Final Report for GNC12-161
There is a critical need to identify natural anthelmintics for food animal production because of the increased resistance of intestinal parasites to commercial anthelmintics and the inability to use commercial anthelmintics for certified organic food production. Condensed tannins (CT) and flavonoids have been investigated and shown varied efficacy as natural anthelmintics. This research was done to investigate the effects of utilizing by-products of the juice and wine making industries, pomegranate husk (PH) and grape pomace (GP), which both contain these bioactive compounds of interest. An extraction was done on both by-products to determine the concentration of CT available. Pomegranate husk varieties of interest, Parifanka and Desertnyi, contained approximately 1.49 and 2.02% CT on a dry matter (DM) basis, respectively. Grape pomace varieties, Shiraz and Cabernet Sauvignon, contained 4.83 and 3.68% CT, respectively.
In vitro batch culture was conducted in a slope ratio design to determine the effects of the by-products on dry matter degradation (DMD) when compared to and mixed with a control, alfalfa hay. Both varieties of GP had lower (P <0.05) DMD at 96 h with greater than 70% dry matter remaining (DMR), however both varieties of PH had similar digestibilities as alfalfa with approximately 40% DMR. There was an inverse response in DMD when GP was mixed with the ground alfalfa hay; as the proportion of GP to alfalfa increased, the DMD decreased (P < 0.05). Parifanka PH had a DMD similar to alfalfa and did not have a significant effect (P > 0.10) on DMD in mixed ratios. Desertnyi PH was observed to have slightly better digestibility than alfalfa, and the DMD decreased with increasing alfalfa.
In vitro parasitology studies were done on stage three larvae of O. ostetagia using extracts of PH and GP. There were several varieties of PH available, so preliminary studies were done to determine two varieties showing highest efficacy on larvae to use in subsequent studies. The Sogidana and Wonderful varieties were used for the PH, and Shiraz and Cabernet Sauvignon varieties were used for GP. Overall, both PH and GP extracts had approximately twice the number of inactive larvae present (P < 0.05) in the well at 24 h when compared to the control, 32 to 41% inactivity versus 17% inactivity, respectively. Grape pomace extracts had a marginally greater (P < 0.05) efficacy on reducing the viability of the parasites than the pomegranate husk extracts at 24 h when observed at 12.5 mg/mL of crude extract. The PH had a higher extractability than GP was able to reach 50 mg/mL of crude extract. The Wonderful variety of PH had the highest (P < 0.05) efficacy against the parasites when compared to Sogidana at the same concentration and against the control.
An egg hatch and larval development study was done on feces from parasitized lambs from different farming practices (organic versus conventional) in the presence or absence of GP extract (38 g CT/kg DM) to evaluate the effects of GP on egg hatchability and larval development. The GP treatment showed a 100% inhibition (P < 0.05) of egg hatch into developing larvae when compared to the control distilled water treatment.
The data from the research conducted has shown that GP from the wine industry and PH have efficacy against larval helminth stages of GIP and GP also has efficacy against egg hatchability and larval development. The PH and GP could potentially have practical application in becoming a natural anthelmintic for small ruminants, but more in depth studies are needed to verify and finalize application methods.
Much emphasis is being placed on decreasing the use of synthetic drugs in food animal production, and there are a limited number of drugs approved by the Food and Drug Administration for some of the minor species (e.g., goats and sheep). Yet, intestinal parasites are among the primary health risks to the growth and survival of small ruminants. In addition, there is a need on further reducing the need of agricultural animals on human-edible foods while increasing the utilization of recycled agricultural waste or by-products. This project investigates improving animal health and production with use of bioactive based products, which, at the same time, might have social and environmental benefits. By using the by-products from juice and wine-making industries, the amount of waste is reduced, which would reduce the cost of waste disposal by the respective industries. This research gives an opportunity to provide a value-added component to the fruit industry and to improve the health, growth, and efficiency of production of small ruminants. The discoveries made from this research provide an opportunity for internal parasite control of animals in organic production practices. These aspects will aid in providing for a more efficient and affordable food system and improving economic viability of food production systems that can lead to economic benefits for both the plant and animal industries, while reducing dependency on chemical anthelmintics to assist in improving animal health and productivity. Utilizing by-products of the juicing and wine making industry is an area of relevance to Ohio, which ranks 10th in grape production, with about 5,700 tons annually harvested, and for small ruminants, with Ohio ranking 13th in sheep production (state with largest production east of the Mississippi River) with about 128,000 head. Ohio also has about 59,000 head of goats, and farmers are showing growing interest in meat goat production. This project has provided an opportunity for the researchers and farmers/producers to work together to gain experiences that can be taken from the lab and incorporated directly into farm practices.
This research is part of the newly growing interest within the scientific community for determining the potential direct impacts of using pomegranate and grape by-product extracts on parasites and sheep. Preliminary research conducted by the coordinator of this project at Tuskegee University showed there is efficacy of pomegranate husk extracts in vitro against helminth parasites. There were very limited data available, however, to ensure that there would be no negative ruminal effects or to demonstrate in vivo efficacy of either pomegranate or grape by-products. There has been quite a bit of work done to support the use of several plant sources containing condensed tannins, flavonoids, and flavonols as natural sources of anthelmintics with regard to small ruminants; however, extensive information was also lacking within the scientific literature on the potential direct impacts of using pomegranate and grape by-product extracts, which also have these bioactive compounds, on internal parasites of sheep. The goal of this research project was to determine effects of these extracts on ruminal DM degradation and against helminth parasites. The overall hypotheses of this research were that GP and PH would significantly reduce the viability of helminth parasites without causing detrimental effects on DM degradation.
The objectives of the study were:
- Extract and quantify the naturally occurring bioactive compounds in pomegranate husk and grape pomace, as well as determine the effects of these bioactive compounds on ruminal microflora and fermentation
- Determine the effects of pomegranate husk and grape pomace extracts on reducing parasite viability in vitro and to determine if extracts will be detrimental to certain life stages of helminth parasites
Plant source preparation for condensed tannin analysis
Pomegranate husks (PH) were obtained from a grove at USDA ARS University of California, Davis. Wine grape pomace (GP) was obtained from a local Ohio Vineyard (Chalet DeBonné Vineyard, Madison, OH). Plant material was dried to ≥90% DM and ground to pass through a 1-mm sieve to ensure maximum surface area for extraction purposes and to use raw material for in vitro fermentation studies. A crude tannin extraction was done on PH and GP using 70% aqueous acetone plus 1 g/L ascorbic acid to obtain most extractable compounds with minimal oxidation of sensitive constituents (Hagerman et al., 2000). A CT analysis was performed on samples using a modified butanol/HCl procedure (Terrill et al., 1992; Jackson et al., 1996) to determine the concentration of CT in the PH and GP and to decide which two varieties from each plant source would be used for subsequent study. Cabernet Sauvignon and Shiraz varieties were used for GP samples, Parifanka and Desertnyi varieties were used for PH samples, and ground alfalfa pellets were used as a control. Samples of alfalfa, both GP varieties, and a mixture of PH varieties were sent to Cumberland Valley Analytical Services (Hagertown, MD) for nutritional analysis and procedural references are provided in Appendix A. Due to limited amount of PH sample needed for analysis, a mixture of all varieties was used to provide enough material to the lab for the procedures.
Extraction of plant material for parasitology studies
Two varieties each of PH and GP were used. Wonderful and Sogidana varieties were used for PH (USDA-ARS University of California, Davis). Cabernet Sauvignon and Shiraz wine grape varieties were used for GP (Debonne Vineyard, Madison, Ohio). The varieties of pomegranate were chosen due to commercial availability of Wonderful cultivars and previous anthelmintic activity of Sogidana cultivar based on preliminary data collected. The grape varieties were chosen due to seasonal limitations. Each of the plant samples were dried at 55°C and ground to pass through a 1-mm sieve. Twenty five grams of each sample was extracted over 24 h with 100 mL of 70% aqueous ethanol with 1 g/L ascorbic acid to enhance extractability and decrease oxidation of sensitive compounds (Hagerman et al., 2000). Excess dried samples were kept in a desiccator at room temperature until needed and extraction stock solutions were kept at 2 to 4°C until needed. Aliquots (1 mL) of each extract were dried using a micro-rotary evaporator. Residues were weighed to determine extract concentrations of stock solutions and dried extracts were reconstituted in a phosphate buffered saline solution (PBS, 0.05 M NaCl, 5% dimethylsulfoxide, and 1 g/L ascorbic acid). Pomegranate husk extraction samples were diluted to concentrations of 6.25, 12.5, 25, or 50 mg/mL; in contrast, GP extraction samples were diluted to 6.25 or 12.5 mg/mL due to decreased extractability and solubility to achieve higher concentrations. The stock solutions of the PH extracts were 60 to 65 mg crude extract/mL, whereas the GP extracts were only extractable at concentrations ranging between 18 to 25 mg crude extract/mL.
Extraction of grape pomace for fecal culture
Dried GP (1.85 kg, > 95% DM) was finely ground (1-mm sieve) and extracted with 70% aqueous ethanol for 24 h. The extract was then strained to remove all liquid from solid material and the residue was discarded. The ethanol was evaporated for extract (< 5% remaining) using vacuum rotary evaporation and the resulting residue was dissolved in distilled water to give a final concentration of 37.5 mg/mL crude extract. This concentration was comparable to the gram amount of CT assumed to be left in fecal matter of lambs (assuming an estimated digestibility of GP at approximately 17%) from a subsequent study after being administered an experimental GP diet containing 45 g CT/kg DM.
In vitro dry matter degradation study
Rumen fluid and solids were obtained from two cannulated Jersey cows prior to morning feeding at the Waterman Dairy Farm in Columbus, OH (Animal Use Protocol #2010A00000176) for the in vitro batch culture experiment. Experimentation was done using a modified procedure described by Piwonka and Firkins (1993). Liquids and solids were obtained from cannulated cows and blended to get a complete innocula with fiber/particle-associated microbes. The experimentation was performed as a randomized complete block design with blocks being time using a 4 x 4 factorial arrangement of concentration and plant source plus an overall control (alfalfa). The concentrations were based on utilizing a slope ratio technique of mixing the ground PH or GP varieties with ground alfalfa pellets. This technique was used to account for the extra fiber from the PH and GP that may interfere with determining degradation over time of those treatments. The treatments were in ratios of 100:0, 25:75, 50:50, or 75:25% for alfalfa:PH or GP varieties. The treatment and substrate totaling 0.5 g were incubated in triplicate and the entire experiment was repeated twice. All treatments were incubated for 0, 6, 12, 24, 48, or 96 h in a shaking water bath at 39°C. After incubation, samples were analyzed for DM remaining (AOAC, 1980).
Third stage O. ostetagia larvae were collected from fresh fecal culture and double baermannized to get a clean, viable stock culture. Larvae were exsheathed using a modified procedure from von Samson-Himmelstjerna et al. (1998). Larvae were placed in 0.2% sodium hypochlorite in saline for 15 min at 37°C. The larvae were then washed three times in warm saline (37°C), and the exsheathment process was verified microscopically. Approximately 100 larvae per well were added in a 96-well plate containing various concentrations of plant extracts. The treatment structure was a completely randomized block design with a 10x4 factorial arrangement of treatments by variety concentration and time plus a control. The replication of plate was considered as a block. Plates were incubated at 39°C for 0, 2, 4, and 24 h. Larvae were visually assessed using an inverted microscope at a given time interval to determine the number of dead (no activity; larvae completely straight), sick (minimal to no activity; larvae slightly curled or noticeable slow muscle movement), or alive (very active movement or tightly curled). Dead and sick larvae were categorized as inactive, but alive larvae were categorized as active. The larval assay was replicated five times (i.e. five blocks) using five wells per concentration per extract variety.
Adult helminth assay (preliminary trial)
Adult N. brasiliensis worms were collected from mice per the protocol described in Camberis et al. (2003). The selection of this species of adult parasite used was due to the perceived similarity to ruminant Trichostrongyle nematodes (Githori et al., 2006) and the ease of rapid propagation. Cultivated adult worms were kept at 37°C in RPMI-1640 media (Sigma-Aldrich Co., St. Louis, MO) containing 1% antibiotic streptomycin/penicillin at a pH = 7.2. For the assay, only PH extracts were utilized due to limited supply of GP extract. The treatment structure was a completely randomized design with a 2x2 factorial arrangement of treatments for PH extract and concentration plus a control. Both varieties of PH extracts were used at 15 and 30 mg/mL after 50% dilution of concentrated extracts with RPMI-1640 + antibiotic media solution in 24-well plates with the control being just RPMI-1640+antibiotic media solution. Approximately 100 adult worms were incubated in extract overnight at 37°C in 5% CO2 jacketed incubator, then observed and counted microscopically for motility, morbidity, and mortality. Plates were done in duplicate with four wells per concentration per variety.
Feces were collected from sheep with known parasite infestation on both commercial and organic farms. Approximately 300 g of feces was randomly collected off the ground from fresh excrement from each farm (n=5, 3 conventional vs. 2 organic) and kept at ambient temperature in insulated containers so that samples could be cultured fresh within 3 h of collection. The samples from each farm were pooled by individual farm to give a well mixed sample to represent each farming practice. Six subsamples were taken from each pooled farm fecal sample for replicates in fecal culture. The feces were then subjected to a control treatment group with no pomace extract added and to a concentrated pomace extract dose. The experimental procedure for fecal culture was done using a procedure from Hall (1987). A fecal egg count (FEC) was done at the initiation of the experiment to determine the number of eggs per gram (EPG) of feces going into culture. This assisted in determining the hatchability of the eggs that develop into larvae. Thirty grams of fecal matter were placed into a 250-mL disposable polystyrene toxicology jar along with 20 mL of water. The feces and water were mixed well to get a slurry that were lightly mixed with 5 g of vermiculite, making sure not to pack the mixture. The lid was loosely placed on the jars and incubated at ambient temperature for 14 d in a dark area. After incubation, the cultures were exposed to light for 1 h, then the culture jars were filled with warm water (30 °C) and inverted into a Petri dish. The moat that was formed was filled with water and the fecal culture was left to stand for 3 to 8 h until larvae collected in the moat. The liquid plus larvae was then pipetted off and placed in a conical centrifuge tube. The larvae were then counted by serial dilution to determine the number of larvae that hatched for each treatment. One to two drops of Lugol’s iodine was added to each slide before counting larvae microscopically to insure an accurate count.
PROC NLIN procedure of SAS 9.3 (SAS Inst., Cary, IN) was used to estimate the degradation kinetics from the DM data using the model % DMR = C + Be-kt, where DMR is the DM remaining, C is the potentially indigestible DM, B is the potentially digestible DM, k is the rate of DM degradation, and t is time. These estimates were analyzed in the PROC MIXED procedure of SAS 9.3 with LSMEANS to determine treatment estimates using a different model per kinetics term of A, B, C, effective digestibility (ED), or k = treatment. A and ED were calculated from the following formulas: A = 100 – B – C and ED = A + B (kd/kd+kp), where kd was the rate of DM degradation and kp was the passage rate at 0.05/h. Linear, quadratic, and cubic effects were determined utilizing polynomial orthogonal contrasts for equally spaced treatments and the contrasts were used to determine DMD treatment differences. Data were significant at P < 0.05 and tendency at 0.05 < P < 0.10.
For the both larval and adult helminth assay, data from each variety at a given concentration were analyzed as individual treatments. Treatments from the larval assay were analyzed with repeated measures of time using PROC MIXED procedure and heterogeneous compound symmetry covariance structure in SAS 9.3 (SAS Inst., Inc. Cary, NC). The five experimental replicates/blocks for the larval assay were analyzed as a random effect, while treatment and time were analyzed as fixed effects. The PROC MIXED procedure of SAS was used without repeated measure for the adult helminth assay because there was only one time point used (~24 h). For the adult helminth preliminary trial, the data were averaged from the quadruplicate measurements per concentration. The two replications of the adult helminth assay were analyzed as a random effect, while treatment was analyzed as a fixed effect. Treatment differences for both larval and adult helminth assays were compared using Fisher’s LSD by analyzing data with LSMEANS using the DIFF option in SAS. Data were reported significant at P < 0.05 with tendencies reported P ≤ 0.10 and P > 0.10 was considered non-significant.
The fecal culture data were analyzed statistically using the PROC MIXED procedure in SAS 9.3 (SAS Inst., Cary, NC, 2011) to determine difference for treatments, farm, farming practices and potential interactions. Farming practice and treatment were analyzed as fixed effects and farm was analyzed as a random effect. The interaction of farm x farming practice and treatment x farm x farming practice were also analyzed as random effects. Treatment differences were compared using Fisher’s LSD by using LSMEANS with the DIFF option in SAS. Statistical significance was reported if P ≤ 0.05, with trends being reported if 0.05 ≤ P ≤ 0.10.
In vitro dry matter degradation study
GP had 92 % DM, but PH was lower at 83.7%. The concentrations of CT (DM basis) in the samples were 1.49, 2.02, 3.68 and 4.83% for Parifanka PH, Desertnyi PH, Cabernet Sauvignon GP, and Shiraz GP, respectively. Alfalfa was used as a negative control in the in vitro DM degradation study and had no detectable CT present.
The estimated DM degradation kinetics are provided in Tables 1, 2, 3, 4 and 5 for the A pool, B pool, C pool, k, and ED, respectively. Both PH treatments contained more (P < 0.05) readily degradable DM (A pool) than the other treatments, even when mixed with alfalfa. The PH varieties had a linear increase (P < 0.05) in the A pool as the portion of PH increased in the samples, whereas Cabernet Sauvignon GP tended to have a linear decrease (P = 0.06) and Shiraz GP numerically followed a similar trend (P = 0.11) as the Cabernet Sauvignon GP as the portion of GP increased in the samples (Table 1). The higher A pool for PH samples is not surprising when the non-fiber carbohydrates in the samples were twice to nearly three times as much than the GP or alfalfa samples. These carbohydrates would be readily degradable by rumen microbes to be used for energy for their growth.
In all samples, expect Parifanka PH (P > 0.10), there was a linear decrease (P < 0.05) in the B pool with greater inclusion of PH or GP in the samples. Shiraz GP had a cubic or skewed decrease (P < 0.05) with Cabernet Sauvignon having a tendency (P = 0.10) for a cubic effect due to the rapid drop in the B pool at inclusion of 100% GP (Table 2). The decrease observed in the GP for the B pool is most likely due to the low degradability associated with GP. GP is a pre-fermented by-product of the wine-making industry so there would be an expectation of a smaller B pool available for DM degradation by rumen microbes. The decrease in B pool for PH is also expected when there is a smaller amount of NDF present in PH samples at 13.5% NDF (DM basis) compared to 38.2% for alfalfa, 54.2% for Shiraz GP, and 43.1% for Cabernet Sauvignon GP (Table not shown).
The GP varieties contained higher (P < 0.05) portions of indigestible DM (C pool) than all other treatments and the C pool linearly increased (P < 0.01) with greater inclusion in the samples. There was no significant effect (P > 0.10) observed with the Parifanka PH, but a cubic effect (P < 0.05) with a tendency (P = 0.10) for a quadratic effect was observed for Desertnyi PH. There was an increase in the C pool as Desertnyi PH was added to the sample until it was included at 75% in which the C pool decreased (Table 3). There is not a clear explanation as to why a skewed increase in the C pool was observed, except the possibility that the C pool was over estimated in SAS from the DMD data. Calculated values of the C pool for Desertnyi PH show that the C pool should have followed a more linear decrease as Desertnyi PH was included in the sample at 25% increments (25% DPH - 40.2%, 50% DPH – 39.7%, 75% DPH – 39.3%) instead of the estimated values from SAS.
There was a cubic effect (P < 0.05) observed for Cabernet GP and Parifanka PH with a tendency (P = 0.06) for Desertnyi PH to have a cubic effect. The cubic or skewed effect of treatment was observed by increases in the rate with 25% inclusion of PH or GP in the sample that decreased drastically with greater inclusion but increased slightly by 100% of PH or GP (Table 4).
The ED values give a better idea of the effects of adding PH or GP to the samples by allowing an estimation of the percent digestibility in the rumen assuming a constant passage rate of 5%/h (Table 5). GP had a negative associative effect on alfalfa DMD that tended to have a linear decrease (P = 0.07) in DMD for Shiraz GP and DMD was linearly decreased (P < 0.05) for Cabernet Sauvignon GP, but when comparing with calculated values for the ratios (data not shown) the alfalfa inclusion added a positive associative effect to GP samples when included up to 50% of the sample for Shiraz GP only. On the other hand, PH had an overall positive associate effect on alfalfa DMD that tended to increase linearly (P = 0.09) and cubically (P = 0.06) for Desertnyi PH and Parifanka PH had a numerically similar trend (P = 0.12) for increasing DMD linearly and cubically (Table 5).
GP had lower degradability observed by lower A and B pools and higher C pool than the other samples, but this was likely due to high levels of lignin associated with the fiber present in the skin, seeds, and stems (Baumgärtel et al., 2007). The Cabernet Sauvignon GP was reported to have an average DM digestibility of 36.8% and to only slightly decrease DM digestibility of alfalfa by 1.6% when Cabernet Sauvignon GP was added at 50% of the diet (Famuyiwa and Ough, 1982), but our research does not support that conclusion. The DM degradability of Cabernet Sauvignon GP was far less than 36.8% (21.3%), and at 50:50 with alfalfa, DM degradability was still lower at 28.6%. In a later study done by Famuyiwa and Ough (1990), they reported the lower digestibility of Cabernet Sauvignon GP as it related to alfalfa/grain mixtures was due to an increased percentage of indigestible cell wall material in the GP samples. They observed Cabernet Sauvignon GP had a cell wall digestibility of 4.1% compared to the 33.6% cell wall digestibility of alfalfa/grain samples (Famuyiwa and Ough, 1990). This would support why there was a decrease in DM degradability with greater inclusion of the GP in samples observed in the ED values (Table 5).
On the other hand, Besharati and Taghizadeh (2009) observed that DM digestibility decreased with increasing GP in diets mixed with alfalfa. The results from our study support their findings. Adding more than 20% GP to a ruminant diet may indeed increase some detrimental effects on DM digestibility, but supplementing at low amounts, such as 5 to 10 % GP in the diet, can lead to beneficial effects (Bahrami et al., 2010).
The PH samples had similar to higher DM digestibility compared to alfalfa and helped to support the results of Shabatay et al. (2008) whereby PH did not alter DM digestibility negatively when incorporated with other feedstuffs for ruminants. Our hypothesis that the DM digestibility would not be negatively compromised, was proven for the PH only but was rejected for GP at concentrations tested. With the inclusion of alfalfa with GP (up to 50%) in diets, GP could assist in countering some of the decreased degradability observed in this trial.
Parasitological in vitro study
Treatment, time, and the interaction were significant (P < 0.01) in the larval assay, so the data were analyzed for differences by treatment within a time point (Table 6). At the beginning of the assay, both pomegranate varieties were different (P < 0.05) from the wine grape pomace except for the lowest concentration of Shiraz. Both the Wonderful and Sogidana varieties started with lower activity than GP varieties or the control which may suggest there is an initial paralysis or death that occurs within minutes of contact with the extracts. By 2 h, this difference between extracts became more obvious with the Shiraz GP having the highest activity and both varieties of PH at 50 mg/mL having the lowest activity (P < 0.05). The GP treatments, regardless of concentration, were not different (P > 0.10) from each other. The PH treatments for both varieties at 12.5 mg/mL were not different (P > 0.10) from the Cabernet Sauvignon GP at concentrations of 6.25 and 12.5 mg/mL. All treatments were lower (P < 0.01) in activity than the control at 2 h. At 4 h, the Shiraz GP continued to have the highest activity (P < 0.05) among the extracts at 38%, and there was no difference observed between concentrations; whereas, the Cabernet Sauvignon had a lower activity (P < 0.05) and was the most effective among the GP varieties, regardless of concentration. There was no difference (P > 0.10) observed between the Cabernet Sauvignon GP and the PH varieties at 12.5 and 25 mg/mL. The PH varieties at 50 mg/mL had the lowest activity (P < 0.05) and best efficacy, but there was no difference (P > 0.10) between PH varieties. All extracts continued to be different (P < 0.01) from the control, which only decreased in activity 16.6% within 24 h. There was no difference (P > 0.10) in activity of extracts between 4 and 24 h but there was a tendency for difference (P = 0.08), which was mostly observed in the Cabernet Sauvignon GP, Sogidana PH, and control. There being no difference in 4 or 24 h was indicative that whatever efficacy was going to be observed, it would take place within 4 h of exposure to the extracts. The interaction of treatment by time could be explained that there was greater efficacy of decreasing larvae viability or health with longer exposure (up to 4 h) of the larvae to the extract; however, there was a slight increase in activity by 24 h, which raised the question if there was some resistance occurring to the extracts or what was the actual mechanism to which the extracts were causing decreased activity. Many of the compounds thought to be in the crude extract of both GP and PH (i.e. flavonoids, flavonols, pelletierine, and CT) has been thought to cause paralysis to helminth parasites initially followed by death, if death occurs (Wibaut and Holstein, 1957; Athanasiadou et al., 2001; Aas, 2003; Min and Hart, 2003; Nguyen et al., 2005; Kerboeuf et al., 2008).
Table 7 shows the preliminary results from the adult helminth assay conducted on N. brasiliensis. PH decreased activity (P < 0.01) of the adults when compared to the control but there were no differences (P > 0.10) for concentration or variety. But once again, it is not clear whether this was just due to paralysis and effects would be reversed once helminths were no longer in the presence of the extracts or if this decreased activity was due to death of the parasite. The main obstacle to this assay was the viability of the helminths decreased, even in the control well, but still not as drastically as the treatment wells. This was thought to be due to adult helminth parasites being more sensitive to in vitro conditions and not as suitable for studies outside the host. In addition to this, N. brasiliensis has a corkscrew or tightly coiled morphology and they like to adhere to each other, so dividing them individually into wells was very labor intensive and caused some of the adults to be lost due to physical damage and were removed from the treatment wells.
The larval studies were a good indication that the extracts of both PH and GP could have potential use in decreasing activity of helminth larvae. Both of the PH extracts resulted in the best efficacy at 50 mg/mL. If GP can be extracted to a similar concentration, it is possible for there to be greater efficacy than the pomegranate when such low concentrations still have an effect. For the adult helminth parasite assays, there still needs to be more experimentation done to improve the assays and the parameters need to be further worked out to improve viability of the adults for N. brasiliensis. If the both PH and GP extracts are able to decrease larval viability and PH is able to decrease adult activity, then it may be possible to decrease transmission of the infestation by potentially decreasing infective larval stages and also decreasing infestation by allowing paralyzed or dead adults and larvae to be passed out of the host, but this must be tested in further experimentation. Overall, the PH and GP extracts show promise in helping to reduce helminth infestation for larvae and PH in reducing viability of adult helminths, but more studies need to be conducted for adult parasites. The trial results proved that our hypothesis that the PH and GP extracts would decrease viability and increase death in larval helminth stages when compared to the negative control of no treatment.
In all culture jars for the GP extract, there was 100% inhibition of egg hatch into larvae (P < 0.01) (Data not shown). On the contrary, in all of the control jars that did not receive treatment, there was an abundance of larvae present. There were more larvae counted than the number of eggs estimated at the beginning of the experiment. This result is to be expected because there is no way of counting all eggs present in fecal samples, no matter how well mixed the sample can be. This is why most FEC are estimates of the average number of EPG of feces and is usually why more than one count is done.
There was no difference (P > 0.10) in farm or farming practice, meaning that neither farming practice nor farm had any influence on the treatment being administered or alternatively the treatment was effective in prohibiting larval development, regardless of farming practices on a particular farm. There was also no significant interaction observed between farming practice and the treatment. This was a different result than what was hypothesized in the beginning of the experiment because it was thought that due to the resistance being observed to chemical anthelmintic therapies on conventional farms; it was assumed there would be some resistance observed from these farms against the GP extracts, but this was not the case. Regardless of resistance observed on conventional farms, the extract still inhibited parasite egg hatch and larval development. This result could be attributed all the bioactive compounds present in the GP, such as CT and flavonoids, which are considered to be the most abundant polyphenol in the GP extracts (Lu and Yeap Foo, 1999). Molan et al. (2003) observed 100% inhibition of egg hatchability at concentrations of 1 mg/mL against T. colubriformis parasite eggs and effectively inhibited L3 larval migration at concentrations of 500 µg/mL. The extracts showing the most efficacies against these parasites were the flavan-3-ol galloyl derivatives, with epigallocatchin (a CT) being the most active in both egg hatch and larval development inhibition assays (Molan et al., 2003). The combination of bioactive compounds in the crude extract of GP seems to make it very effective in controlling GIP transmission through contaminated feces.
This surprising result of the GP extract showing efficacy in both farming methods allows for implications that the treatment could be used in feed to help with integrated pest management of decreasing parasite burden in farm animals. There is still further research that needs to be done to validate this implication before it can be fully put into practice, but it provides promise for natural means to help reduce GIP in agricultural animals on both organic and conventional farms.
Educational & Outreach Activities
The research conducted for this project was presented at the 35th Annual OEFFA conference in Granville, OH. This conference is Ohio’s largest sustainable food and farm conference featuring keynote speakers and several presenters on sustainable agriculture research, outreach, and programs for farmers, extension agents, and educators. A video recording of this presentation is available through NCR-SARE's YouTube channel at: https://youtu.be/gdpQWoPM8kU
The data from the research conducted has shown that grape pomace, a by-product from the wine industry, and pomegranate husk has efficacy and could potentially have practical application in becoming a natural anthelmintic for small ruminants but more in depth studies need to be conducted to verify and finalized application parameters. Pomegranate husk has shown similar digestibility kinetics as alfalfa and could potential be used as a new feedstuff that would add extra benefits of helping control gastrointestinal parasitism in ruminants. Grape pomace fermentation experiments, on the other hand, have shown that inclusion in the diet greater than 25% can lead to decreased digestibility of the diet. Overall, there is potential for added health benefits for both by-products in ruminant diets.
Due to the preliminary nature of this research, there was no farmer adoption of any of the methods presented in this work. However, there were several farmers and extension agents at the conference and during the project that expressed interest in wanting more information to potentially use grape pomace on farm for helping in controlling parasite transmission among their farm animals. This shows implication that there is a increasing interests in implementing certain aspect of this research in actual on-farm practices once more conclusive animal trials can be completed.
Areas needing additional study
There are several aspects of this research that need additional study. Conducting a protein binding capacity assay on the grape pomace and pomegranate husk would be beneficial because CT are known to bind to protein and assessing the binding capacity of the tannins in both GP and PH would help better understand if they are any effects on the digestibility of protein in the feed.
The in vitro parasite study examining effects of the PH and GP extracts provides promise but needs to be expanded. The extracts should be fractionated and tested for efficacy to determine what compound(s) is responsible for observed efficacy in decreasing viability of the parasites or if efficacy is best observed from crude extraction. Extracts should be tested to determine if the bioactive compounds in the extracts are killing the parasites or just paralyzing them. Studies should be designed to observe the same parasites when the extract is present and then once the extract is removed. This will determine if the extract is –cidial or –static, which either way could be beneficial to host assisted parasite removal. It would also be beneficial to examine effects of extracts on other economically important parasites, such as Haemonchus contortus.
There should also be additional studies conducted regarding the fecal culture to determine whether the GP is actually inactivating eggs and killing the parasite larvae or if it is just inhibiting growth. Learning more about the mechanism of action in the fecal culture on the eggs and developing larvae would assist in understanding how to use grape pomace or its extracts as a way to break the transmission cycle of certain parasites.
The next step in this project should be to conduct animal trials to determine if the by-products will work in an animal model as effective as or better than the in vitro parasitology models. The only drawback is to formulate a feed that would have the proper concentration of condensed tannin and flavonoids while providing optimum nutrition for parasitized lambs.