Geographic Location

The current research includes two experiments, both of which had a field phase and a laboratory phase. The field phase was carried out in the Experimental Unit for Feedstock Cattle in the Dry Tropics In the Department of Veterinary Medicine and Zoo technics at the Autonomous University of Sinaloa, located in the fields of Livestock Los Migueles, S.A of C.V., located at 24° 51’ N, 107° 26’ W, 57 masl; average annual temperature is 24.8 °C, maximum temperature 33.3 °C, minimum16.3 °C; the average annual rainfall is 665.6 mm and a dry tropical climate prevails (^{García, 1981}; ^{INEGI, 2009}). The laboratory phase was carried out at the Parasitology Laboratory, Department of of Veterinary Medicine and Animal Science Autonomous University of Sinaloa, in Culiacan, Sinaloa.

All of the calves in the two experiments were treated according to the recommendations in the Guide for the Care and Use of Agricultural Animals in Research and Teaching (FASS, 2010), and with adherence to the official Mexican Normative Guidelines (NOM-051-ZOO-1995; Human treatment in animal mobilization, NOM-062-ZOO-1999. Technical specifications for the production, care and use of laboratory animals).

Experiment 1

This experiment evaluated the influence of the addition of 6 g kg^-1 DM of hydrolysable and condensed tannin extract to the diet of calves at the beginning of their fattening in terms of the level of nematodes. For this, 30 Brahman calves (227 ± SE 13.9 kg) were bought from the semi-mountainous region of Mocorito, Sinaloa, which were then transported 150 km to the experimental site. The calves were fed Sudan hay (Sorghum sudanese) and alfalfa hay (Medicago sativa L.) for five days. On the sixth day after their arrival, the calves were identified with an ear tag, weighed and vaccinated with bacteria to prevent diseases caused by Clostridia, Histophilus somni (Ultrabac7-somubac; Pfizer) and Mannheimia haemolytica (OneShot; Pfizer). In groups of five, the calves were randomly distributed in six stalls with dirt floor (6×12 m) with a 24 m², a lineal feeder bunker of 2.4 m and a waterer of 0.6 m.

The calves were fed with free access only to one diet (Table 1), along with permanent availability to clean and fresh water. The adaptation period was 21 d to: establish social order, familiarity with the installations, adaptation to the experimental hand ling, along with normalizing food consumption, since food consumption is low in feedlot calves when they first arrive to the stockyard, especially in the first 7 d (^{Hutcheson and Cole, 1986}; ^{Fluharty and Loerch, 1996}). There is a less feces excretion and the nematode egg count per gram of feces can be overestimated.

After 21 d of adaptation, the calves were individually weighed and feces samples of each calf were taken during three consecutive days because the females do not oviposit every day and the sample would not reflect with precision the quantity of excreted eggs (Fernández et al., 2008; ^{Silva-Díaz et al., 2015}). The feces were collected at 0600 h directly from each calf’s rectum using a sterile latex glove; they were labeled, and then taken to the laboratory. In the laboratory the number of nematode eggs per gram of feces (EGF) was quantified individually using the McMaster method (^{Sandoval et al., 2011}; ^{Morales et al., 2013}), for which a saturated glucose solution was added to a McMaster tube, then 2 g of feces was added, and after homogenizing by agitation, a gauze filter was inserted to filter the contents of the tube. Afterwards, using a dropper the filtered liquid was obtained and deposited in McMaster chamber. The liquid was allowed to settle for 5 min, at which time the counting of the eggs began using an optical microscope with a 10x objective lens (Carl Zeiss Axiostar, USA). The total number of eggs found in the two sections of the chamber were multiplied by 100, and then divided by 2, and the result was registered as EGF (^{Sandoval et al., 2011}; ^{Morales et al., 2013}).

After this period of adaptation, the treatments were randomly applied in the groups of calves. 1) Control: reception diet without tannin extracts (TE); 2) Control plus 6 g kg^-1 DM of condensed TE (TC); and 3) Control plus 6 g kg^-1 DM de hydrolysable TE (TH). The condensed TE, extracted from the quebracho tree was provided as a stabilized preparation of TE Bypro^® (INDUNOR; Buenos Aires, Argentina), whereas the hydrolysable TE, extracted from chestnut (Castanea sativa), was provided as the stabilized preparation NutriP^® (SilvaFeed; Turin, Italy); both extracts contained 70 % of tannins. The dosis of 6 g kg^-1 DM of TE was selected based on the results from ^{Rivera Mendez et al. (2016)}. The daily dosis of tannins per pen was dissolved in 1 kg of ground maize, which was added to the feed at the moment that it was put in the feeder bunker. The premix of maize and food was manually mixed. In the pens with control group of calves, 1 kg of ground maize was given to homogenize the energy intake in all of the treatments. The treatments were applied during 28 and the calves were weighed on day 1 and on day 28. Three days after the treatments ended, fecal samples were taken from each calf and the same procedure (as mentioned above) was carried out.

Experiment 2

Based on the results from Experiment 1 and according to the concentration of tannins added the diet, along with its effect on the number of nematodes present in feces, a second experiment was conducted in which only TH was used in a dosis that was 2.5 times more, as well as doubling the number of experimental units. The TH dosis was fixed at 15 g kg^-1 DM of the DM, based on the results of the experiment conducted by ^{Krueger et al. (2010)}, who point out that the this TH level did not improve the weight gain nor did it affect the feed consumption, for which it a was considered a safe level for the calves in our experiment.

In this experiment, 40 Brahman (212±SE 8.8 kg) calves were used, bought at the semi-mountainous region of Guamúchil, Sinaloa, and transported to Livestock Los Migueles. The calves were kept in a pen with a dirt floor (27×30 m) with 27 m of a linear feeder bunker made of cement, 4 m of a linear waterer equipped with float ball, and 162 m² of roofing constructed with sheet metal. Upon their arrival, the animals calves were fed with Sudan hay (Sorghum Sudanese) and alfalfa hey (Medicago sativa L.) for 5 d. Fifteen days after the calves’ arrival, they were identified with an ear tag, weighed, and received the same management of preventative medicine as described in Experiment 1. In groups of five, the calves were randomly put into eight pens with dirt floor, (6×12 m) with 24 m² of roofing, a lineal feeder bunker measuring 2.4 m and a watering measuring 0.6 m. The calves were fed free access with the diet described in Table 1. After a 3 d adaptation period in the pens, feces samples (on day 4) were collected for three consecutive days, and the same procedure as described in Experiment 1 was carried out. On day 21 after the arrival, treatments were randomly assigned to the calves: 1) reception diet without TE (control) and 2) reception diet plus 15 g kg^-1 DM of hydrolysable tannin extract (TH).

The source of HT was the same and the treatments were given similar to Experiment 1. The treatments were given during 28 d, with the first treatment being applied on day 1. After the 28th day, feces samples were collected directly from the calves’ rectum during three consecutive days, and the same procedure (as described in Experiment 1) was carried out. The average calculations from those three days were considered as the EGF excretion of each calf. The calves were weighed 6 d before the treatment began and 6 d after concluding the application (40 d in total). The objective of this system of management was to reduce the interference on the calves’ weight by the manipulation during the 3 days in order to obtain feces samples.

Statistical analysis

Because the values of the results of the number of nematode eggs per gram of feces did not present statistical normality, (p≤0.01), these results were normalized before the analysis by their transformation to values of Log₁₀ (n+40) in Experiment 1 and to Log₁₀ (n+17) in Experiment 2, where n represented the observed value. With the results of weight gain and EGF, an ANOVA analysis was carried out for a completely random design (^{Hicks, 1973}). The mathematical model (^{Hicks, 1973}) was: Y _ij =m+t _i +e _ij . The measurements of the treatments were compared using the DMS test (p≤0.10). All of the statistical analysis was done using the version 9 of Statistix^® 9 (2007).

Experiment 1

The gastrointestinal nematode eggs found at the beginning of the experiment correspond to the genera Cooperia sp (100 %), Trichostrongylus sp (71.33 %), Haemonchus sp (63.67 %), Bunostonum sp (6.67 %), Oesophagostomum sp (6.67 %) and Ostertagia sp (3.33 %). The large proportion of Haemochus sp and Cooperia sp verify these genera are indeed dominant in Mexico (^{Olivares et al., 2006}; ^{Fernandéz-Figueroa et al., 2015}). Both are of universal distribution (^{Coles et al., 2006}; ^{De Graef et al., 2013}) and represent a serious problem for cattle production in the U.S. (^{Gasbarre et al., 2009}; ^{Borloo et al., 2013}), South America (^{Borges et al., 2012}; ^{Seo et al., 2015}), Europe (^{Demeler et al., 2009}; De Graef et al., 2013), Africa (^{Pfukenyi et al., 2007}) and Asia (^{Sato et al., 2014}).

Table 2 shows the influence of the feed with tannin extracts on the number of nematode eggs per gram of feces in calves beginning their fattening process in the stockyard, while Table 3 shows the results of weight gain. The decrease in the number of EGF of Haemonchus (p=0.10) due to the inclusion of TH indicates that when the hydrolysable tannins reached the abomasum, the acidic pH that predominates in this organ (^{Brake et al., 2014}) allowed the TH to disassociate from the tannin-protein complexes that would have formed in the rumen (^{Casciola et al., 2009}; ^{Beserra et al., 2011}), which could allow the HT or its products from acid hydrolysis, like ellagic and hexahydroxydiphenic acids (^{Torres et al., 2008}; ^{Vázquez et al., 2012}), join to the cuticle of the Haemonchus that is found in the abomasum, alter its properties and avoid its vital functions (^{Niezen et al., 1995}; ^{Casciola et al., 2009}; ^{Vázquez et al., 2012}).

The absence of the effect of the condensed tannins in the excretion of the Haemonchus eggs can be related to the dosis used in our experiment, even though the anthelmintic effect of the condensed tannins is documented (^{Min and Hart, 2003}; ^{Calderón Quintal et al., 2010}). However, this effect is evident only if grazing ruminants consume the equivalent of 20 g kg^-1 DM of TC in the DM of the diet (^{Butter et al., 2000}), an amount three times the one used in our experiment. The appearance of an apparently higher number of EGF of Haemonchus on day 28 compared with on day one is due to the females’ oviposition between dissimilar days, and as such the values can oscillate between a certain range (Fernández et al., 2008; ^{Silva-Díaz et al., 2015}). In Nellore calves, who are resistant to nematodes, there are oscillations up until four times the initial value in a trial of 105 d (^{Bricarello et al., 2007}).

The excretion of Cooperia was not modified for the addition of the two types of tannins (p=0.60). The absence of the effect of the addition of 6 g kg^-1 MS of TH in the amount of EGF of Cooperia, can be related to the location of this nematode, which is found in the small intestine (^{Borloo et al., 2013}; ^{Karanikola et al., 2015}), contrary to the Haemonchus that is found in the abomasum. The pH conditions that are found in the small intestines of bovines tend to be slightly acidic (^{Brake et al., 2014}); the tannins arrive at this organ and it is probable that the TH and TC from complexes with both foodborne proteins (^{Arévalo, 2008}; ^{Beserra et al., 2011}; ^{Brake et al., 2014}), and enzymes belonging to the digestive tract, and with intestinal epithelial cells (^{Frutos et al., 2004}). Thus, it can be assumed that the amount of TH would be enough to circulate with the components of the Hamonchus cuticle in the abomasum, but not enough to interact with Cooperia in the intestine, and as such the amount of TH included in the diet would not be sufficient for them to manifest, in the event that the effect of the TH on the Cooperia EGF excretion. The absence of the TC effect in the Cooperia EGF excretion, is consistent with the assertion of ^{Butter et al. (2000)}, that 20 g kg^-1 DM of TC is required in the diet in order to reveal the anthelmintic effect; for which the dosis of 6 g kg^-1 used in our experiment in TC and TH would not be sufficient to evidence an anthelmintic activity in the nematodes of the calves’ intestine. The quantity of Trichostrongylus EGF was neither modified for the types of tannins (p = 0.99), and the intestinal location of this parasite (^{Coles et al., 2006}; ^{De Graef et al., 2013}) follows the same type of logic as Cooperia. The excretion date of Oesophagostomum, Bunostonum and Ostertagia EGF were not analyzed because since the beginning of the test they were only present in the Control group and the numbers were very low (2 a 5 EGF); additionally the eggs of these were not found in any of the calves after the application of the treatment.

The final weight (p=0.30) and the weight gain (p=0.86) were not modified by the treatments. The addition of 6 g kg^-1 MS of TH decreased the Haemonchus excretion (p=0.10), by 58%, but did not affect the excretion of eggs of Cooperia (p=0.60), or those of Trichostrongylus (p=0.99), which infested all of the calves, and whose presence would be sufficient to affect the weight gain, which did not happen.

Even though nematode infestation is frequently related to a calf ’s lower ability to gain weight (^{Sutherland and Leathwick, 2011}; ^{Lem et al., 2014}), this effect is not always present in experiments. In Angus stee (initial weight of 196 kg), in feedloct and inoculated with Cooperia and Ostertagia larvae, there was no difference in weight gain between those that were dewormed or not with Tiabendazol, even though the anthelmintic significantly reduced the nematode egg count (^{Ames et al., 1969}). In pastures, there was no difference in weight gain between the Nellore calves with less than 50 Haemonchus and Cooperia eggs per gram of feces (EGF), compared to those with more than 500 EGF (^{Bricarello et al., 2007}); moreover, the level of infestation with nematodes did not change the weight gain in grazing calves (^{Seo et al., 2015}).

Experiment 2

The gastrointestinal nematodes’ eggs found belong to the following genera: Haemonchus sp (82.5 %), Cooperia sp (75 %), Trichostrongylus sp (25 %) and Oesophagostomum sp (15 %). Like Experiment 1, the predominant genera were Haemochus sp and Cooperia sp. Table 4 shows the influence of the addition of TH to the diet on the number of nematode eggs in calves at the beginning of intensive fattening, while Table 5 shows the influence on weight gain.

The addition of 15 g kg^-1 DM of TH during the 28 days decreased Haemonchus EGF (p=0.03) by 61 % and the number of Cooperia EGF (p=0.04) by 68 % when compared to the Control. The use of the dosis of 15 g kg^-1 DM of TH showed the anthelmintic activity of the tannins reported in grazing ruminants (^{Athanasiadou et al., 2005}; ^{Calderon-Quintal et al., 2010}; ^{Quijada et al., 2015}), and that is attributed to the tannin’s capacity to interact with the cuticle proteins of the nematodes interfering in their vital processes (^{Niezen et al., 1995}; ^{Otero e Hidalgo, 2004}; ^{Casciola et al., 2009}; ^{Vázquez et al., 2012}). These results suggest that the increase in the amount of TH in the calves, increased the ability of the tannins to interact with the nematodes both in the abomasum as well as in the small intestine, and they are consistent with the argument that anthelmintic activity of the TH depends on the dosis (^{Bachaya et al., 2009}).

The absence of the TH impact in final weight (p=0.25) and in weight gain (p=0.64) is an expected result, given that ^{Krueger et al. (2010)} did not observed effect on these variables when 15 g kg^-1 DM of TH was provided to heifers in intensive fattening. The positive effect on weight gain in cattle in intensive fattening was only observed when TH concentrations were 6 g kg^-1 DM of MS or less (^{Rivera-Mendez et al., 2016}).

As was already mentioned, a decrease in the number of nematode eggs does not always reflect an increase in weight of fattening cattle, in feedlot or grazing (^{Ames et al., 1969}; ^{Bricarello et al., 2007}; ^{Seo et al., 2015}).