1. Introduction

The worldwide need for a sufficient and regular milk supply is predicted to rise by 2030 35%, owing primarily to growing global requirements [1, 2]. To meet this ever-increasing need, many countries have invested heavily in increasing their dairy production in recent decades [3, 4]. Milk production strategies face many socioeconomic and environmental challenges, including the hazards associated with the antibiotics and growth hormones used in animals [5]. These challenges have prompted a global quest for natural eco-friendly feed additives to increase cow milk supply [6–8].

Dairy cows’ gut microbiota is crucial in energy intake and nutrition. As a result, it should be considered a target for improving milk quality, milk yield, and dairy cow health [9]. Probiotics are live microorganisms that, when given appropriately, regulate the gut microbes’ balance, enhance the animals’ growth, and boost their resistance to diseases [10]. The commonly accepted goal in probiotics production is to raise the proportion of beneficial bacteria in the gut to promote effective nutrient utilization while leaving harmful bacteria behind, increasing dairy production [11]. The most frequently used probiotic organisms are Streptococcus thermophilus, Lactobacillus spp., Bifidobacterium spp., and fungal species [12].

Various factors determine the probiotic’s effectiveness, including the microbial species composition, the dose of each preparation, the delivery route, the kind of diet, and the feeding duration [13]. One of the most important factors is the dosage used in each given probiotic treatment and/or the specific combination of probiotic organisms as single- or multistrain [14]. Several recent reports verified the efficacy of multistrain probiotic formulation rather than a single strain in improving the health and growth of poultry and aquatic species [15]. Improved effectiveness with some multistrain preparations may be related to the synergistic and additive effects and symbiotic interaction between the organisms involved [16]. Despite extensive research on the benefits of probiotics in dairy products, commonly with single-strain preparations [17, 18], the results of the effects of multistrain on dairy cows are scanty in the literature.

Given the above data, we hypothesized that feeding a mixture of probiotics initiated from the prepartum stage could enhance dairy cow health and lactation performance. Consequently, the present study aimed to evaluate the influence of multistrain probiotics formulation (MPF) containing Lactobacillus spp., Streptococcus thermo phallus, Bifidobacterium spp., and some fungal species as a functional feed additive during the late gestation and early lactation period on the lactation performance of postpartum crossbred (Holstein Friesian × Balady) dairy cows. To further assess the safety of this MPF product, blood biochemical indices of kidney and liver functioning, hematological parameters, and protein profile were analyzed.

2. Materials and Methods

3. Results

3.1. Lactation Performance and Feed Utilization

Table 2 and Figures 1(a) and 1(b) present the results for milk production, milk composition, DMI, and feed utilization of dairy cows that were supplemented with different doses of MPF. The cows’ daily milk production, 3.5% FCM, and ECM in the MPFI and MPF2 groups were higher (P < 0.001) than those of the control group. The MPF-supplemented cows also exhibited significantly greater milk energy output, DMI (expressed as a percentage of body weight or kg per day), and yields of solids-not-fat, lactose, ash, protein, total solids, and fat compared to the control group (P < 0.001). DMI (kg/d) significantly increased in dairy cows that consumed MPF from parturition to day 84 of the trial period (Figure 1(b)). However, lower concentrations of milk solids-not-fat, fat, total solids, and protein were observed in cows fed MPF-containing diets compared to the control group (P < 0.001). The supplementation of MPF significantly (P < 0.001) enhanced feed conversion, as indicated by kg of DMI per kg of milk and kg of DMI per kg of ECM (Table 2). Additionally, a significant interaction effect was observed regarding the concentrations of milk protein (P = 0.017) and solids-not-fat (P = 0.015), with a trend observed for total solids (P = 0.062) and lactose (P = 0.096; Figures 2(a)–2(d)).

Table 2. Lactation performance of dairy cows supplemented with a multistrain probiotic formulation (MPF) over the first 12 weeks of lactation.

	Treatment (Trt)			SEM	P value
	Control	MPF1	MPF2		Trt	Week	Trt × week
Milk production (kg/d)
Milk	9.79c	12.90b	14.71a	0.21	<0.001	<0.001	0.748
3.5% fat-corrected milk	10.71c	13.36b	14.70a	0.19	<0.001	<0.001	0.794
Energy-corrected milk (ECM)	9.64c	11.75b	12.98a	0.16	<0.001	<0.001	0.971
Milk composition (g/kg milk)
Total solids	126.37a	118.77b	116.56b	0.62	<0.001	0.003	0.062
Solids-not-fat	86.13a	82.05b	81.82b	0.42	<0.001	0.002	0.015
Fat	40.24a	36.72b	34.74b	0.39	<0.001	0.790	0.815
Protein	32.05a	28.61b	27.92b	0.35	<0.001	0.019	0.017
Lactose	45.73	45.20	45.63	0.19	0.399	0.001	0.096
Ash	8.34	8.25	8.28	0.06	0.805	0.103	0.444
Somatic cell count (×105)	145.83	124.45	126.50	3.56	0.081	0.875	0.871
Milk energy content (MJ/kg)	3.06a	2.84b	2.76b	0.04	<0.001	0.027	0.266
Milk yield (kg/d)
Total solids	1.24c	1.53b	1.71a	0.02	<0.001	<0.001	0.983
Solids-not-fat	0.85c	1.06b	1.20a	0.02	<0.001	<0.001	0.955
Fat	0.39c	0.47b	0.51a	0.04	<0.001	<0.001	0.848
Protein	0.32c	0.37b	0.41a	0.04	<0.001	<0.001	0.338
Lactose	0.45c	0.58b	0.67a	0.01	<0.001	<0.001	0.908
Ash	0.08c	0.11b	0.12a	0.01	<0.001	<0.001	0.128
Milk energy output (MJ/d)	29.95c	36.53b	40.36a	0.51	<0.001	<0.001	0.973
Dry matter intake (kg/d)	12.55c	13.41b	14.03a	0.07	<0.001	<0.001	0.952
Dry matter intake (% BW)	2.70b	2.94a	2.86a	0.02	<0.001	<0.001	0.927
Feed conversion (kg/kg)
Kg DMI/kg milk	1.30a	1.05b	0.96b	0.09	<0.001	<0.001	0.842
Kg DMI/kg ECM	1.32a	1.16b	1.09b	0.08	<0.001	<0.001	0.944

• ^a–cDifferent lowercase superscript letters in the same column indicate that the difference between groups is significant (P < 0.05). SEM: standard error of the mean. The experimental treatments were with no MPF supplementation (control group) or with the addition of MPF at 0.5 g/cow/d (MPF1 group) or 1 g/cow/d (MPF2 group).

3.2. Hematological and Serum Biochemical Measurements

Most hematological indicators, including Hb, RBCs, MCHC, MCH, MCV, platelet count, and leukogram parameters, did not alter significantly (P > 0.05) between the two MPF groups and the control group (Table 3). However, cows receiving MPF supplements had higher levels of Hct (P = 0.011). Most of the hematological measurements had no statistically significant interactions between treatment and time. Nevertheless, the cows administered MPF had a slightly lower percentage of monocytes (T × W; P = 0.077) than the control group 28 days after calving (Figure 3(a)).

Table 3. Changes in hematological parameters of cows supplemented with a multistrain probiotic formulation (MPF) during the dry period and early lactation.

	Treatment (Trt)			SEM	P value
	Control	MPF1	MPF2		Trt	Day	Trt × day
Erythrogram
RBCs, 106 × µL	3.05	3.28	3.32	0.07	0.216	0.064	0.376
Hb (g/dL)	9.19	9.85	10.02	0.16	0.075	0.240	0.471
Hct (%)	31.05b	34.87a	34.47a	0.61	0.011	0.089	0.456
MCV (fL)	103.78	107.58	106.79	3.34	0.867	0.019	0.508
MCH (pg/dL)	30.34	30.21	30.69	0.45	0.894	0.047	0.567
MCHC (g/dL)	30.03	28.35	29.17	0.58	0.418	0.004	0.876
Platelet count (103/µL)	259.9	230.0	283.5	20.41	0.514	0.034	0.433
Leukogram
WBCs (103/µL)	12.82	13.80	11.70	1.25	0.794	0.236	0.509
Lymphocyte (%)	51.96	56.40	57.00	2.55	0.636	0.173	0.455
Monocytes (%)	3.27	3.32	3.14	0.12	0.788	0.658	0.077
Neutrophils (%)	43.83	39.38	38.88	2.61	0.695	0.228	0.439
Eosinophils (%)	0.58	0.57	0.63	0.06	0.847	0.000	0.996
Basophils (%)	0.29	0.27	0.29	0.02	0.956	0.010	0.950

• ^a–bDifferent lowercase superscript letters in the same column indicate that the difference between groups is significant (P < 0.05). SEM stands for the standard error of the mean; Hb, hemoglobin; RBCs, red blood cells; Hct, hematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; WBCs, white blood cells. The experimental treatments were with no MPF supplementation (control group) or with the addition of MPF at 0.5 g/cow/d (MPF1 group) or 1 g/cow/d (MPF2 group).

Serum biochemical measurements are shown in Table 4, and changes in urea and BUN are presented in Figures 3(b) and 3(c). Serum concentrations of total cholesterol (P = 0.041), LDL (P = 0.002), and VLDL (P = 0.001) were lower; and urea (P = 0.07) and BUN (P = 0.07) tended to be lower in the MPF cows than in the control cows. Yet, serum HDL concentrations (P = 0.055) tended to be higher than the control cows (Table 4). Furthermore, the concentrations of blood IgG tended (P = 0.096) to be increased in the blood of the MPF2 group. Cows that were given MPF supplements had a lower serum concentration of urea and BUN at 84 days of parturition (T × W; P = 0.01; Figures 3(b) and 3(c)).

Table 4. Changes in selected measures in the serum of cows supplemented with a multistrain probiotic formulation (MPF) during the dry period and early lactation.

	Treatment (Trt)			SEM	P value
	Control	MPF1	MPF2		Trt	Day	Trt × day
Total protein (g/dL)	5.88	5.99	6.17	0.10	0.924	0.642	0.913
Albumin (g/dL)	3.09	3.08	3.24	0.07	0.769	0.108	0.478
Globulin (g/dL)	2.74	2.91	2.93	0.08	0.905	0.783	0.508
Albumin/globulin	1.20	1.08	1.12	0.05	0.723	0.159	0.350
Total cholesterol (mg/dL)	76.50a	72.58ab	67.00b	1.54	0.041	0.072	0.777
Total triglycerides (mg/dL)	87.42	85.92	82.17	1.27	0.263	0.487	0.758
High-density lipoprotein (mg/dL)	40.75	45.58	44.41	0.81	0.055	0.635	0.810
Low-density lipoprotein (mg/dL)	21.52a	16.31b	15.11b	0.90	0.002	0.006	0.661
VLDL (mg/dL)	14.23a	10.70b	9.15b	0.59	0.001	0.260	0.124
Urea (mg/dL)	33.58	31.80	28.94	1.01	0.070	0.134	0.010
Blood urea nitrogen (mg/dL)	15.67	14.84	13.51	0.47	0.070	0.134	0.010
Immunoglobulin G (mg/mL)	29.38	27.99	35.56	1.26	0.096	0.864	0.649

• ^a–bDifferent lowercase superscript letters in the same column indicate that the difference between groups is significant (P < 0.05). SEM stands for the standard error of the mean; VLDL, very low-density lipoprotein. The experimental treatments were with no MPF supplementation (control group) or with the addition of MPF at 0.5 g/cow/d (MPF1 group) or 1 g/cow/d (MPF2 group).

4. Discussion

In the current work, the MPF use considerably increased the milk yield and the yield of major milk components, including total solids, protein, solids-not-fat, lactose, fat, and ash, while slightly suppressing the SCC and blood monocytes. The favorable effects are most likely due to an enhanced ruminal microbiota due to MPF probiotic addition. For example, some ruminal bacteria such as L. plantarum [27], Bifidobacterium bifidum [28], and S. thermophilus [29] have been described as strong proteolytic bacteria, and microbially derived amino acids can account for as much as two-thirds of the amino acids absorbed by the small intestine of a ruminant. To that end, these microorganisms in the intestine produce the microbial crude protein, which greatly affects the amount and quality of metabolizable protein absorbed in the small intestine. As a result of its conversion to milk protein, the metabolizable protein ultimately affects the milk production yield [30]. These strains are the main components of the used MPF. Additionally, the populations of lactobacillus, bacillus, and total bacterial count were significantly increased due to MPF administration. The milk output observed in our study (ranging from 9.79 to 14.71 kg/d) reflects the productivity levels typical of crossbred cows in our region [31].

It is worth noting that in our study, there was a decrease in the concentration (g/kg milk) of major milk components, including total solids, protein, solids-not-fat, and fat, despite an increase in their yield. This phenomenon can be attributed to a combination of factors, such as cattle genotype, diet composition, the dilution effect resulting from increased milk production associated with MPF supplementation [32], and potentially a reduction in the mammary blood flow rate [33]. The higher DMI observed in our study with MPF supplementation is in line with previous findings. Nocek et al. [34] reported an increased DMI of 2.6 kg/day in dairy cows supplemented with a combination of E. faecium and Saccharomyces cerevisiae probiotics from 3 weeks prepartum to 10 weeks postpartum. Similarly, Kembabazi et al. [35] found that supplementing lactating dairy cows with mixed strain probiotics during the early stage increased DMI. The ability of probiotics to improve DMI in ruminants can be attributed to several factors. One possible mechanism is the enhanced proliferation and growth of ruminal cellulolytic bacteria, which leads to improved fiber degradation and nutrient utilization. Probiotics can also help prevent ruminal acidosis, a condition that negatively impacts feed intake and overall rumen function [36]. Additionally, the supplementation of dairy cattle with probiotics has been shown to increase the frequency of feed intake, fiber degradation, and feed digestibility in the rumen [37].

The slight reduction in SCC and blood monocytes in cows subjected to MPF administration suggests that this probiotic product can decrease inflammation in the mammary glands of dairy cows, leading to an increase in the milk production, as evident in our findings. In this regard, a relationship was recorded between the composition of blood monocyte types and postpartum infectious diseases in dairy cows like mastitis [38]. Besides, Eger et al. [39] reported that the blood cell counts of the different monocyte subgroups were higher in cows with postpartum mastitis. Additionally, numerous research proposed that low SCC values are associated with higher milk production [40, 41], a finding we observed in the present study.

The fecal bacterial composition of cows fed MPF demonstrated a considerable increase in the beneficial populations of lactobacillus and bacillus, whereas a significant reduction in the count of Clostridium, Salmonella, coliforms, and E. coli proposing that these probiotic supplements reinforced an improved defense mechanism of the rumen and intestine, possibly decreasing the hazard of pathogens’ colonization. Comparably, Guo et al. [42] demonstrated that using MPF-containing B. animalis, L. casei, B. cerevisiae, and S. faecalis did not disturb the overall fecal microflora at the phylum level but increased the beneficial Prevotella abundance while decreased the opportunistic pathogens Dorea in Holstein’s calves. In this regard, many of the Lactobacillus probiotic strains inhibit the growth of pathogens and aid in the body’s natural defenses against infections by producing metabolites such as diacetyl, hydrogen peroxide, acetoin, and bacteriocins [43, 44]. Because of MPF’s beneficial effect on commensal microbial community composition, rumen fermentation efficiency may be increased, leading to maximized milk production [45]. Besides, several studies have demonstrated that probiotics play an indirect influence in increasing milk production by raising the digestive efficiency and feed conversion ratio of dairy animals [46, 47]. This role was evident in the results of the current study, as the MPF supplementation significantly enhanced the feed conversion and also indicated a trend toward increased digestibility coefficients of DM and OM.

Marinating the dairy cow’s health status is needed for good milk production and quality during lactation. In the current experiments, the safety of MPF as a feed supplement in dairy cows has been determined using multiple biochemical indicators in the animals’ blood. Serum urea and BUN levels were thought to predict renal function in dairy cows accurately [48]. Total protein, globulin, albumin, and BUN levels in the serum of animals have been utilized extensively to indicate the amount of energy and protein required for production [49]. The recorded normal range of altogether shows the conditions for optimal production. BUN might increase the following water deprivation [50]: thirst, diarrhea [51], urinary diseases [52], pregnancy toxemia [53], and acidosis [54], which were not the case for these cows. Yet, compared with the cows fed nonsupplemented diets, the cows that were fed MPF-supplemented diets had lower urea and BUN, indicating a better kidney function [48] and a positive impact on nitrogen proficiency and energy metabolism [55].

Probiotics serve as immunological adjuvants, stimulating IgG synthesis [56]. In the current study, MPF-fed cows had an increasing tendency of the serum IgG level. Extensive evidence supports the significant impact of the rumen microbiome on the development and functioning of both innate and systemic immunological components [57]. Hence, improving the fecal bacterial composition recorded in the current study could be responsible for enhancing immunoglobulin levels [57, 58].

Lipid metabolism and alterations in serum lipid and lipoprotein profiles, especially HDL, constitute a fundamental component of dairy cows’ energy metabolism and physiology [59]. HDL-C is the chief lipoprotein for transferring cholesterol in cows [60]. Energy balance is maintained, and oxidized fatty acids are kept to a minimum by keeping HDL-C-rich lipoprotein profiles in dairy cows [61]. HDL-C may effectively predict energy balance for distinguishing healthy cows from diseased ones. Cows suffering from fatty liver and milk fever exhibited lower mean HDL-C levels than healthy dairy cows [62]. In the current experiment, serum HDL concentrations in MPF-fed cows tended to be higher than that of the control cows. VLDL, LDL, and total cholesterol serum concentrations were lower in the MPF-fed cows than in the control cows. The different probiotic strains in MPF could be responsible for this improved lipid profile. For instance, numerous Lactobacillus strains have reduced TC and TG concentrations in rats [63, 64]. Likewise, B. bifidum significantly reduced serum total cholesterol and LDL levels and slightly increased serum HDL in rats [65].

5. Conclusions

The overall results showed that supplementing lactating cows with 0.5 g or 1 g of MPF/cow/day during late gestation and early lactation improved milk production and feed efficiency. Both MPF doses increased blood Hct levels and reduced LDL, total cholesterol, and VLDL levels without affecting other blood parameters. MPF supplementation also led to higher feed digestibility and favorable shifts in bacterial populations, promoting beneficial microbes while reducing harmful ones. The cows supplemented with 1 g/day of MPF demonstrated the highest milk production and the most significant reduction in harmful bacterial populations in the feces. Utilizing these advantageous microbes presents a natural and consequence-free strategy for enhancing dairy production. More research is needed to understand how MPF affects the metabolic and physiological processes of dairy cows throughout the transition period.

Ethical Approval

The animal study protocol was approved by the Ethics Committee of Institutional Animal Care and Use Committee of Zagazig University (approval code: ZU-IACUC/2/F/320/2022).

Conflicts of Interest

The authors declare that there are no conflicts of interest.