Animals and diet

The research was conducted under the guidelines approved by the Academic Committee of the Department of Animal Science of Ethics, Biosafety and Animal Welfare of the UAEM Amecameca University Center of the Autonomous University of the State of Mexico. The experiment was conducted in the rabbit metabolic area.

Forty weaned kits (30 days old), California X New Zealand breed (506.52 ± 120.47 g), were used, which were assigned in individual cages in five treatments (n= 8); with levels of addition of the polyherbal mixture at 0.0, 200, 400, 600 and 800 mg kg^-1 of dry matter (DM) (BioCholina Powder®, Technofeed Mexico), an additive composed of Trachyspermum ammi, Achyranthes aspera, Azadirachta indica, Citrullus colocynthis and Andrographis paniculata), which contains 16 g kg^-1 of total choline conjugates¹⁶, the five groups being homogeneous in terms of weight, which was considered as initial weight (P>0.05).

The experimental diet was formulated to meet the nutritional requirements of fattening rabbits¹⁷ (Table 1), however, choline requirements were kept below (1,130 mg kg^-1). The polyherbal formula was added to the formulated balanced feed, mixed and subsequently pelletized to offer it to rabbits ad libitum, as well as clean and drinking water. The experiment lasted 34 days, with seven days of adaptation, the diets of the treatments were isoenergetic and isoproteic.

The following productive variables were determined, initial weight (0d), final weight (34d) and daily weight gain, as well as the record of the feed offered and rejected, to obtain voluntary consumption on a wet basis and its subsequent adjustment to dry matter; in addition, the feed conversion (kilos of feed to obtain one kilogram of live weight) was obtained.

Sample collection and analysis

At 65 days of age of the rabbits, the slaughter was carried out, with previous fasting of 24 h. The management of the carcasses followed the methodology proposed by Peiretti and Mineri¹⁸. During the slaughter, the weight of the hot carcass, the gastrointestinal tract including spleen and mesenteric tissue, liver and kidneys (abdominal organs), as well as heart and lungs (thoracic) were recorded. The carcasses were stored at 4 °C for 24 h and weighed again to obtain the weight of the cold carcass; to perform the meat quality measurements, the Longissimus dorsi muscle was dissected in its lumbar portion¹⁹.

Meat quality

Analyses were performed on the Longissimus dorsi muscle at 24 h postmortem; measurements were made in duplicate for each of the variables. The pH was taken at the level of the 5^th lumbar vertebra with a penetration potentiometer (Hanna instruments H199163)²⁰. Color measurements were taken with a colorimeter (Konica Minolta, trichromatic) and readings were reported as: L* (luminosity), a*(red), b* (yellow), used to obtain C* (chroma) and H* (Hue)²¹. The water retention capacity (WRC) was determined through loss of water by dripping and by pressure²² and also the loss of water by cooking²³.

To determine the economic impact, the cost of the kit, cost of each ingredient of the diet, including the herbal mixture per treatment dose, were considered, with prices of 2020; all these as expenses of the activity. For income, the sale price of live animals and carcass was calculated based on the local market; once the income and expenses were obtained, the profit (income-expenses) was obtained for the live animal, in hot and cold carcass; finally, the income-expense ratios (income-expenses) were calculated, which allowed to know the rate of return in percentage units, where it is indicated that an income-expense rate less than 1 represents losses, equal to 1 indicates that there were no profits, while rates greater than 1 are indicative of profits. All economic values are indicated in US dollars at an exchange rate of $1USD to $20.03MX dated February 10, 2021.

Statistical analysis

The results were analyzed with a completely randomized design, using the R²⁴ software, using initial weight as a covariable for the productive variables and using orthogonal polynomials to obtain linear and quadratic effects to evaluate the effects of the polyherbal additive²⁵ with a significance level of P<0.05.

Productive response

According to the results obtained, there were no significant effects on feed consumption and daily weight gain due to the addition of polyherbal mixtures, despite the fact that these additives are considered as phytogenic additives with the characteristic of stimulating the production of digestive enzymes, such as trypsin and amylase, with the ability to optimize the absorption of nutrients and consequently improve productive and carcass responses⁶, however, in this study, these benefits were not observed, a situation similar to that presented by other authors¹², in a study carried out in lambs fed choline conjugates at doses of 0 and 4 g day^-1, no better response was found in the added treatment with respect to the control on final weight (28.63 - 28.68 kg) or daily weight gain (106.44 - 107.98 kg). Similarly, in two studies carried out in rabbits using various plants, they did not observed improvements in production parameters despite being plants rich in secondary metabolites^26,27, since it is reported that the metabolites present are not sufficient to modify the microbiota or the conditions of the gastrointestinal tract.

On the other hand, the relationship between feed conversion and weight gain was not affected by the doses of polyherbal mixtures, values that agree with what was reported by another paper¹⁰, an study in which there was a linear effect (P=0.07) in conversion but not in weight gain (P>0.10) in growing lambs, using a polyherbal formula, as well as in the results of Selvam et al²⁸ in chickens fed an herbal mixture (0.0, 500 and 1,000 g^-1 t^-1 of feed), and they obtained values of 1.58 in the control treatment and of 1.47 and 1.48 in the added groups.

The addition of polyherbal mixtures showed a positive effect on the variables of feed consumption and daily weight gain, since rabbits with the lowest body weight were in the control group (27.7 g d^-1), which is deficient in choline with 1,130 mg d^-1, when the minimum requirement is 1,130 mg kg^-1 DM. In this regard, in studies where choline deficiency has been experimentally induced, growth retardation, anemia, muscular dystrophy and death have been observed²⁹, a situation that is confirmed in this experiment.

Although the active mechanism of choline chloride is still unclear, excess choline has been shown to negatively affect animal yield; one of the hypotheses that may explain the results obtained is that, when exceeding the ability to metabolize choline at the cellular level, an accumulation of phosphocholine occurs, a situation that could be occurring in treatments with 600 and 800 mg (27.2 and 26.3 g d^-1 respectively), which are above the necessary requirement for rabbits at this stage of growth¹⁶.

An experiment conducted on quails at doses of 1,000, 1,500, 2,000 and 2,500 mg kg^-1 of choline showed a greater weight gain between d 7 to 21 of age (1.95, 2.10, 2.98, 2.48 g), as well as better conversion (5.29, 5.44, 3.68, 4.35); this situation contrasts with what was observed in the present research, probably because the addition of polyherbal mixtures could have modified the digestive physiology, acting on the cecal microbiome, since the digestive system of the rabbit is adapted to the fermentation of plant epithelial cells in the cecum³⁰, while in birds, these digestive fermentation processes do not occur.

Although in this study no hematological analyses were performed to know the hepatic functioning due to the addition of polyherbal mixtures, various studies have observed the variation of the response when using herbal formulas; in a research³¹, they found that liver weight had no relationship in the production of specific enzymes (ALT and AST), as they showed no changes between the groups when choline (1,000 mg kg^-1) was added as a lipotropic agent in broilers, likewise⁹, they observed a decrease in serum concentrations of AST in broilers fed choline (1 g kg^-1) and lectin (0.5 g kg^-1). It is worth mentioning that the use of various plants participates in the metabolism of lipids and cholesterol³², a situation that modifies the general metabolism of the animal and therefore the productive response, it is possible that the plants that make up the polyherbal formula have effects on lipids and these in turn can modify the absorption of nutrients.

Meat quality

According to the pH results obtained, significant differences were found between the treatments added with polyherbal mixtures compared to the control (P<0.05), presenting values ranging from 5.5 for the control and 5.8 as a maximum for the added groups. These results are consistent with what was reported by others³³, where they found pH values of 5.8 for meat of rabbits fed different parts of the Tithonia tubaeformis plant. The optimal pH at 24 h postmortem for glycolytic muscles, such as the Longissimus dorsi muscle of the rabbit, is between 5.3 and 6.0³²; this parameter is one of the most important indicators for assessing meat quality, as it is associated with appearance, color (myoglobin chemical reactions) and water absorption³⁴.

The parameters obtained of the variable b* of meat color are lower (between 3 and 4.7) than those reported³⁵ in rabbits fed flavonoids contained in alfalfa, at doses of 0.0, 400, 800, 1,200 mg kg^-1 and find values that range between 6.1 and 6.6. As mentioned by Selvam et al²⁸, where they reported values of 8.34 and 10.16 for variable b of rabbits fed different parts of the Tithonia tubaeformis plant. The coordinate b* that represents the yellow pigment in meat, this value was affected in the treatments with the addition of the polyherbal mixture, which could be due to a series of nonenzymatic browning reactions that occur in the oxidation products of lipids and amines in the phospholipid head groups or the amine in the protein³⁶. Likewise, the presence of antioxidant compounds such as phenols and flavonoids contained in polyherbal mixtures could be responsible for the color differences observed. The results obtained in this experiment in terms of color indicate a meat with a slight coloration that tends to brown and is nonexudative, a situation that is not desirable since rabbit meat is pale nonexudative³⁷.

In several studies^26,27 that involved the use of plants in the feeding of rabbits, it was observed that the incorporation at low levels of plants with high antioxidant potential and with phenolic compounds, they do not have the ability to modify the quality of the meat, a situation similar to that reported in this experiment, this because the volumes added are low.

Economic impact

The addition of 200 mg kg^-1 DM of the polyherbal formula generated the highest live weight and the best feed conversion in this experiment, such a situation was reflected in the economic indicators; when adding this dose, the greatest profits and income-expense ratios were obtained, since it can be indicated that it was the optimal dose for these animals. In one study³⁸, it was observed that the addition of plants with immunostimulant potential generated economic benefits in the production of rabbits, a situation similar to that reported in this research; this effect may be due to the fact that polyherbal formulas contain a large number of metabolites, and these generate beneficial modifications in the gastrointestinal tract, or in the absorption of nutrients, a situation that generates better feed conversions. In the same sense, in a study conducted in dairy cattle, it was observed that the addition of a polyherbal formula rich in choline improved economic indicators due to the fact that milk production was improved and the number of pathology events was decreased³⁹.