defaunated animals normalized relative to those in faunated animals (1.00)
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The presence or absence of rumen protozoa is correlated with ruminal fermentation characteristics and methane production. Removal of rumen protozoa (defaunation) increases the bacterial population density, the efficiency of bacterial protein synthesis and the rate of nitrogen flow to the duodenum, especially when the feed is deficient in protein relative to energy content. Though carbohydrate digestion of plant cell walls is reduced by defaunation, improving protein supply and livestock productivity together with a reduction in methane production may be more important. However, defaunation of cattle is more difficult than for sheep and this is thought to be attributable to different anatomical structures between the ovine and bovine rumen.
Keywords: cattle, defaunation, protozoa, microbial protein, methane
Protozoa were first discovered by Leeuwenhoek in 1675, and various protozoa were identified as ciliates as their locomotion was made by the means of small hairs or cilia over the surface of the body. Through evolution, as ruminants consumed grass and water, the protozoa established in stomachs of ruminants and adapted to utilise this habitat and the rumen protozoa were first described in 1843 (Hungate 1966). Anaerobic rumen ciliates are extremely abundant, ranging from 104 to 106 cells/mL of rumen liquor and are capable of engulfing bacteria and digesting plant materials such as cellulose and other structural carbohydrates (Finlay and Esteban 2013; Esteban et al 2014).
Rumen protozoa are important, but not essential in the rumen ecosystem and to the well-being of host animals (Williams and Coleman 1992; Newbold et al 2015). Removal of rumen ciliate protozoa (defaunation) has led to reported increases in growth rate and live weight gain of ruminants (Bird and Leng 1978; Bird et al 1979; Eugène et al 2004; Newbold et al 2015) especially when the feed is deficient in protein relative to energy content. In addition, rumen protozoa are significant hydrogen (H2) producers and synthesise mainly acetate and butyrate rather than propionate (Williams and Coleman 1992). Defaunation is therefore expected to induce a greater proportion of propionate in the ruminal volatile fatty acids (VFA; Eugène et al 2004). The reduced methane (CH4) emissions caused by defaunation also reported by several authors (Whitelaw et al 1984; Hegarty 1999; McAllister and Newbold 2008; Newbold et al 2015) may reflect reduced H2 availability by removing endosymbiotic methanogens (Finlay et al 1994; Tokura et al 1997; Finlay and Esteban 2013). This paper aims at reviewing extensive publications related to methods of defaunation of the rumen and compile recent studies and reviews of defaunation impacts on ruminal fermentation, enteric methane production and animal productivity.
Rumen ciliates are not observed in newborn animals, but rumen ciliates are passed from mother to offspring by direct transfer of saliva containing the active protozoa (Stewart et al 1988). Fonty et al (1986) found protozoa appeared in lambs in the following order: Entodinium (15-20 days), Polyplastron, Eudiplodinium (20-25 days) and Isotricha (50 days).
Therefore, rumen ciliate protozoa are not present in animals at birth, enabling protozoa-free animals to be established by separating offspring from their mothers (Ivan et al 1986). Bryant and Small (1960) reared calves isolated from birth which did not have ciliate protozoa until they were inoculated with rumen contents at 24 weeks of age. Eadie and Gill (1971) separated lambs from their dams at 2 days of age and maintained protozoa-free lambs for 61 weeks during the length of the experiment. Dehority (1978) also isolated lambs for almost a year without protozoa until the sheep were inoculated with rumen contents to faunate them. In addition, Hegarty et al (2008) established a flock of ciliate-free lambs born from defaunated ewes and the lambs remained protozoa-free for an extended period of time while grazing. It seems probable young ruminants can be reared free of protozoa when isolated after birth, but it is time-consuming and it does not apply to adult ruminants.
Capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) show strong protozoal toxicity and are useful rumen defaunating agents (Matsumoto et al 1991). Matsumoto et al (1991) observed that rumen protozoa, except Entodinium spp., were undetectable after 3 days of feeding 30 g of hydrated coconut oil (CO) containing 52% lauric acid. Feeding 250g of refined CO to beef heifers reduced rumen protozoal population by 62% (Jordan et al 2006) and protozoal populations in beef heifers were decreased by 63% and 80% by 300 g/d CO after 45 and 75 days, respectively (Lovett et al 2003). Machmüller (2006) reported a reduction in rumen protozoa by 88 and 97% when feeding sheep with 3.5 and 7% CO, respectively. Rumen protozoa were reduced to half of the original population by cottonseed, with holotrich and cellulolytic protozoa apparently lost from the rumen of sheep and only Entodinium spp. remained (Dayani et al 2007). A single drench of readily available vegetable oil (Seng et al 2001; Nhan et al 2007) has delivered short term suppression of rumen protozoa populations leading to productivity advantages. This suggests that short term suppression may be a valuable management tool during periods of high protein demand, such as weaning and lactation when the rumen is normally unable to meet protein animal demand (Ĝrskov and Robinson 1981).
Chemicals administered into the rumen by using an oesophageal tube or through a rumen fistula have been shown to eliminate the rumen protozoa. A chemical dosing method was described by Becker (1929) who fasted goats for three days and at the end of 72 hours, goats were dosed with 50 mL of 2% copper sulphate for two consecutive days. Jouany et al (1988) repeated this method and reported 50% of treated sheep died of copper poisoning and only one sheep was protozoa-free for 93 days. This protocol was therefore considered dangerous and unreliable.
Rumen ciliate protozoa are susceptible to surface-active agents and these agents provide an effective protocol for defaunation of the rumen. In sheep, defaunation has been successful with sodium 1-(2-sulfonatooxyethoxy) dodecane (Bird et al 2008; Hegarty et al 2008; Nguyen et al 2016) or sodium lauryl sulfate (Santra et al 2007a). In cattle, removing protozoa with chemical treatment appears more challenging (Machmüller et al 2003). Diocyl sodium sulfosuccinate (Manoxol OT) used by Abou Akkada et al (1968) and non-ionic surfactants such as nonyl-phenol ethoxylate (Teric GN9) used by Bird and Leng (1978) were not successful in rendering cattle free of ciliate protozoa for a prolonged period of time. Bird and Light (2013) and Nguyen and Hegarty (2017) defaunated the rumen by feeding cattle with coconut oil distillate rich in lauric acid to suppress rumen protozoa for at least 7 days before three days of orally dosing with sodium 1-(2-sulfonatooxyethoxy) dodecane.
Rumen protozoa account for as much as half the total microbial biomass in the rumen and up to 50% of total fermentation products (Williams and Coleman 1992; Newbold et al 2015). Removing protozoa from the rumen, therefore, may result in modifying ruminal digestion of plant cell walls and starch which are considered to be the two main sources of energy supply for ruminants (Jouany and Martin 1997).
The reduced total VFA concentration following defaunation was presented in Figure 1. The lower total VFA concentration in the defaunated rumen could be due to a reduced ruminal digestibility of fibre components of the diet (Newbold et al 2015), leading to a reduced rate of VFA production (Nguyen et al 2015). The absence of rumen protozoa, therefore, can lead to a 5-15% reduction in carbohydrate digestion of plant cell walls (Jouany et al 1988).
The effects of defaunation on the molar proportions of VFA are not entirely consistent within the literature (Williams and Coleman 1992) and it is also seen in this review (Figure 1). An increased molar proportion of propionate in the defaunated rumen was evident in many existing reviews (Jouany et al 1988; Hegarty 1999; Eugène et al 2004). However, other studies showed that butyrate and acetate proportions were generally increased (Machmüller et al 2003; Bird et al 2008) and proportion of propionate generally decreased after defaunation (Machmüller et al 2003; Hegarty et al 2008). A higher proportion of acetate and lower proportion of propionate in the VFA of defaunated animals was also reported when animals were fed low-quality diets (Bird 1982).
Perhaps the greatest consequence of defaunation for the rumen ecosystem and fermentation chemistry is reduced predation of bacteria and an increased bacterial population. This increases microbial protein outflow and in turn increases animal productivity, especially where low protein diets are limiting animal production (Williams and Coleman 1992; Newbold et al 2015). A lower concentration of NH3 in the defaunated rumen was the most consistent effect of defaunation reported in data averaged across studies by Nguyen et al (2015, 2016, 2017, 2018) and in the published reviews (Figure 1). A decrease in rumen NH3 level is a consequence of the absence of protozoa reducing both bacterial predation and the degradation of feed-protein in the rumen (Williams and Coleman 1992). The higher nitrogen flows into the duodenum results from an increase in feed nitrogen and microbial nitrogen flow (Ushida and Jouany 1990), leading to increased supply of amino acids to the host.
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| Figure 1. Rumen metabolite concentration and methane production in the rumen fluid of defaunated animals normalized relative to those in faunated animals (1.00) |
Enteric methane represents a loss of 5 to 7% of gross energy intake, equivalent to a CH4 yield of 16 to 26 g CH4/kg of dry matter consumed (Hristov et al 2013). This review supports the conclusion that the removal of rumen ciliate protozoa reduces CH4 production (Figure 1). Nguyen et al (2018) first measured daily CH4 production and CH4 yield of defaunated and faunated sheep while grazing and reported a tendency towards a lower CH4 yield in defaunated sheep (p = 0.07). However, the percentage emission reduction was variable and mechanisms by which CH4 emissions are reduced by defaunation are not clear. Hegarty (1999) proposed four possible mechanisms by which defaunation induces a lower CH4 emissions, being; (1) reduced DM fermentation in the rumen, (2) decreased endosymbiotic methanogens associated with rumen protozoa, (3) modified ruminal VFA profile with increased molar proportion of propionate and decreased availability of H2, and (4) increased oxygen pressure in rumen fluid.
By removing rumen protozoa, defaunation must eliminate the ecto- and endo-symbiotic habitats for physically associated methanogens (Finlay et al 1994; Tokura et al 1997; Kumar et al 2013). However, as CH4 production is not always decreased by defaunation (Kumar et al 2013), alternative methanogen populations may arise and replace those of the protozoa-associated methanogens (Morgavi et al 2012). The changes in the methanogenic community following defaunation are inconsistent among studies (McAllister and Newbold 2008; Mosoni et al 2011; Morgavi et al 2012; Kumar et al 2013). Reduced CH4 production following defaunation observed may be due to reducing the most active CH4 methanogens in the rumen and the substitution of other methanogenic populations which are less able to utilise H2 to produce CH4. Another possibility is that in the absence of protozoa other populations of microbes establish or increase in the rumen that may enable alternative sinks for H2 that have a higher affinity for H2 than do methanogens (Fonty et al 2007).
The demand for products from livestock, especially in the tropics where forage is often deficient in protein content, is increasing. The requirement to produce at least 70% more food in order to feed 9 billion people by 2050 (World Bank 2008) is a major challenge to animal production. The world livestock population has surged in many developing countries in response to this rapid growing demand for livestock products (FAO 2006). In Asia, the majority of ruminants are fed protein-deficient diets from locally produced and available by-products due to increasing competition for feed between human consumption and monogastric livestock demands (Devendra and Leng 2011).
Defaunation of the rumen offers an opportunity to optimise the productivity of ruminants in such protein-scarce environments. This review demonstrates a consistent effect of defaunation to increase microbial protein outflow which increases protein supply to the host for live weight gain and wool production (Figure 2). A small decrease in DM intake and DM digestibility due to defaunation was not associated with reduced animal growth. A greater microbial protein supply by defaunation may lead to greater efficiency of nutrient utilisation for absorption compared to conventional animals (Newbold et al 2015). These positive effects of defaunation on animal growth are often seen with poor quality roughage diets that are low in fermentable carbohydrate and rumen degradable nitrogen for the growth of rumen microbes (Nguyen et al 2015).
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| Figure 2. Dry matter (DM) intake, digestibility, microbial protein outflow, live weight gain and wool growth of defaunated ruminants normalized relative to those of faunated ruminants (1.00) |
In pen-feeding studies, defaunated lambs showed 18% faster growth rate and greater wool growth and wool fibre diameter over faunated or refaunated lambs offered a 50:50 concentrate and roughage ration (Santra et al 2007b). Birth weight of lambs born from defaunated ewes was 13% heavier than from faunated ewes on single-born lamb and pre-weaning growth rates were 10% and 14% heavier in lambs reared free of ciliate protozoa for both single and twin-born lambs, respectively (Hegarty et al 2008). On high energy and low protein diets, defaunated cattle grew at a 43% faster rate than faunated cattle on the same intake (Bird and Leng 1978) and lambs without rumen protozoa showed significantly increased growth rates and efficiency of utilisation of feed when fed a low level of protein.
In grazing studies, Bird and Leng (1984) observed a greater rate of body weight gain (23%) and wool growth (19%) in defaunated compared to faunated lambs grazed on a green oats pasture. Protozoa-free lambs born from defaunated ewes were significantly (4-8%) heavier than were lambs born from faunated ewes measured from 2 months of age to 5 months of age and wool growth was also greater in protozoa-free lambs grazed on fescue dominant pastures (Hegarty et al 2000). Nguyen et al (2018) reported defaunated sheep showed higher average daily gain (~4%) compared with faunated sheep while grazing, but this was not statistically significant.
Despite the fact that elimination of rumen protozoa has shown potentially positive impacts on improving animal productivity and reducing enteric CH4 emissions from ruminants, there are no defaunation methods that are safe, effective and practically applicable for commercial enterprises.
It seems likely that coconut oils can be used to control protozoa in the rumen, but not completely eliminate all of them. Bird and Light (2013) and Nguyen and Hegarty (2017) fed cattle with coconut oil distillate rich in lauric acid to reduce rumen protozoa concentration before three days of orally dosing with sodium 1-(2-sulfonatooxyethoxy) dodecane. This procedure was reported as successful in defaunating the rumen of cattle. However, the dosing procedure may still require repeating for permanent defaunation. This defaunation technique suppresses animal intake for an average of 10 days, but it is still not clear what period of time is required for the microbial ecosystem to fully stabilise.
The finding of a small but significant protozoal population in the omasum (Nguyen and Hegarty 2019) supports the hypothesis of Towne and Nagaraja (1990) that protozoa residing in the omasum could provide a reservoir of organisms that re-infect the rumen after defaunation treatments have ceased. This presents a commercial challenge to use of oral or in-feed defaunating treatments and suggests an antiprotozoal compound that is carried in blood before diffusing into the gastrointestinal tract may be required to successfully defaunate cattle commercially. Since no such compounds are available, this is a major constraint to defaunation of bovines in the tropical regions where protein deficiency is limiting the productivity of ruminants.
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Received 12 February 2020; Accepted 27 February 2020; Published 1 April 2020