to month, ranging from 24.7 to 13.3 outdoors and 22.5 to 17.5 indoors; that is, no type of stress was imposed on the examined animals (Table 1).

The DWG of the goat kids varied across treatment groups and time intervals. Significant differences were observed among groups over 15-day intervals. Notably, the control group exhibited less weight gain than the groups administered LF or NAC. Among the treated groups, those administered LF at 200 mg/ml exhibited relatively consistent and greater weight gain than those treated with the other concentrations (Table 2).

Table 2. Daily weight gain (DWG) of the kids in the 7 treatment groups during the study period.

Treatments	DWG (kg)
	After 15 days	After 30 days	After 45 days	After 60 days
Control	0.058 ± 0.012^b	0.048 ± 0.02^c	0.091 ± 0.018^b	0.055 ± 0.01^c
LF50	0.106 ± 0.019^ab	0.13 ± 0.025^ab	0.1 ± 0.04b^c	0.094 ± 0.012^ab
LF100	0.114 ± 0.009^a	0.076 ± 0.016^bc	0.096 ± 0.012^b	0.102 ± 0.01^ab
LF200	0.116 ± 0.017^a	0.134 ± 0.012^ab	0.114 ± 0.008^a	0.086 ± 0.011^abc
NAC50	0.082 ± 0.005^ab	0.06 ± 0.017^c	0.062 ± 0.027^c	0.062 ± 0.02^bc
NAC100	0.054 ± 0.008^b	0.142 ± 0.02^a	0.102 ± 0.01^ab	0.056 ± 0.021^bc
NAC200	0.1 ± 0.033^ab	0.036 ± 0.015^c	0.106 ± 0.017^ab	0.126 ± 0.016^a
P value	0.042	0.022	0.018	0.037

^{a ,b,c}The mean values placed in rows with different superscript letters are significantly different (P ≤ 0.05). The values are expressed as the means ± SEs. LF50, LF100, and LF200: Lactoferrin groups with 50, 100, and 200 mg/l, respectively. NAC50, NAC100, and NAC200: N-acetyl cysteine groups at 50, 100, and 200 mg/l, respectively.

Blood neutrophil and lymphocyte counts were monitored at 20, 40, and 60 days after treatment initiation for the seven groups. The control group generally displayed lower counts than the treated groups. Higher concentrations of LF or NAC (200 mg/l) resulted in substantially greater counts of neutrophils and lymphocytes than did lower dosages (Table 3). The LF group had the highest neutrophil count; however, the NAC group had a greater lymphocyte count throughout the 60 days.

Table 3. Blood neutrophil and lymphocyte counts of the kids in the 7 treatment groups during the study period.

Treatments	Neutrophils (cell/µl)			Lymphocytes (cell/µl)
	After_20d	After_40d	After_60d	After_20d	After_40d	After_60d
Control	1016 ± 4^a	1024 ± 2.45^a	1116 ± 2.45^a	3120 ± 73.48^a	3320 ± 226.72^a	3984 ± 272.06^a
LF50	2026 ± 2.45^b	1038 ± 3.74^a	1128 ± 10.2^a	3760 ± 169.12^b	4380 ± 73.48^b	5256 ± 88.18^b
LF100	2600 ± 244.95^b	3400 ± 244.95^b	3740 ± 269.44^b	4480 ± 115.76^c	5160 ± 163.1^c	6192 ± 195.71^c
LF200	5400 ± 748.33^c	4800 ± 374.17^c	5280 ± 411.58^c	5420 ± 111.36^d	5820 ± 37.42^d	6984 ± 44.9^d
NAC50	1040 ± 13.04^a	1044 ± 14.35^a	1136 ± 12.88^a	5640 ± 24.49^d	6140 ± 97.98^d	7368 ± 117.58^d
NAC100	2018 ± 8.6^b	1400 ± 244.95^a	1560 ± 261.92^a	6320 ± 149.67^e	6960 ± 150.33^e	8352 ± 180.4^e
NAC200	2046 ± 14^b	2200 ± 200^d	2420 ± 220^d	7160 ± 269.44^f	8200 ± 374.17^f	9840 ± 449^f
P value	0.008	0.007	0.001	0.007	0.006	0.001

^{a ,b,c}The mean values placed in rows with different superscript letters are significantly different (P ≤ 0.05). The values are expressed as the means ± SEs. LF50, LF100, and LF200: Lactoferrin groups with 50, 100, and 200 mg/l, respectively. NAC50, NAC100, and NAC200: N-acetyl cysteine groups at 50, 100, and 200 mg/l, respectively. Sig.: significance value.

Monocyte and eosinophil counts varied across the treatment groups throughout the study duration. The control group showed comparatively lower counts. Groups receiving higher doses of LF displayed increased counts of monocytes, with the 200 mg/l concentration group showing the highest counts among the treatment groups. The NAC treatment groups had lower counts of eosinophils than did the control and LF groups (Table 4).

Table 4. Blood monocyte and eosinophil counts of the kids in the 7 treatment groups during the study period.

Treatments	Monocytes (cell/µl)			Eosinophils (cell/µl)
	After_20d	After_40d	After_60d	After_20d	After_40d	After_60d
Control	170 ± 13.04^a	126 ± 6^a	163.8 ± 7.8^a	550 ± 22.36^a	880 ± 51.48^a	1144 ± 66.9^a
LF50	306 ± 8.72^b	428 ± 41.28^b	556.4 ± 53.66^b	410 ± 24.49^b	620 ± 25.5^b	806 ± 33.1^b
LF100	408 ± 19.6^c	528 ± 9.7^c	686.4 ± 12.6^c	300 ± 15.81^c	510 ± 10^c	663 ± 13^c
LF200	568 ± 18.28^d	630 ± 17.32^d	819 ± 22.52^d	230 ± 12.25^d	430 ± 12.25^d	559 ± 15.9^d
NAC50	198 ± 2^a	164 ± 5.1^ae	213.2 ± 6.63^ae	160 ± 18.71^e	280 ± 25.5^e	364 ± 33.1^e
NAC100	236 ± 2.45^e	192 ± 3.74^ef	249.6 ± 4.86^ef	106 ± 1.87^f	140 ± 10^f	182 ± 13^f
NAC200	270 ± 5.48^e	224 ± 10.3^f	291.2 ± 13.38^f	60 ± 10^f	90 ± 10^f	117 ± 13^f
P value	0.001	0.007	0.008	0.007	0.001	0.008

^{a ,b,c}The mean values placed in rows with different superscript letters are significantly different (P ≤ 0.05). The values are expressed as the means ± SEs. LF50, LF100, and LF200: Lactoferrin groups with 50, 100, and 200 mg/l, respectively. NAC50, NAC100, and NAC200: N-acetyl cysteine groups at 50, 100, and 200 mg/l, respectively. Sig.: significance value.

Variations in total leukocyte count and hemoglobin level in the blood among the different treatment groups. The treated groups consistently demonstrated higher leukocyte counts and hemoglobin levels than did the control group. Notably, the groups receiving higher doses of LF exhibited progressively increased leukocyte counts and hemoglobin levels over the course of the study (Table 5).

Table 5. Blood total leukocyte count (TLC) and hemoglobin level of the kids in the 7 treatment groups during the study period.

Treatments	TLC (cell/µl)			Hemoglobin (g/dl)
	After_20d	After_40d	After_60d	After_20d	After_40d	After_60d
Control	4384 ± 298.4^a	5669.6 ± 147.5^a	7370.5 ± 191.8^a	8.52 ± 0.21^a	8.66 ± 0.22^a	9.61 ± 0.24^a
LF50	7112 ± 62.5b^f	8119.2 ± 140.8^b	10555 ± 183.1^b	10.03 ± 0.05^bc	11.06 ± 0.4^b	12.28 ± 0.45^b
LF100	8332 ± 131^c	9931.2 ± 271.4^c	12910.6 ± 352.8^c	10.56 ± 0.13^b	12.36 ± 0.18^c	13.72 ± 0.2^c
LF200	10328 ± 390.4^d	11360 ± 370.9^d	14768 ± 482.2^d	14.34 ± 0.43^d	13.8 ± 0.2^d	15.32 ± 0.22^d
NAC50	5944 ± 136.6^e	6815.2 ± 125.4^e	8859.8 ± 163^e	9.16 ± 0.04^e	9.56 ± 0.08^e	10.61 ± 0.09^e
NAC100	6680 ± 64.5^b	7475.2 ± 51.1^f	9717.8 ± 66.5^f	9.6 ± 0.08^ce	9.94 ± 0.02^e	11.03 ± 0.03^e
NAC200	7392 ± 85.2^f	8864.8 ± 55.6^g	11524.2 ± 72.2^g	9.84 ± 0.02^c	10.1 ± 0.03^e	11.21 ± 0.04^e
P value	0.002	0.004	0.003	0.010	0.002	0.004

^{a ,b,c}The mean values placed in rows with different superscript letters are significantly different (P ≤ 0.05). The values are expressed as the means ± SEs. LF50, LF100, and LF200: Lactoferrin groups with 50, 100, and 200 mg/l, respectively. NAC50, NAC100, and NAC200: N-acetyl cysteine groups at 50, 100, and 200 mg/l, respectively. Sig.: significance value.

Compared with those in the control group, the total protein and globulin levels in the treatment groups, especially those administered higher concentrations of LF or NAC, were increased. Notably, the total protein concentration was greater in the 200 mg/ml LF treatment group than in the other groups (Table 6). The NAC group had higher globulin levels.

Table 6. Serum total protein and globulin levels of the goat kids in the 7 treatment groups during the study period.

Treatments	Total protein (g/dl)			Globulin (g/dl)
	After_20d	After_40d	After_60d	After_20d	After_40d	After_60d
Control	5.404 ± 0.084^a	5.104 ± 0.113^a	5.47 ± 0.059^a	2.582 ± 0.051^a	2.178 ± 0.14^a	2.032 ± 0.088^a
LF50	6.144 ± 0.099^b	5.604 ± 0.018^bd	6.02 ± 0.041^b	2.724 ± 0.03^ab	2.438 ± 0.016^b	2.276 ± 0.03^9b
LF100	6.742 ± 0.04^c	5.79 ± 0.034^b	6.33 ± 0.055^c	2.926 ± 0.061^b	2.516 ± 0.021^b	2.468 ± 0.055^bc
LF200	6.792 ± 0.229^c	6.052 ± 0.165^c	6.586 ± 0.068^d	3.39 ± 0.032^cd	2.762 ± 0.044^c	2.8 ± 0.024^de
NAC50	5.674 ± 0.056^ad	5.394 ± 0.04^d	5.672 ± 0.047^e	3.222 ± 0.068^d	2.614 ± 0.027^bc	2.68 ± 0.036^ce
NAC100	5.864 ± 0.031^bd	5.534 ± 0.026^de	5.852 ± 0.053^f	3.618 ± 0.045^c	2.944 ± 0.034^d	2.962 ± 0.049^d
NAC200	6.502 ± 0.071^c	5.678 ± 0.022^be	6.154 ± 0.038^b	3.472 ± 0.191^cd	3.05 ± 0.021^d	3.26 ± 0.166^f
P value	0.011	0.004	0.006	0.004	0.012	0.004

^{a ,b,c}The mean values placed in rows with different superscript letters are significantly different (P ≤ 0.05). The values are expressed as the means ± SEs. LF50, LF100, and LF200: Lactoferrin groups with 50, 100, and 200 mg/l, respectively. NAC50, NAC100, and NAC200: N-acetyl cysteine groups at 50, 100, and 200 mg/l, respectively. Sig.: significance value.

The treated groups generally displayed lower levels of ALT and creatinine than did the control group. In particular, the groups administered higher doses of NAC had lower ALT and creatinine levels than the other groups (Table 7).

Table 7. Serum alanine transaminase (ALT) and creatinine levels of the goat kids in the 7 treatment groups during the study period.

Treatments	ALT (mg/dl)			Creatinine (mg/dl)
	After_20d	After_40d	After_60d	After_20d	After_40d	After_60d
Control	39 ± 2.429^a	33 ± 0.447^a	34.6 ± 1.631^a	1 ± 0.055^a	1 ± 0.045^a	1.12 ± 0.037^a
LF50	35.2 ± 2.01^a	31.4 ± 0.748^a	31 ± 0.707^b	0.88 ± 0.02^b	0.88 ± 0.02^b	0.94 ± 0.024^b
LF100	30.4 ± 0.678^b	28.6 ± 0.6^b	29.2 ± 0.2^bc	0.82 ± 0.02^b	0.84 ± 0.024^b	0.88 ± 0.02^bc
LF200	27.18 ± 0.364^bc	25.8 ± 0.374^c	27.8 ± 0.374^cd	0.72 ± 0.02^c	0.82 ± 0.02^b	0.86 ± 0.024^c
NAC50	25.8 ± 0.374^c	25.14 ± 0.05^cd	25.6 ± 0.6^de	0.72 ± 0.02^c	0.74 ± 0.024^c	0.78 ± 0.02^d
NAC100	24.4 ± 0.245^cd	23.4 ± 0.812^d	23.4 ± 0.51^e	0.64 ± 0.024^c	0.74 ± 0.024^c	0.76 ± 0.024^d
NAC200	21.8 ± 0.97^d	20 ± 1.265^e	18.8 ± 1.715^f	0.64 ± 0.024^c	0.68 ± 0.02^c	0.66 ± 0.024^e
P value	0.001	0.001	0.002	0.002	0.004	0.001

^{a ,b,c}The mean values placed in rows with different superscript letters are significantly different (P ≤ 0.05). The values are expressed as the means ± SEs. LF50, LF100, and LF200: Lactoferrin groups with 50, 100, and 200 mg/l, respectively. NAC50, NAC100, and NAC200: N-acetyl cysteine groups at 50, 100, and 200 mg/l, respectively. Sig.: significance value.

4. Discussion

All the goat kids in the study were housed under uniform conditions at the facility, with environmental adjustments made to prevent both cold and heat stress. The assessment of heat stress primarily considers factors such as dry bulb temperature and relative humidity (RH%), which can be combined to determine the THI (Goma & Phillips, 2022). The present study focused on evaluating the effects of LF and NAC on DWG and hematological parameters in goat kids under controlled environmental conditions. Table 1 shows the climatic trends observed during the trial months, revealing seasonal variations in climate factors that could affect the trial outcomes and should be controlled within the indoor microclimate, there are no difference between indoor microclimate for the six months.

The results showed that DWG varied significantly throughout different time periods and supplement doses. Table 2 shows that, in comparison to those in the control group, the DWG in the treatment groups, especially those treated with LF at a concentration of 200 mg/ml and NAC, significantly changed after 15, 30, 45, and 60 days. After 60 days of LF therapy, the data in Table 2 show a considerable increase in DWG, which was in line with the findings of earlier studies by Hu et al. (2021) and Ciji and Akhtar (2021). LF is a glycoprotein that binds iron and has been shown to have antibacterial and immunomodulatory effects on a variety of pathogens (Gupta & Prakash, 2017; Selim et al., 2021). The diverse functions of LF in different body secretions and immunological modulation have been highlighted (Coccolini et al., 2023). Interestingly, it has shown promise in boosting immunological responses and controlling iron intake, which might favorably impact weight gain (Purba et al., 2022; Venkata Rao et al., 2022). NAC supplementation also improves feeding behavior and may act as a precursor to glutathione synthesis, as indicated by its correlation with increased DWG (Ripani et al., 2022; Suong et al., 2022). Lowering bacterial biofilms and lowering inflammatory responses are two clear benefits of NAC’s antibacterial qualities and effect on oxidative stress (Shiozawa et al., 2020; Yi et al., 2015).

There were significant changes in the neutrophil, lymphocyte, monocyte, eosinophil, total leukocyte count (TLC), and hemoglobin percentages (Tables 3–6). LF and NAC both exhibit immune-modulating properties, favoring white blood cell counts and having a beneficial impact on hematological parameters to boost immunity (Costagliola et al., 2021; Poles et al., 2021). The possible regulatory effects of LF on the immune system are highlighted by its influence on leukocyte composition and the relative quantity of neutrophils and lymphocytes in the bloodstream (Abed et al., 2020). Lf is an important contributor to mammalian innate immunity, as it can inhibit the growth of many pathogens. With the outbreak of the COVID-19 pandemic, studies on the effects of Lf on COVID-19 have sharply increased. Given the well-documented antiviral properties of Lf (e.g. Berlutti et al., 2011) we believe that experiments in this direction should be highly encouraged. Lf is well-known to block viral entry by interacting with heparan sulfate proteoglycans of the host cells and/or with surface components of viral particles (Rosa et al., 2022).

Even though Lf was first isolated from milk, it is also found in most exocrine secretions and in the secondary granules of neutrophils. Antimicrobial and anti-inflammatory activity reports on lactoferrin identified its significance in host defense against infection and extreme inflammation. Anticarcinogenic reports on lactoferrin make this protein even more valuable. Compared to those in the other groups, the LF200 group had increased monocytes and TLC levels, however NAC200 group had decreased eosinophils. Previous research has demonstrated the antibacterial effect of LF on a range of infections (Bruni et al., 2016). Additionally, its regulatory effects on the immune system have been emphasized (Venkata Rao et al., 2022). On the other hand, the NAC group displayed the lowest eosinophil numbers and the greatest lymphocyte counts. In line with the noted changes in white blood cell counts, NAC also exhibited bacteriostatic effects and was found to be effective against bacterial biofilms (Shiozawa et al., 2020; Suong et al., 2022).

The results of this study are consistent with the notion that LF administration can modulate the immune system and perhaps suppress bacterial development (Gupta & Prakash, 2017), and they also reinforce the immunomodulatory effects of NAC (Elnagar et al., 2022; Ebeid et al., 2024; Kasem et al., 2022; Lasram et al., 2015).

The use of LF and NAC resulted in a substantial increase in DWG, hemoglobin, TLC, and other hematological parameters. This improvement was particularly noticeable at higher doses of LF in terms of the percentage of hemoglobin. These results are consistent with previous reports that LF has immunostimulatory characteristics, strengthens mucosal barriers, and augments innate and adaptive immune responses (Costagliola et al., 2021; Singh et al., 2023). Further supporting the potential use of LF in bovine disease mitigation is its beneficial effects on hematopoiesis, cellular targets, cytokine release, and NK cell functionality (Singh et al., 2023).

More research has been performed on the mechanisms underlying the effects of these supplements. The antioxidant qualities of NAC were emphasized, as was its function as a precursor to glutathione, which is necessary for a number of physiological processes (Poles et al., 2021; Saleh et al., 2023; Yi et al., 2015). Elzoghby et al. (2020) highlighted the diverse effects of LF, which include immunological regulation, antibacterial activity, and antiviral properties. These actions aid in improving immune responses and preventing disease.

Furthermore, the favorable effects of LF on DWG and kid immunity were confirmed by its observed impacts on leukocyte composition and the structure of the gastrointestinal villi, without changing cytokine signaling (Cooper et al., 2013). Notably, the capacity of LF to control leukocytes in circulation and its interactions with receptors shed light on its immunomodulatory activities.

The greater eosinophil counts in the control groups suggested that LF and NAC had a lessening effect. Through their antimicrobial functions, both LF and NAC can decrease the number of eosinophils; LF has antiviral and antibacterial qualities while promoting the growth of lactobacilli (Elzoghby et al., 2020; Mallaki et al., 2021). However, NAC has demonstrated promise in reducing influenza-induced inflammation and increasing the synthesis of glutathione (Kasem et al., 2022; Omar et al., 2024).

The ability of LF to boost immunity and preserve goat kid health was demonstrated by its notable effects on serum globulin and total protein concentrations, as well as its ability to maintain creatinine and ALT levels within normal ranges (Yan et al., 2018).

During the trial period, the serum globulin concentration was greater in the NAC group. In addition, the advantages of NAC for preventing oxidative damage and hepatotoxicity have been highlighted, as have its drawbacks and possible negative consequences (Liou et al., 2021; Kamal et al., 2024). Significantly, over the course of the experiment, NAC improved liver and renal function, as indicated by decreases in the serum ALT and creatinine levels.

Although LF and NAC have promising effects on immunity and weight gain, their therapeutic efficacy and limitations need to be carefully considered. Yan et al. (2018) and Liou et al. (2021) highlighted the effects of NAC on hepatotoxicity and its potential limits in therapeutic windows.

The study’s conclusions are consistent with previous ones, highlighting the immune-modulating properties and possible therapeutic uses of LF and NAC in promoting weight gain and boosting immunity in young goats. To investigate the mechanics and wider applications of these materials, additional studies are needed.

5. Conclusion

In conclusion, the administration of NAC and LF had beneficial effects on the immune system and weight gain of young goats. These results are consistent with earlier studies, suggesting their potential contributions to improving veterinary clinical results. However, for their efficient use in animal husbandry procedures, a thorough grasp of their processes and therapeutic limitations is essential.

Authors’ contributions

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by Mohamed E. El-Sharawy, Yasser S. Hussein, Abdelhamid Saeed Abo El-Enin, Ahmed SH. Shams, Rashed A. Alhotan, Elsayed Osman Hussein, Branislav Galik, Kamal M.A., Ahmed A. Saleh, Soliman M. Soliman, Sara Mahmoud Omar, Tarek A. Ebeid, Ahmed M. I. Mustafa, Ibrahim Atta Abu El-Naser. The first draft of the manuscript was written by Mohamed E. El-Sharawy, Kamal M.A., Ahmed A. Saleh, Sara Mahmoud Omar, Tarek A. Ebeid, and all authors commented on previous versions of the manuscript. All the authors have read and approved the final manuscript.

Ethical statement

The study was approved by the Ethics Committee of Local Experimental Animals Care Committee and conducted following the guidelines of Cairo University, Egypt (no. Vet CU 25122023860).

Acknowledgements

The authors thank the support of the Animal science department, faculty of Agriculture, Kafrelsheikh University; Laboratory reagents used during the study were provided by the Laboratory of Veterinary Hygiene and Management Department, Faculty of Veterinary Medicine, Cairo University.

Disclosure statement

The authors declare that they have no competing interests.

Data availability statement

The data presented in this study are available from the corresponding authors upon reasonable request.

Additional information

Funding

This research has been supported by the Researchers Supporting Project (grant no. RSPD2024R581) and King Saud University, Riyadh, Saudi Arabia.

Notes on contributors

Mohamed E. El-Sharawy

Mohamed E. El-Sharawy PhD in animal physiology, Animal Production Department, Faculty of Agriculture, Kafrelsheikh University, Egypt.

Yasser S. Hussein

Yasser S. Hussein PhD in animal physiology, Animal Production Research Institute, Ministry of Agriculture, Dokki, 12618, Egypt.

Abdelhamid Saeed Abo El-Enin

Abdelhamid Saeed Abo El-Enin PhD in animal physiology, Animal Production Research Institute, Ministry of Agriculture, Dokki, 12618, Egypt.

Ahmed S. H. Shams

Ahmed S. H. Shams PhD in animal breeding, Animal Production Research Institute, Ministry of Agriculture, Dokki, 12618, Egypt.

Rashed A. Alhotan

Rashed A. Alhotan PhD in animal nutrition, Department of Animal Production, College of Food & Agriculture Sciences, King Saud University, P. O. Box 2460, Riyadh 11451, Saudi Arabia.

Elsayed Osman Hussein

Elsayed Osman Hussein PhD in poultry nutrition. AlKhumasia for Feed and Animal Products Riyadh- Olaya - Al Aqareyah 2 - Office 705 PO 8344, Riyadh 11982.

Branislav Galik

Branislav Galik PhD in animal nutrition Institute of Nutrition and Genomics, Slovak University of Agriculture in Nitra, Slovakia. Trieda A. Hlinku 2, 94976 Nitra.

M. A. Kamal

M. A. Kamal PhD in animal nutrition Department of Veterinary Hygiene and Management, Faculty of Veterinary Medicine, Cairo University, 11221, Giza, Egypt.

Ahmed A. Saleh

Ahmed A. Saleh PhD in poultry nutrition Department of Poultry Production, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt.

Soliman M. Soliman

Soliman M. Soliman PhD in animal nutrition Department of Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Cairo University, 11221, Giza, Egypt.

Sara Mahmoud Omar

Sara Mahmoud Omar PhD in animal and human nutrition Nutrition and Food Science Department, Faculty of Home Economics, AL-Azhar University, Egypt.

Tarek A. Ebeid

Tarek A. Ebeid PhD in poultry physiology Department of Poultry Production, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh 33516, Egypt.

Ahmed M. I. Mustafa

Ahmed M. I. Mustafa PhD in animal nutrition Animal Production Research Institute, Ministry of Agriculture, Dokki, 12618, Egypt.

Ibrahim Atta Abu El-Naser

Ibrahim Atta Abu El-Naser PhD in Animal, Poultry and Fish Department of Animal, Poultry and Fish Production, Faculty of Agriculture, Damietta University, 34517, Egypt