REPRODUCTIVE EFFICIENCY OF NELLORE ( BOS INDICUS ) COWS SUBJECT TO BOTH FTAI AND HOMEOPATHIC SUPPLEMENTATION

The aim of this work was to determine the effect of homeopathic supplementation on both ovarian dynamics and conception rate in Nellore cows subjected to fixed-time artificial insemination (FTAI). Cows (n = 150) were randomly distributed to the control (CG) and the homeopathy group (HG). The HG cows were supplemented with Pró-cio in the mineral salt for 60 days and both experimental groups were further subjected to FTAI. Cows were evaluated for ovarian dynamics (n = 16), progesterone (P4) concentration (n = 16), and conception rates (n = 150). Ovarian dynamics determined by ultrasonography and showed similar findings for CG and HG, respectively. Thus follicular diameter (8.7 ± 1.0 mm vs. 10.0 ± 0.8 mm), mean pre-ovulatory follicle volume (0.46 ± 0.15 mL vs. 0.61 ± 0.12 mL), and mean follicular growth (3.65 ± 1.41 mm vs. 4.60 ± 1.21 mm) did not differ between groups. Moreover, corpus luteum diameter was similar between groups (CG: 16.28 ± 0.7 mm vs. HG: 15.6 ± 0.8 mm; P > 0.05), although P4 levels did differ (CG: 2.55 ± 0.85 ng mL vs. HG: 6.52 ± 1.19 ng mL; P < 0.05). The conception rate after FTAI was not affected by homeopathic supplementation (CG: 74.67 %, and did HG: 77.33 %; P > 0.05). In conclusion, the homeopathic supplementation Pró-cio increases P4 concentrations but does improve the reproductive efficiency of Nellore cows subject to FTAI.


INTRODUCTION
The efficiency of cattle production systems is directly related to its adequate nutritional, sanitary, and reproductive management (FERREIRA-SILVA et al., 2017;TATCHER, 2017).The application of assistedreproduction technologies increases the overall herd genetic merit by delivering more offspring from the most productive animals (HANSEN, 2014).For such approaches, the control of estrous cycles is paramount and is typically attained by hormone-based protocols (BISINOTTO et al., 2014;STEVENSON;BRITT, 2017).
There is a growing necessity worldwide to increase the fertility rates of commercial beef cattle herds, concomitant with the need for reducing costs while diminishing the utilization of hormone-based protocols.Therefore, the aim of this work was to evaluate the ovarian dynamics and conception rates in Nellore cows subjected to both homeopathic supplementation with Pró-Cio ® and fixed-time artificial insemination (FTAI).

Location
Experiments were carried out in Currais, Piauí state, Brazil.The geographic coordinates of the farm are latitude 09º 00' 25" South and longitude 44º 24' 39'' West.The altitude is 320 m, with a mean annual temperature of 26.4 °C, and mean annual rainfall of 996 mm 3 .Animals were raised in cultivated pastures (i.e., Brachiaria brizantha and Andropogon gayanus) and had free access to water and mineralized salt.

Animal Selection and Management
Multiparous Nellore (Bos indicus) cows used had aged from 3 to 5 years, had delivered within 60 days, and displayed a body condition score between 2.5 and 3.5 in a 1 to 5 scale, as described by BROSTER and BROSTER (1998).The cows were subject to clinical and gynecological examination as recommended by GRUNERT and BIRGEL (1982).Based on these criteria, selected cows (n = 150) were randomly allocated to experimental groups (i.e., Control Group -CG and Homeopathy Group -HG).
The homeopathic supplement (Pró-Cio ® ; Real H Nutrição e Saúde Animal) was added to the mineralized salt (20 g per animal each day) to HG cows throughout the experiment (60 days).After the initial 30 days of homeopathic supplementation (adaptation period), cows were subject to an FTAI protocol.
On day 0 (D0), cows of both groups received an intravaginal device (Sincrogest ® ; Ourofino Agronegócio) containing 1 g progesterone and 2mg estradiol benzoate (Sincrodiol ® ; Ourofino Agronegócio) by an intramuscular (IM) shot.On day 8 (D8), cows were treated with 500 IU (IM) equine chorionic gonadotropin (Sincro eCG ® ; Ourofino Agronegócio), 500 µg Cloprostenol (Cloprostenol Sódico; Sincrocio ® ; Ourofino Agronegócio), and 1.0 mg EB at the moment of intravaginal device removal.Forty-four hours after intravaginal implant removal, the FTAI was carried out with frozen-thawed semen from bulls of proven field fertility (semen successfully used for FTAI).The conception rate was determined 40 days after FTAI, as previously described by Pereira et al. (2016).The conception rate was calculated by dividing the total number of pregnant cows by the total number of cows subject to FTAI and further multiplied by 100. Figure 1 is a schematic representation of the experimental design of the study.

Ovarian dynamics
The ovarian dynamics was carried out by ultrasonography.One technician performed all analysis using a single device (Sono Vet R3, Madison) equipped with a linear transducer (7.5 MHz).The ovarian activity analysis was performed in eight cows of each group, as previously described (SILVA FILHO et al., 2015).The traits that were analyzed: number of follicles, mean diameter (vertical and horizontal mean diameters) of the dominant follicle, codominant and pre-ovulatory follicles, follicular volume, and further corpus luteum diameter (if found).The exams were carried out at the onset of homeopathic supplementation and in sevenday intervals afterward until estrous synchronization for FTAI.During synchronization, the examinations were carried out on days D0, D4, D8, and D10 and ten days after the FTAI protocol (for corpus luteum measurement).

Progesterone Analysis
Blood samples were obtained by jugular vein puncture with a 21 G needle into heparincontaining tubes (Becton Dickinson, Rutherford, NJ).The blood was centrifuged (2,500 g for 10 minutes) for plasma retrieval and stored at -20 °C.The determination of progesterone (P4) concentration was performed by chemiluminescence (Coat-a-Count; Diagnostics Products Corporation).The coefficient of variation of the test was 4.25 %, and the minimum detectable level was 0.08 ng mL -1 .

Statistical Analysis
The data generated during each exam was analyzed using the SPSS 16.0.Initially, the data was subject to both critical and consistency analysis (frequencies, distributions, and homogeneity).Moreover, pre-ovulatory follicle diameter and volume, follicular growth between D4 and the day of FTAI, the quantity of recruited follicles on D4, and corpus luteum diameter 10 days after FTAI were determined by analysis of variance (ANOVA).The pregnancy rate and frequency of cows with a new follicular wave were evaluated by the chi-square test.The significance level was 5 %.

RESULTS
Table 1 contains the data relative to the ovarian activity of HG and CG cows.It can be observed that mean POF diameter, mean POF volume, follicular growth (between D4 and D10, and FTAI day), and mean corpus luteum diameter after FTAI did not differ between groups (P > 0.05; Table 1).No statistical difference was found between groups (P > 0.05).
The distribution of ovarian follicles according to their size is shown below (Figure 2).There was no difference (P > 0.05) between groups for follicle size interval.Moreover, mean follicular growth during the FTAI protocol was also determined (Figure 3).  Figure 4 displays the data relative to mean corpus luteum (CL) diameter and P4 concentration in both HG and the CG at day 10 post-FTAI.No difference was found (P > 0.05) between groups for CL diameter, but P4 levels were higher (P < 0.05) on the HG than in the CG.The conception rate the HG was 77.33 % (58/75) and 74.67 % (56/75) in the CG, with no difference (P > 0.05) between groups.

DISCUSSION
The diameter of pre-ovulatory follicles correlates with ovulation potential (SARTORI et al., 2001;GIMENES et al., 2008), with CL diameter (VASCONCELOS et al., 2001), and ultimately with conception rate (PERRY et al., 2007).Collectively, these parameters interplay to establish cow fertility and thus lead to determine herd overall reproductive performance.
The data described above showed that homeopathic treatment with Pró-cio ® did not lead to increased follicular development.Therefore, it can be ruled out that the protocol does not induce affect the cyclicity status.This reasoning is not supported by findings from RAJKUMAR et al. (2006), who found that a homeopathic supplement could be used for reverting cow anestrus.The increased estradiol concentration could be due to the addition of different plants (i.e., Pulsatilla spp.and Aletris farinosa) to this homeopathic supplement (BOERICKE, 2001;DUKE et al., 2002).In buffaloes, combinations of homeopathic products also reduced anestrus incidence (KUMAR et al., 2004).
Pre-ovulatory follicles in HG cows reached 7.5 mm or more, while follicles from some animals had reached 12 mm.This fact was suggestive that conception rates would be increased by improving ovulation rates, since dominant follicle dilation in Bos indicus occurs when reaching 7 mm (GIMENES et al., 2008).This reasoning is also supported by the relationship between pre-ovulatory follicle size, CL diameter and the P4 production potential.Within smaller follicle sizes, the resulting lower P4 concentration would not enough for blocking prostaglandin synthesis by the endometrium (VASCONCELOS et al., 2001) and thus inhibit premature luteolysis (BRIDGES et al., 2005).
The number of follicles that were recruited on D4 was not increased by the homeopathic supplementation.Comparison with previous data is somewhat difficult due to the varying methodological approaches and definition of minimum antral follicle size (FERREIRA et al., 2004;STARBUCK-CLEMMER et al., 2007;SÁ FILHO et al., 2008).However, some reports have pointed out to differences in reproductive physiology between Bos indicus and Bos taurus (i.e., mean recruited follicles and dominant follicle size).Such differences could be due to some physiological parameters, such as higher IGF-I and insulin serum concentrations and elevated FSH-responsiveness (SILVA-SANTOS et al., 2011;SARTORI;BARROS, 2011).
The follicle size in HG was higher than the CG cows, but CL was indistinguishable between groups.More recent reports have shown that the P4 production is not exclusively dependent on CL diameter, but is also influenced by its vasculature.In Bos indicus cows, the estrus P4 concentration is approximately 1 ng mL -1 but can reach 4.5 ng mL -1 ten days later (ADEYEMO;HEATH, 1980).Since P4 concentrations differed between groups, independently of CL diameter, other factors (e.g., blood flow fluctuations, the presence of luteotropic or luteolytic factors, and different availability of P4 precursors) may have accounted for such difference (WILTBANK, 1994).
Conception rates were not affected by the homeopathic supplementation.This result may be context-dependent or breed-dependent since Penteado et al. (2005) found the homeopathic product may increase cyclicity and pregnancy rates in dairy cattle.Another important factor is that eCG favors pregnancy rates after FTAI in postpartum cows (BARUSELLI et al., 2004) and thus may have masked the beneficial effect of homeopathic supplementation.Another applausive concern is that some animals may not have ingested enough homeopathic supplement during the experiment.However, this fact can be ruled out due to two reasons.Firstly, cows subjected to homeopathic supplementation displayed higher P4 levels, suggesting a physiological response from all cows under such conditions.Secondly, supplementation of mineral sat with different compounds influences reproductive traits in both cows and bulls (JUCHEM et al., 2010;REIS et al., 2014).
In conclusion, the homeopathic supplementation Pró-cio increases P4 concentrations but does improve the reproductive efficiency of Nellore cows subject to FTAI.
Company for providing the homeopathic product, to CNPq for the Master's degree scholarship (PFMPS), and to Pasqual Rotilli for providing the animals and the farm instructor for the study.M.T.M. is a PNPD-CAPES fellow.

Figure 3 .
Figure 3. Follicular growth during the FTAI protocol (from day 4 until day 10) in Nellore cows (Bos indicus) under homeopathic supplementation with Pró-Cio (HG) or the control group (CG).

Table 1 .
Mean values ± SEM for recruited follicles on D4, diameter and volume of the pre-ovulatory follicle on D10, follicular growth between D4 and D10, and corpus luteum diameter at day 10 post-IATF in Nellore cows under homeopathic supplementation with Pró-Cio (HG) and the control group (CG).