The global livestock industry faces challenges to achieve higher productivity due to the increasing demand for animal protein. Increasing the human population with a ban on antibiotic growth promoters requires alternatives in the form of functional foods, nutraceuticals, or phytochemicals. Garlic (Allium sativum) has been reported helpful for poultry production due to its physiological, antimicrobial, energizing and hepatoprotective effects; it stimulates the immune, digestive and growth promoter systems (Al-Shuwaili  et al., 2015).
       
The Spanish ecotype "Morado de Las Pedroñeras"is a crop in constant use for human consumption and has been used in animals for some years in Europe, it has been shown to have an antibiotic and anticancer effect due to its high level of thiosulfinate and allicin, which are the active principles of garlic (Pérez-Ortiz  et al., 2020). However, the impact of garlic on the productive variables in chickens is variable gender-wise (Lee et al., 2017). Therefore, this study aims to determine the effect of supplementation with Spanish purple garlic on the productive indicators related to growth in females and males since, to our knowledge, there are no studies that have evaluated it.

The experiment was carried out at the Veterinary Medicine Faculty, National Autonomous University of Mexico, Mexico City, during the first semester of 2020. The Institutional Subcommittee approved all procedures of this study (protocol number 094) to compliance with the care and use of animals in poultry production and according to the international recommendations (Meluzzi and Sirri, 2009).
 
Experimental birds and diets
 
From 1 day of age, ninety-eight clinically healthy broiler chicks Cobb Vantress 500 were distributed with an utterly randomized design (50% female and 50% male) with average body weight 39.5±0.84 g, into four dietary treatments groups having three repetitions for each treatment (6/7 birds).
       
Zooallium® [Z], a product standardized to 65% purple garlic ecotype from Las Pedroñeras, Cuenca, Spain, was used in this study. Birds were fed either conventional (T₀) diets or supplemented diets (T₁ to T₃) with 0.1, 0.2 and 0.3% purple garlic. Birds were reared in vertical battery cages within the experimental isolation unit during 40 days of growth and kept under uniform management conditions.
       
All the birds were weighed weekly in the morning before feeding and watering until they were 40 days old. The performance of the experimental birds in terms of body weight gain (BWG), feed intake (FI), average daily weight gain (ADG), feed conversion ratio (FCR) and total profit (TP). All variables were recorded by sex (female and male birds).
 
Gut morphometry
 
At the end of the experiment, three birds were randomly selected per group and sacrificed by cervical dislocation (EFSA, 2019) to study intestinal morphometry. Jejunum and duodenum sections were collected and fixed in 10% formalin. Histological sections were stained with hematoxylin and eosin staining. Ten randomly selected villi were measured on each slide per microscopic field from each section. To measure the length of the villi, eyepiece lenses (10X) and objective lenses (3.2X) were used to measure the width of the villi. The lengths of the villi were measured from the tip of the villi to the level of the bottom of the villus crypt (depth). Villus width was defined as the distance from the outer epithelial border to the exterior of the opposite epithelial border along a line passing through the vertical midpoint of the villi. Intestinal morphometry was only performed in female bird samples.
 
Statistical analysis
 
The sample size calculation was carried out using published data of Chicken bodyweight considering a and b values of 0.05 and 0.80, determined on the JMP® 10 program. Productive variables were analyzed using PROC GLM (version 9.3; SAS Ins. Inc., Cary, NC, USA), morphometry data were analyzed using ANOVA. The Tukey-Kramer test was used for specific contrasts in all analyses mentioned above. The normality of the variable and the homogeneity of the variations between the groups were previously evaluated to analyze the results. The criteria for statistical significance were P<0.05.

Effect of garlic on the productive variables of chickens
 
In this study, the potential effect of the diet with concentrate supplemented with different doses of purple garlic on the growth promotion and intestinal morphometry of female and male Cobb Vantress 500 broilers were evaluated. Broilers supplemented for 40 days with 0.2% and 0.3% of garlic reached a weight significative at the end of this period, a weight significative (P<0.05) more significant than that achieved with the other groups, however, there was no significant difference in feed conversion among four groups. The effect of feeding different levels of purple garlic powder on the performance of broilers is shown in Table 1. The productive parameters did not differ significantly (P<0.05) among four groups in females. However, the BWG and TP in the T₂ and T₃ groups in males were considerably higher than the other groups. At the numerical level, the productive parameters: ADG and FCR were better in the T₂ and T₃ groups than in the other groups (Table 1).


       
According to what has been published, the effect of garlic on the productive variables of chickens is variable: Choi et al., (2010), who administered to poultry for 35 days, with 1%, 3%, 5% garlic powder, did not find a significant effect on productive parameters; instead, Javandel et al., (2008) used diets supplemented with garlic flour and only in the highest dose (2%), they observe an increase in feed conversion. With such small amounts of 0.2% and 0.3% of purple garlic, the present study achieved the goal of achieving a higher weight than the groups that did not receive a garlic supplement. In the works of Puvaca et al., (2014), garlic improved the feed conversion rate with 0.5% garlic powder in the diet of broiler chicken. With a dose of 5% garlic, Al-Shuwaili et al., (2015) reported improved live bird weight and feed conversion when administered for eight weeks. On the other hand, Karangiya et al., (2016) concluded that a 1% dose of garlic works as a growth promoter in chickens. With a lower amount of purple garlic from ²Las Pedroñeras², the present work achieved this objective, so a low-medium dose was able to improve the productive variables.
 
The female bird´s body weight
 
Did not differ significantly on any recorded day. However, the body weight in the males of the T2 and T3 groups was significantly higher (P<0.05) on day 40 compared to the other groups (Table 2).


 
The body weight in the male broilers
 
Was significantly lower (P<0.05) in the control and T1 groups than in other groups and the female groups. Chicken genders have been documented to affect glycan and lipid metabolism, related to growth performance and different-sex hormones generally influence metabolic processes (Varlamov et al., 2015). In chickens, like other species, there is an effect of sex on the growth rate; absolute sexual dimorphism is reached in chickens at 14 days of age, from which point its value increases in the males. In this early growth stage, corresponding to the pre-inflection phase of the growth curve, bodyweight shows an exponentially increasing pattern. Male and female broilers have different initial growth potentials; for example, males use food more efficiently than females, from week 5 to 8. This explains why the males reached greater weight at day 40 than the females in groups T₂ and T₃ not so in groups T₀ that before day 40 the males showed a greater weight than the females, however at day 40, they were 6% less heavy than females. In the T1 group from day 2 to 40, the females were heavier than the males.
       
The available information shows that garlic can improve production parameters. However, according to the available literature, the different results obtained with the administration of garlic could be due to a difference in the administered dose, the ecotype of the garlic, the sex of the chicken.
 
Gut morphometry
 
The statistical analysis of the morphometry of the duodenum showed that values of mean villi height were significantly (P<0.05) lower in T₁ and T₂ compared with other groups. The values of duodenum mean villi width were no differences between the groups. The jejunum values of mean villi height in T1 were lower than the other groups, the mean T3 group values were significantly (P<0.05) lower than the T₂ group. (Fig 1). Four experimental groups observed a non-significant difference was observed in the total intestine, duodenum, jejunum and ileum lengths among four experimental groups (Fig 2).



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Duodenum is the first part of the small intestine where 95% of the fat is digested; in the jejunum, the major nutrients are digested and absorbed (Svihus, 2014). In the current study, the statistical analysis of the morphometry showed that the values of the mean height of the villi of the duodenum were significantly (P<0.05) lower in T1 and T2 than in the other groups. Furthermore, the jejunum values of the mean height of the villi in T1 were lower than in the other groups; the mean jejunal villus width values in all treatment groups were significantly (P<0.05) lower than in the control group. Therefore, villi height and width have been positively related to increased nutrient uptake (Oladele et al., 2012) or compensatory mechanism. The morphometry of the villi is reported to change during chick development and the greatest dimension of the villi is observed at 20-22 days (Arce et al., 2008). Unfortunately, in the present study, it was impossible to sample the chickens at this stage and could it be done until the end of the study (40 days) and only carried out in female birds. The present work supports the use of purple garlic from Las Pedroñeras, Spain (standardized commercial product) supplemented in the feed of broilers in a daily concentration of 0.2-0.3% as a natural alternative and viable tool in broilers for growth promotion.
       
The type and presentation of the garlic used may be related to the results obtained. A standardized commercial product of 65% Spanish purple garlic powder was used in the present study. In other studies, garlic bulbs purchased from the local market have been used or supplements. It is recognized that different garlic ecotypes and variations in processing could alter the chemical form of the biologically active components (Dubey et al., 2010; Singh, et al., 2014; Singh, 2015). The garlic ecotype ²Morado de Las Pedroñeras² showed numerous differences with black garlic in the volatile profile, ascorbic acid content, sugar and polyphenols, antioxidant capacity and composition of phenolic acids and flavonoids (Martínez-Casas  et al., 2017). In addition, the highly unstable thiosulfinates, such as allicin (considered responsible for most of the pharmacological activity of crushed raw garlic cloves), disappear during processing and are quickly transformed into various organosulfur components (Lawson and Hunsaker, 2018).
       
As mentioned previously, a factor that potentially influences garlic’s growth performance is gender (Javandel et al., 2008). However, garlic’s exact mechanism of action as a growth promotor is uncertain.

Purple garlic supplemented at a dose rate of 0.2 and 0.3% in the broiler diet has a significant growth promoter in male broilers.

We thank the companies JR SUAREZ MONEDERO S.L. (Zooallium) for the partial financing to carry out this work. We thank CEIEPAv-FMVZ-UNAM, PhD. Pilar Pérez, MSc. Ernesto Ávila and Dr. Arturo Cortes for the support received.