Effects of Diet and Hindgut Defaunation on Diet ...

Effects of diet and hindgut defaunation on diet digestibility and microbial concentrations in the cecum and colon of the horse B. E. Moore and B. A. Dehority J ANIM SCI 1993, 71:3350-3358.

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Effects of Diet and Hindgut Defaunation on Diet Digestibility and Microbial Concentrations in the Cecum and Colon of the Horse' B. E. Moore2 and B. A. Dehority3 Department of Animal Science, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster 44691-4096

ABSTRACT:

The effects of diet and hindgut defaunation (removal of protozoa from the hindgut) on diet digestibility (Trial 1) and on total and cellulolytic bacterial and fungal concentrations in the cecum and colon (Trial 2 ) were investigated. A highforage ( H F ) diet, 90% alfalfa hay-10% concentrate, or a higher-concentrate ( H C ) diet, 60% alfalfa hay-40% concentrate, was limit-fed. In Trial 1, defaunation resulted in a slight decrease in DM digestibility ( P < . l ) and had no effect on cellulose digestibility. Dry matter digestibility was higher ( P < .001) with the HC diet; however, no differences were observed in cellulose digestion. For the faunated periods, protozoal concentrations were similar in the cecum and greater in the colon for both diets ( P < .05). A diet x location

interaction was observed for the genera Buetschlia and Blepharocorys. In Trial 2, defaunation had no effect on either total or cellulolytic bacterial concentrations in the cecum or colon. Total bacterial concentrations were higher ( P < .06) in the colon when ponies were fed the HC diet. Defaunation did not affect total fungal concentrations in the cecum; however, fungal concentrations in the colon were slightly higher ( P < .1) when the ponies were defaunated. Diet had no effect on total or cellulolytic fungal concentrations. Both total and cellulolytic fungal concentrations were approximately 10-fold higher in the colon than in the cecum ( P < .01). Protozoa do not seem to play an essential role in the fermentation of feedstuffs in the equine hindgut.

Key Words: Equine, Protozoa, Fungi, Bacteria, Digestion, Hindgut

J. Anim. Sci. 1993. 71:3350-3358

Introduction Although numerous studies have been conducted to determine the contribution of protozoa t o digestion in the rumen (Hobson and Jouany, 1988; Williams and Coleman, 19881, very few studies have investigated the role of protozoa in equine hindgut digestion. Their role may differ from that of the ruminal protozoa, because the ciliates that live in the cecum and colon of the horse are primarily members of families different from those of ciliates that occur in the rumen. Nutrients available to these hindgut protozoa are also different, as a result of an initial digestion of feedstuffs in the foregut. With different feeds, the substrates available for fermentation would be expected to change.

ISalaries and research support provided by state and federal funds appropriated to the Ohio Agric. Res. and Dev. Center, The Ohio State Univ., manuscript no. 3-93. aPresent address: Alltech, Inc., 3031 Catnip Hill Pike, Nicholasville, KY 40356. 3T0 whom correspondence should be addressed. Received January 15, 1993. Accepted July 21, 1993.

Very little is known about the interrelationships between the different microbial populations in the equine hindgut. The influence of diet on bacterial and protozoal populations has been investigated to a limited degree (Kern et al., 1973; Goodson et al., 1988), but dietary effects on fungi are unknown. Possible competition for substrates among these populations in the equine hindgut has not been investigated to date. The objectives of the present study were 1) to determine the effect of defaunation on digestion of DM and cellulose in the pony hindgut, using a high-forage or a higher-concentrate diet and 2 ) to evaluate the effect of these two variables, defaunation and diet, on total and cellulolytic bacterial and fungal concentrations in the cecum and colon.

Materials and Methods Animals Five pony geldings ranging from 5 to 10 yr of age were surgically prepared with cecum and colon fistulas and fitted with cannulas or Pezzer catheters

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(size 45F). Anesthesia and surgical procedures were carried out under an approved animal care and use protocol. The ponies were treated with an anthelmintic (Ivermectin; Merck and Co., Rahway, N J ) and vaccinated for eastern and western encephalomyelitis, rhinopneumonitis, tetanus, and influenza A1 and A2 before the start of the Latin square digestion trial. Two additional ponies, a colon fistulated gelding and a cecal fistulated mare, served as donors of protozoa throughout Trials 1 and 2. Experimental ponies were kept in a heated barn in individual stalls with smooth walls and walked or lunged once a week when possible. Water was provided free choice. The animals were treated with the anthelmintic between trials. Donor ponies were treated with the anthelmintic every other month.

Defamation and Refaunation The procedures used for defaunation were an adaptation of the methodology described by Orpin (1977) for defaunation of the ovine rumen with dioctyl sodium sulfosuccinate ( DSS) . They found that although bacterial concentrations were decreased during the treatment phase they returned t o normal values within 11 to 21 d. The diet used during defaunation ( D diet) consisted of .68 kg of alfalfa pellets and .23 kg of whole shelled corn fed at 0930, and the same diet plus .23 kg of supplement (P-193 or P-l93C, Table 1) was fed at 1630. Ingredient weights were on an as-fed basis. This diet was fed on d 1and 2. On each day, after the morning feeding, 1 L of mineral oil was infused into the cecum after allowing as much cecal fluid as possible to drain out through the cannula. On d 3, the morning feed was given, but only .23 kg of supplement was fed in the evening. After the evening feeding, 120 mL of a 5% solution of DSS (Butler Company, Columbus, OH) was infused into

both the cecum and colon (240 mL total). On d 4 the animals were not fed in the morning and only .23 kg of supplement was given in the evening, at which time the DSS treatment was repeated. The ponies were fasted the morning of d 5 and returned to the D diet in the evening. On d 6, the normal D diet was fed in the morning and cecum and colon samples were taken. If no protozoa were found, an additional .23 kg of alfalfa hay was added t o the evening D diet until treatment diets were fed. All defaunated animals remained protozoa-free for the entire treatment period. Faunation was accomplished with a mixture of cecum contents from one donor pony and colon contents from the second donor pony. After removing as much ingesta as possible from each pony, approximately 500 mL of anaerobic dilution solution ( ADS), kept at body temperature, was infused into the organs to wash out as much of the remaining ingesta as possible. The ingesta from both ponies were combined, a subsample was taken for counting the protozoa, and the remaining contents were divided equally and placed into the cecum and colon of the defaunated experimental pony.

Determination o f Protozoal Concentrations All cecum and colon samples were taken 30 min after the morning feeding. When ponies began the period defaunated and were to remain that way, samples were obtained three times per week to check for the presence of protozoa. When ponies were undergoing defaunation during the 1st wk of the period, samples were obtained on d 6 and 8 to evaluate success of the procedure and then three times weekly as above. Animals that needed to be refaunated were infused as described previously on d 1 of wk 1. Numbers of protozoa were then determined on d 7 and 14. All protozoal samples were preserved by diluting

Table 1. Composition of hay and supplements for Trials la and 2b Ingredient, %

P-192

P-193

P-192C

P-193C

Hay 1

Hay 2

Corn Soybean meal Molasses Trace mineral saltC Monosodium phosphate Magnesium oxide Se (201 mg of S e k g ) Vitamin A (30,000 IU/g) Vitamin D (3,000 IU/g) Vitamin E ( 4 4 IU/g) Chemical composition Cellulose CP

78.61 10.18 4.38 4.38 1.75 .44 .09 .09 .09

82.23 13.13 2.52 1.08 .43 .43 .ll .02 .02 I02

15.23 62.19 10.42 4.48 5.18 1.79 .45 .09 .09 .09

65.56 29.71 2.57 1.11 .45 .44 .ll .02 .02 .02

-

-

-

__

-

-

-

__

-

-

-

-

-

-

4.95 7.76

3.47 13.30

8.34 29.60

5.37 19.19

26.22 16.89

32.29 14.38

~

__ _.

-

~~

aTrial 1 used hay 1 and P-192 (90% h a y ) , P-193 (60% hay) for three ponies; hay 2 and P-192C (90% hay), P-193C (60% h a y ) for one Pony. bTrial 2 was done half with hay 1and P-192 (90% hay), P-193 (60% h a y ) and half with hay 2 and P-192C (90% hay), P-193C (60% hay). Tontains > 93% NaC1, .35% Zn, .28% Mn, ,1758 F, .035% Cu, .007% I, and ,0079’~Co. Downloaded from jas.fass.org by guest on July 12, 2011

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with an equal volume of 50% formalin (18.5% formaldehyde). Cecum and colon samples were obtained either by opening the cannulas and allowing contents to flow into a plastic beaker, removing contents with a spatula, or by using a wide-mouth, 60-mL syringe with a plastic tube attached and obtaining the sample by suction. The colon sample from one pony had to be obtained by use of a plastic infusion rod attached to a 60-mL syringe. Samples from animals fitted with Pezzer catheters were obtained by suction, using a syringe when possible, or the catheter was removed and the tube with syringe was used. All instruments were rinsed with hot water between samplings and, if necessary, defaunated animals were sampled before faunated ones t o prevent transfer of protozoa. Protozoa were counted using the procedure of Dehority (1984) and identified to the generic level based on the descriptions of Hsiung (1930). Essentially, the procedure involves staining with Brilliant green, dilution with 30% glycerol solution, and counting in a Sedgwick-Rafter counting chamber.

Diets Two diets were fed, either a high-forage (HF) diet of 90% alfalfa hay/lO% concentrate, or a higherconcentrate ( HC) diet of 60% alfalfa hay/40% concentrate (Table 1 ) . These diets were selected to provide two different energy intakes. The amount of feed offered was based on 1.5%of the BW of the combined average of all ponies in the trial, in an attempt to ensure total feed consumption. Energy intake of the ponies fed the HF diet was above maintenance, and all animals gained weight during the experimental periods. Both diets contained 430 k 10 g of CP and were balanced to meet or exceed the NRC (1989) requirements for the equine. Because of unexpected difficulties in surgical procedures for fistulation and the need to replace a pony halfway through the trial (because of a prolapsed cecum), not all animals completed the trial on the same hay. The three original animals completed Trial 1 on hay 1 and concentrates P-192 ( H F diet) and P-193 (HC diet). The replacement pony was fed hay 2 and concentrates P-192 C ( H F diet) and P-193 C (HC diet). Although the analysis of hay 2 was very similar to that of hay 1, the concentrates P-192 C and P-193 C were adjusted to approximate the diets for the original three animals. The same two diets used in Trial 1 were fed in Trial 2 (i.e., the HF and HC diets). The first two periods of this trial used hay 1 with P-192-HF or P193-HC. The last two periods used hay 2 with P192C-HF or P-193C-HC (Table 1). In both trials, animals were fed once daily at 1000 f 30 min. By feeding once per day, it was anticipated that diurnal variation in microbial concentrations would be minimal. Preliminary studies indicated that under this schedule, protozoal concentrations in the

cecum and colon did not vary much in the first several hours after feeding. On this basis, all further samples for microbial concentrations were taken 30 min after feeding. This should be a relatively stable period with regard t o fermentation in the given organ because the new feed would not yet have passed into the hindgut. Goodson et al. (1988) found only minimal differences in bacterial and protozoal concentrations in the pony cecum just before feeding and 5 h after feeding. Daily DM intake across both trials was .27 kg of concentrate and 2.43 kg of hay for the HF diet and 1.08 kg of concentrate and 1.62 kg of hay for the HC diet.

Trial 1 : Effect of Defaunation on Digestibility Experimental Design. A digestibility trial was conducted using a 2 x 2 factorial arrangement of treatments in a 4 x 4 Latin square design with four animals and four treatments (faunated or defaunated, HF or HC diet). Each treatment was administered to each animal over a 28-d period, for a total of 112 d for each pony. All animals were randomly assigned t o treatments and began the trial in the defaunated state. In Period 1, during the first 7 d, the ponies either remained defaunated or were inoculated on d 1 with protozoa. During the first 7 d of the subsequent periods, according to treatment, ponies were defaunated or inoculated with protozoa on d 1. Except when the pony was being defaunated, the animals were kept on the previous diet until d 8. Two weeks were then allowed for diet adjustments, and on d 21 the animals were placed in a digestion crate for 5 d. In the middle of Trial 1, a cecal prolapse occurred in one of the ponies and it was replaced with the reserve (fifth) animal. The digestibility trial was analyzed as a 2 x 2 factorial in a 4 x 4 Latin square (SAS, 1988). The model contained effects due t o animal, period, defaunation, diet, and defaunation x diet interaction. Fecal samples (Trial 1) were collected, weighed, subsampled (10% removed), and frozen daily during the 5-d collection period. These samples were combined, and along with all diets fed, were analyzed for DM (AOAC, 1984) and cellulose (Crampton and Maynard, 1938). Cellulose and DM disappearance were determined by difference. Samples for determination of protozoal concentrations were taken on d 21, 23, and 25. Two additional samples, taken on d 22 and 24 during one digestibility trial period for each animal, were used to evaluate day-to-day variation over a 5-d period. Trial 2: Effect of Diet and Defaunation on Bacterial and Fungal Concentrations Experimental Design. This experiment was conducted with three of the pony geldings used in Trial 1 and the same ponies served as donors of protozoa. A microbiology study was conducted to determine


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the effects of defaunation and diet on total and cellulolytic bacterial and fungal concentrations in the cecum and colon. Variables in a 2 x 2 factorial arrangement of treatments in a randomized complete block design were 1) HF diet, 2 ) HC diet, 3 ) faunation, and 4) defaunation. The experimental design was confounded by time; however, this was not considered to be a relevant factor. Each treatment was administered to each animal for 28 d, for a total of 112 d. Defaunation and refaunation procedures were as described for Trial 1 and 2 wk were again allowed for dietary adjustment. Data were analyzed as a 2 x 2 factorial in a randomized complete block design, with ponies serving as blocks (SAS, 1988). The model contained effects due to blocks, diet, defaunation, and the diet x defaunation interaction. During the last week of the period, cecum and colon samples were taken twice, usually on d 22 and 24, for determination of protozoal, bacterial, and fungal concentrations. A third sample for measuring protozoal concentrations only was taken on either d 21, 23, or 25. Concentrations of total and cellulolytic bacteria and fungi were determined by the methods of Dehority et al. (1989) and Obispo and Dehority ( 19921, respectively. Protozoal concentrations were determined as previously described.

Total and generic distribution of protozoa were affected both by location (cecum vs colon) and diet (Table 3). For both diets, total protozoal concentrations were greater in the colon than in the cecum ( P < .05). The cecal protozoal population was not affected by diet ( P > .1);however, feeding the HC diet resulted in an increase in numbers of protozoa in the colon ( P < .05). There were diet x location interactions ( P < .03) for two genera of protozoa, and these showed opposite trends. Numbers of Buetschlia were greater in the cecum than in the colon when the HC diet was fed ( P < .05) but were greater in the colon than in the cecum when the HF diet was fed ( P < .05). Numbers of Blepharocorys were not significantly greater in the colon than in the cecum when the HC diet was fed ( P > . l ) ;however, when the HF diet was fed, numbers were greater in the cecum than in the colon ( P < .05). Protozoal concentrations varied considerably within individual ponies sampled over five consecutive days (Table 4). Larger mean total protozoal concentrations and a greater range in concentrations were observed in the colon (52.64 to 131.2 x 103/g) than in the cecum (30.48 to 77.28 x 103/g). Coefficients of variation averaged 45.7 and 76.0% for total protozoal concentrations in the cecum and colon, respectively.

Trial 2 Results

Trial 1 No interaction between defaunation and diet was detected ( P > .1). The amount of DM digested decreased slightly ( P < . l ) when the animals were defaunated (Table 2). Cellulose digestibility was not affected by defaunation. Both the weight of DM digested and percentage of DM digestion were greater ( P < .001) when the HC diet was fed than when the HF diet was fed. The amount of cellulose digested was greater ( P < .001) when the animals were fed the HF diet; however, percentage of cellulose digestion was not different between the diets.

No significant differences were found in total and cellulolytic bacterial concentrations in the cecum and colon with respect to defaunation (Table 5 ) . Cecum total bacterial concentrations tended to be slightly greater when the animals were in the defaunated state, whereas total bacterial concentrations in the colon showed the opposite trend. Although not significantly different, the concentration of total bacteria across treatments tended to be greater in the colon than in the cecum, with means of 237.5 and 102.9 x 107/g, respectively. Cellulolytic bacterial concentrations in both the cecum and colon showed a tendency to increase with defaunation. The mean concentrations of cellulolytic bacteria in the cecum and colon were 2.7 and 7.8 x 107/g, respectively.

Table 2. Main effects of defaunation and diet on diet digestibility in the ponya Dietb Item DM intake, kg DM digested, kg DM digested, % Cellulose intake, kg Cellulose digested, kg Cellulose digested, %

Faunated

Defaunated

13.76 9.53c 69.27' 3.00 1.57 53.16

13.61 9.19d 67.4Bd 2.94 1.53 52.21

HC 13.78 10.07e 73.0Se 2.50e 1.3E~~ 54.23

HF

SE

13.59 8.66f 63.66f 3.44f 1.76f 51.14

.ll .12 .66 .03 .04 1.51

aFour ponies and four treatments; 2 x 2 factorial in a 4 x 4 Latin square design. bHC = 609% hay-40% concentrate diet; HF = 90% hay-lO%, concentrate diet. Intakes are the total for the 5-d collection period. C,dMeans under protozoa status in the same row without a common letter in their superscript differ ( P < .1). e,fMeans under diet in the same row without a common letter in their superscript differ ( P < .001). Downloaded from jas.fass.org by guest on July 12, 2011

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Table 3 . Effect of diet on concentration and generic distribution of protozoa in the cecum and colon of the ponya H F ~

HCb Item

Cecum

Colon

Cecum

Colon

SE

Total, x 103/mL Genera, %

52.82'

133.53de

55.25'

97.71df

27.77

Buetschlia Cycloposthium Blepharocorys Paraisotricha

44.34c 19.57 24.87 .95 10.27

15.44d 31.50 46.42 1.59 5.12

16.68' 10.90 66.76' 0 5.62

50.87d 15.86 25.02d .35 7.91

9.13g 10.50 13.15g .94 4.20

Unknown

aValues represent the means of 12 observations. bHC = 60% hay-40% concentrate; HF = 90% hay-10% concentrate. 'vdFor a given diet, means in the same row followed by different superscripts differ ( P < ,051. e,fIn colon contents, protozoal concentrations were greater ( P < .05) when the ponies were fed HC compared to H F gDiet x location interaction ( P < ,031.

The HF diet resulted in a slight increase ( P > .1)in the concentration of total bacteria in the cecum compared with the HC diet (Table 6). In contrast, an increase in total bacterial concentrations ( P < .06) was observed in the colon when the HC diet was fed. Concentrations of cellulolytic bacteria in the cecum and colon were not affected by diet. Protozoal concentrations in the cecum of the ponies were not affected by diet (Table 7). However, in the colon, total protozoal concentrations were increased slightly ( P < . 1 ) when the HC diet was fed. In general, total and cellulolytic fungal concentrations seemed to be slightly greater in both the cecum and colon when the animals were defaunated (Table 8). However, only the difference in total fungal concentrations in the colon approached significance ( P < .1). No differences ( P > . 1 ) due to diet were observed in concentrations of total and cellulolytic fungi either in

the cecum or colon, even though the mean values for total and cellulolytic fungi nearly doubled when the HF diet was fed compared with the HC diet (data not presented). Highly significant ( P < .01) differences in total and cellulolytic fungal concentrations were observed between the cecum and colon (Table 9). Concentrations of both total and cellulolytic fungi in the colon were more than 10 times greater than concentrations in the cecum.

Discussion Removal of protozoa from the hindgut of the pony (defaunation) resulted in only a slight decrease ( P < . 1)in the overall digestibility of DM and had no effect on cellulose digestion. Thus, it seems that protozoa are not essential for fermentation of ingesta in the cecum

Table 4. Variation in the generic composition of cecum and colon protozoa determined in four ponies, each sampled at the same time for five consecutive days Pony Genus

1

2

3

4

3.12 f 1.9gb 2.88 k 2.14 24.40 k 12.25 0 .08 f .18 30.48 f 13.17

5.36 f 4.65 5.20 k 2.86 33.76 f 25.65 0 4.00 i 2.80 48.32 f 24.47

27.92 f 12.64 5.04 k 3.18 38.88 k 10.80 0 5.44 f 4.15 77.28 f 15.79

4.08 f 3.42 1.92 f 1.91 55.76 k 38.22 0 4.16 i 4.67 65.92 f 45.31

40.00 12.00 5.44 .64 2.40 60.48

2.40 k 2.47 7.52 L 3.23 37.28 f 54.29 0 5.44 f 3.85 52.64 k 57.85

47.22 f 38.79 21.12 f 18.77 60.64 f 16.16 0 2.24 k 1.31 131.20 k 43.41

102.56 i 83.85 .72 f .71 24.25 f 8.07 0 1.28 k 1.75 128.65 f 89.62

Cecuma

Buetschlia Cycloposthium Blepharocorys Paraisotricha Unknown Total

BuetschEiu Cycloposthium Blepharocorys Puruisotricha Unknown Total

k 58.33

7.09 6.16 1.43 3.62 k 55.19

f f f f

aNumber x lo3 per milliliter of contents. bMean and standard deviation. Downloaded from jas.fass.org by guest on July 12, 2011

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Table 5. Main effects of defaunation on total and cellulolytic bacterial concentrations in the cecum and colon of poniesa

Table 7. Main effects of diet on cecum and colon protozoal concentrations in poniesa Dietb

Total bacteria x 107/g

Protozoal status

x 107ig

Cecum Faunated Defaunated SE

91.67 114.08 19.25

Faunated Defaunated

324.36 150.67 129.28

2.64 13.02 7.11

aEach mean is based on six values (each value is the mean of two determinations per pony on each of the two different diets).

and colon of the equine. This evidence would also suggest that the protozoa possess no unique digestive enzymes. Although reports on the effects of defaunation in ruminants vary, the results observed in ponies tend to follow patterns similar to those observed in defaunated ruminants, in which only slight decreases in ruminal digestibility have been detected (Hobson and Jouany, 1988). Because no effect on cellulose digestion was observed with defaunation, it may be concluded that either the protozoa are not very important in cellulose digestion or that the bacteria and fungi simply take over this role when the protozoa are removed. In reality, the extent of cellulose digestion may be more a function of availability of this substrate from the intact forage than of the type of cellulolytic microorganisms involved. The increase in DM digestibility that occurred with the HC diet was as expected, because more digestible substrate is

Table 6. Main effects of diet on total and cellulolytic bacterial concentrations in the cecum and colon of poniesa

Diet'

Total bacteria 1071~

91.92 113.83 19.25

HC HF SE

400.5OC 74.53d 129.28

Colon 12.22 3.44 7.11

aEach mean is based on six values (each value is the mean of two determinations per pony, with and without protozoa). bHC = 60% hay-40% concentrate; HF = 90% hay-10% concentrate. c,dMeans for the colon in the same column with different superscripts differ ( P < .06).

85.17' 65.37d 5.08

present in the 40% concentrate. Other investigators working with various equine diets have reported similar results (Hintz et al., 1971a,b). Although adding concentrate increases total digestibility of a diet, no effect was observed on protozoal concentrations in the cecum. This study corroborates the findings of previous investigators (Kern et al., 1973; Goodson, 1981). When ponies were fed 25% oats and 75% clover or timothy hay vs 100% forage, no increase in protozoal concentrations in the cecum was observed (Kern et al., 1973). When an all-concentrate diet (corn and soybean meal) was compared to an allalfalfa diet, protozoal concentrations declined in the cecum (Goodson, 1981). Conversely, the addition of concentrate to the diet in the present study caused an increase in colon protozoal concentrations. Adam (1953) found that addition of oats to a grass diet had no effect on ciliates in the cecum but increased counts in the ventral colon. In the same study, addition of oats to a hay diet caused cecal protozoal concentrations to decline, whereas in the ventral colon no effect was observed. These results were obtained from a

Table 8. Main effects of defaunation on total and cellulolytic fungal concentrations in the cecum and colon of poniesa

Protozoal status 3.04 2.28 1.01

53.27 59.07 3.78

aEach mean is based on three values (each value is the mean of two determinations per pony). 'HC = 60% hay-40% concentrate; H F = 90% hay-10% concentrate. 'SdMeans in the same column with different superscripts differ ( P < .1j.

Cellulolytic bacteria 107/~ Cecum

HC HF SE

Colon 103/g

HC HF

SE 1.91 3.41 1.10

Colon

SE

Cecum

Cellulolytic bacteria

Total fungi x 102ig

Cellulolytic fungi x l02ig Cecum

Faunated Defaunated SE

2.65 4.39 1.71

Faunated Defaunated

23.39' 52.61' 12.24

2.43 2.65 1.57 Colon

SE

20.60 38.83 15.30

aEach mean is based on six values (each value is the mean of two determinations per pony on each of the two different diets). b,cMeans for the colon in the same column with different superscripts differ ( P < .1j.


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Table 9. Concentrations of total and cellulolytic fungi in the cecum and colon of the ponya

Location

Total funq

Cellulolytic fungi 102ig

Cecum Colon

SE

3.52b 38.13' 6.30

2.54b 30.17' 6.04

aEach mean is based on six values (each value is the mean of two determinations per week for each pony on each of two different diets, with and without protozoa). b,CMeans in the same column with different superscripts differ tP < ,011.

single pony. When the energy level of the diet is increased in ruminants, protozoal concentrations in the rumen usually increase. This response has been observed in sheep (Nakamura and Kanegasaki, 1969; Grubb and Dehority, 1975) and in cattle (Abe et al., 1973; Dehority and Mattos, 1978). The lack of response of pony cecum protozoa t o the addition of concentrate to the diet may be due to the rate of passage of ingesta through the cecum, because an increase in concentration was noted in the colon. It has been found that feeding an all-forage diet to ponies results in the cecal fluid volume turning over approximately three times per day, whereas feeding 100% concentrate causes the fluid volume to turn over five times per day (Goodson, 1981). Thus, adding concentrate to the diet may result in increased fluid turnover in the cecum. Assuming that a portion of the protozoa move with the fluid, as demonstrated in the rumen (Ankrah et al., 19901, more protozoa would be expected to leave the cecum. This would mask any increase in concentration resulting from more available substrate and could possibly explain why adding concentrate has no effect on cecal protozoal concentrations but results in an increase in colon protozoal concentrations. A diet x location interaction was observed for two genera of protozoa in Trial 1. Presumably, the major factors that control protozoal concentrations are generation time, available substrate, and turnover time. Buetschlia concentrations were largest in the cecum when the HC diet was fed and largest in the colon when the HF diet was fed. This may indicate a response to increased quantities of highly available carbohydrates. The increase in Buetschlia concentrations in the cecum would suggest a relatively short generation time for this genus, which would prevent washout. A longer retention time in the colon than in the cecum (Argenzio et al., 1974) may explain why increased concentrations occurred in the colon when the 90% hay diet was consumed (i.e., the amount of readily available carbohydrates in the cecum is decreased and retention time in the colon is long enough to allow utilization of the structural carbohydrates).

A diet x location interaction was also observed for the genus Blepharocorys, but the concentration changes were opposite those for Buetschlia. Feeding the HC diet resulted in increased concentrations in the colon, whereas the HF diet resulted in higher cecal concentrations. If the average time for microbial cell division in a continuous fermentation system exceeds 69%' of the turnover time, washout will occur (Hungate, 1966). Thus, if Blepharocorys had a somewhat longer generation time, one might expect concentrations to rise in the colon, if indeed concentrate increases turnover in the cecum (Goodson et al., 1988). In turn, if cecal turnover time is decreased on a forage diet, then higher concentrations of this genus might be expected to occur in the cecum. Based on defaunation studies conducted in ruminants (Veira, 1986), an increase in bacterial concentrations in the hindgut would be expected when protozoa are removed. The slight increase in concentration observed in the cecum when the ponies were in the defaunated state followed this trend. In contrast, concentrations of bacteria present in the colon were almost twice as high when protozoa were present as when protozoa were absent. Because it has been documented that protozoa prey on bacteria in the rumen (Williams and Coleman, 19881, it was expected that this would also be true in the equine hindgut and that bacterial concentrations would increase when the predators were removed. No explanation can be offered at this time for the present results. With defaunation, cellulolytic bacteria tended to increase in concentration both in the cecum and colon, as might be expected. Feeding the HC diet resulted in an increase ( P < .06) in total bacterial concentrations in the colon, as well as a slight increase ( P < .1)in colon cellulolytic bacterial concentrations compared with concentrations when the HF diet was fed (Table 6). Numerous studies in ruminants have shown that significant increases in rumen bacterial concentrations occur when concentrate diets are fed (Maki and Foster, 1957; Bryant and Robinson, 1961; Leedle et al., 1982). Presumably, this is simply the result of increased available substrate levels. According to Tisserand ( 199 11, unpublished results of Bellet indicated that feeding corn or oats to horses results in higher concentrations of cecal bacteria; oats had a greater effect than corn. Kern et al. (1973) also found significant increases in cecal bacterial concentrations when diets consisting of 25% oats were fed, compared with an all-forage diet of timothy or clover hay. Feeding an all-concentrate diet consisting of corn and soybean meal also increased cecal bacterial concentrations compared with feeding an all-alfalfa hay diet (Goodson, 1981). The present study contrasts with these reports, in that cecal bacterial concentrations were not increased by feeding the HC diet. The differences between this and previous trials could be a


DIGESTIBILITY A N D MICROBIAL STUDIES I N PONIES

reflection of the percentage of concentrate vs forage fed (i.e., 10 and 40% concentrate diets in the present trial compared with 100% forage or 100% concentrate in the studies of Goodson [19811 and Tisserand [1991]). Kern et al. (1973) compared an all-forage to a 25% concentrate diet. Defaunation resulted in a nonsignificant increase in total fungal concentrations in the cecum. Cellulolytic fungal concentrations were unaffected, and a slight increase in both total and cellulolytic fungal concentrations in the colon was observed ( P < .11. Defaunation also seems to increase concentrations of ruminal fungi (Romulo et al., 1986; Ushida et al., 1989). The increase could be because the fungi are no longer consumed by the protozoa or because of an increase in available substrate. As in this trial, the increase in ruminal fungal concentrations reported by Ushida et al. ( 1989) was not significant. Recent studies have strongly indicated that the decrease in numbers of ruminal fungi are due to protozoal predation and not to a n increase in available nutrients (Newbold and Hillman, 1990). Wide daily variations in the concentration of hindgut protozoa have been reported previously from ponies fed the same diet (Adam, 1951, 1953). However, not all these differences could be accounted for by sampling technique. Variability in the protozoal concentrations of samples taken at the same time on five consecutive days and for each of four different ponies in this trial corroborates previous findings. Greater variation was observed in the colon than in the cecum, which might be explained on the basis of higher concentrations and a greater overall range in concentrations in the colon. It could also be due to sampling, in that cecal contents were easier to obtain because of their higher liquid content, and larger sample volumes were usually obtained from this site. Because colon samples are considerably less fluid, it is difficult t o obtain appreciable quantities of ingesta from this location. Further digestibility studies with other forages and concentrates, and possibly different forage to concentrate ratios, would probably be helpful in evaluating our present results. In addition, determination of both rate of fluid and particulate matter passage in the cecum and colon would provide insight into the interpretation of our digestibility results and protozoal generic distributions in the faunated animals.

Implications The present study has provided direct evidence that the protozoa contribute no unique capabilities to fermentation in the equine hindgut. Whether they may have some role in providing essential nutrients t o the other microbes (bacteria and fungi) or back to the host itself cannot be evaluated on the basis of these

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short-term trials. Enumeration of bacterial and fungal concentrations in the cecum and colon of the pony, and the effect of diet on their concentrations, provides a base for evaluating desirable modifications of the normal fermentation. This type of information may eventually help decrease the incidence of digestive disorders and improve the health and productivity of the horse.

Literature Cited Abe, M., H. Shibui, T. Iriki, and F. Kumeno. 1973. Relation between diet and protozoal population in the rumen. Br. J. Nutr. 29:197. Adam, K.M.G. 1951. The quantity and distribution of the ciliate protozoa in the large intestine of the horse. Parasitology 41:301. Adam, K.M.G. 1953. In uiuo observations on the ciliate protozoa inhabiting the large intestine of the horse. J . Gen. Microbiol. 9: 376. Ankrah, P., S. C. Loerch, and B. A. Dehority. 1990. Sequestration, migration and lysis of protozoa in the rumen. J . Gen. Microbiol. 136:1869. AOAC. 1984. Official Methods of Analysis (14th Ed.). Association of Official Analytical Chemists, Arlington, VA. Argenzio, R. A., J. E. Low, D. W. Pickard, and C. E. Stevens, 1974. Digesta passage and water exchange in the equine large intestine. Am. J. Physiol. 226:1035. Bryant, M. P., and I. M. Robinson. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446. Crampton, E. W., and L. A. Maynard. 1938. The relation of cellulose and lignin content to the nutritive value of animal feeds. J . Nutr. 15:383. Dehority, B. A. 1984. Evaluation of subsampling and fixation procedures used for counting rumen protozoa. Appl. Environ. Microbiol. 48:182. Dehority, B. A,, and W.R.S. Mattos. 1978. Diurnal changes and effect of ration on concentrations of the rumen ciliate Charon uentriculi. Appl. Environ. Microbiol. 36:953. Dehority, B. A,, P. A. Tirabasso, and A. P. Grifo, Jr. 1989. Mostprobable-number procedures for enumerating ruminal bacteria, including the simultaneous estimation of total and cellulolytic numbers in one medium. Appl. EnLlron. Microbiol. 55:2789. Goodson, J. 1981. Effects of an abrupt change in ration, from all forage to concentrate, on the microbial populations and ecology of the pony cecum. Ph.D. Dissertation. The Ohio State Univ., Columbus, Goodson, J., W. J . Tyznik, J. H. Cline, and B. A. Dehority. 1988. Effects of a n abrupt change from hay to concentrate on microbial numbers and physical environment in the cecum of the pony. Appl. Environ. Microbiol. 54:1946. Grubb, J . A,, and B. A. Dehority. 1975. Effects of a n abrupt change in ration from all roughage t o high concentrate upon rumen microbial numbers in sheep. Appl. Microbiol. 30:404. Hintz, H. F., R. A. Argenzio, and H. F. Schryver. 1971a. Digestion coefficients, blood glucose levels and molar percentage of volatile acids in intestinal fluid of ponies fed varying forage-grain ratios. J. Anim. Sci. 33:992. Hintz, H. F., D. E. Hogue, E. F. Walker. Jr., J. E. Lowe, and H. F. Schryver. 1971b. Apparent digestion in various segments of the digestive tract of ponies fed diets with varying roughage-grain ratios. J . Anim. Sci. 32:245. Hobson, P. N.; and J.-P. Jouany. 1988. Models, mathematical and biological of the rumen function. In: P. N. Hobson ( E d . ) The Rumen Microbial Ecosystem. pp 461-511. Elsevier Applied Sciences, London. Hsiung, T. S. 1930. A monograph on the protozoa of the large


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intestine of the horse. Iowa State J. Sci. 4:359. Hungate, R. E. 1966. The Rumen and Its Microbes. Academic Press, New York. Kern, D. L., L. L. Slyter, J. M. Weaver, E. C. Leffel, and G. Samuelson. 1973. Pony cecum vs. steer rumen: The effect of oats and hay on the microbial ecosystem. J. h i m . Sci. 37:463. Leedle, J.A.Z., M. P. Bryant, and R. B. Hespell. 1982. Diurnal variations in bacterial numbers and fluid parameters in ruminal contents of animals fed low- or high-forage diets. Appl. Environ. Microbiol 44:402. Maki, L. R., and E. M. Foster. 1957. Effect of roughage in the bovine ration on types of bacteria in the rumen. J. Dairy Sci. 40:905. Nakamura, K., and S. Kanegasaki. 1969. Densities of ruminal protozoa of sheep established under different dietary conditions. J. Dairy Sci. 52:250. Newbold, C. J., and K. Hillman. 1990. The effect of ciliate protozoa on the turnover of bacterial and fungal protein in the rumen of sheep. Lett. Appl. Microbiol. 11:lOO. NRC. 1989. Nutrient Requirements of Horses. ( 5 t h Ed.). National Academy Press, Washington, DC. Obispo, N. E., and B. A. Dehority. 1992. A most probable number method for enumeration of rumen fungi with studies on factors

affecting their concentration in the rumen. J. Microbiol. Methods 16:259. Orpin, C. G. 1977. Studies on the defaunation of the ovine rumen using dioctyl sodium sulphosuccinate. J. Appl. Bacteriol. 43: 309. Romulo, B. H., S. H. Bird, and R. A. Leng. 1986. The effects of defaunation on digestibility and rumen fungi counts in sheep fed high fibre diets. Proc. Aust. SOC.Anim. Prod. 16:327. SAS. 1988. SAWSTAT@User’s Guide (Release 6.03). SAS Inst. Inc., Cary, NC. Tisserand, J. L. 1991. Microbial digestion and its consequences for feeding in the horse. In: D. Fielding and R. A. Pearson ( E d . ) Donkeys, Mules and Horses in Tropical Agricultural Development. pp 73-78. Univ. of Edinburgh, U.K. Ushida, K., H. Tanaka, and V. Kojima. 1989. A simple in situ method for estimating fungal population size in the rumen. Lett. Appl. Microbiol. 9:109. Veira, D. M. 1986. The role of ciliate protozoa in nutrition of the ruminant. J. Anim. Sci. 63:1547. Williams, A. G.,and G. S. Coleman. 1988. The rumen protozoa. In: P. N. Hobson ( E d . ) The Rumen Microbial Ecosystem. pp 77-129. Elsevier Applied Science, London.


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