Pge 1 of 5 Protozo n igestive trt prmeters of the impl Authors: Dirk G. Booyse 1 Burk A. Dehority 2 Affilitions: 1 Deprtment of Antomy n Physiology, University of Pretori, 2 Deprtment of Animl Sienes, Ohio Stte University, USA Corresponene to: Dirk Booyse Emil: ooyse@op.up..z Postl ress: Privte Bg X04, Onerstepoort 0110, Dtes: Reeive: 02 Mr. 2011 Aepte: 23 My 2011 Pulishe: 12 Ot. 2011 How to ite this rtile: Booyse, D.G. & Dehority, B.A., 2011, Protozo n igestive trt prmeters of the impl, Onerstepoort Journl of Veterinry Reserh 78(1), Art. #327, 5 pges. oi:10.4102/ojvr. v78i1.327 2011. The Authors. Liensee: AOSIS OpenJournls. This work is liense uner the Cretive Commons Attriution Liense. Intestinl ontents were ollete from eight impl t three ifferent lolities uring the winter hunting seson (2005 2009), s well s from nother 24 nimls from one-yer tril t gme frm lle Ditholo (2003 2004). Gs proution, protozo ounts n severl other physiologil prmeters were mesure from oth rumen n eum or olon ontents. Only higher ophryosolei n Isotrihie speies of protozo were ounte n ientifie. Ostroinium grile ws present in ll 32 impl. mggii ws present in 31 nimls n imple n (either eutum or utum) in 30 nimls. Dsytrih ruminntium ws present in only 11 of the impl. Conentrtions of protozo were orrelte with the seson of smple olletion n highly orrelte with the nimls living on the gme frm. Gs proution (ml/g of wet rumen ingest) ws wekly orrelte with protozo onentrtion ut not with the seson of olletion. Introution A totl of 13 protozon speies hve previously een reporte from the rumen of the impl. Dogiel (1925) liste six protozon speies from impl in Keny n lter, in 1932, he oserve five speies in Ugn. Four speies were the sme in oth lotions n the fifth elonge to the sme genus ut ws ientifie s ifferent speies (Tle 1). In, Vn er Wth n Myurgh (1941) reporte the presene of three speies in impl rumens n lter Kleynhns (1977) reore the presene of 11 speies. Four of the speies oserve y Kleynhns, Isotrih prostom, Entoinium prvum, mggii n Dsytrih ruminntium, were new host reors for impl (see Tle 1). Dehority n Oenyo (2003) ientifie protozo in impl from Keny only to the generi level n i not fin ny gener in whih speies h not previously een oserve. Aprt from the ounting n ientifition of protozo, severl other prmeters of the igestive trt of the impl (rumen, smll intestine, eum n olon) were lso investigte n re reporte here for the first time. These inlue ph n temperture of ontents, length of orgn, ontents weight per orgn, gs proution, ry mtter n ensity. Mterils n methos Animls One femle niml ws ollete from frm lle Krooult, 100 km north-west of Pretori (24 43 49.45 S, 27 33 22.88 E), in July 2009, five nimls (three mle n two femle) were from the Loskop Dm Nture Reserve in Mpumlng provine, 200 km est of Pretori (25 25 51.91 S, 29 19 39.80 E), n two mle nimls were from the Rietvlei Nture Reserve, 50 km south of Pretori (25 52 41.17 S, 28 16 17.83 E). In ition, 24 nimls (ll mle) were from one-yer tril from the Ditholo gme frm, 60 km north of Pretori (25 19 43.95 S, 28 19 04.34 E). Fee Impl re mixe feeers n therefore grze s well s rowse. Fee ville to the impl onsists of vegettion types lle ushvels. A ushvel n e esrie s hevily grsse plin, otte y ense lusters of trees n tll shrus. The grsses foun here re generlly tll n turn yellow or rown in winter, whih is the ry seson throughout most of. Sour ushvel onsists of grzing fiels, mostly omprising grsses like Ergrostis urvul, Ergrostis hloromels, Hyprrheni hirt n Cymopogon exvtus. These grsses re fst-growing plnts in the summer, ut ie rpily in winter, turning hr n tsteless. Sour grss speies n, however, reover quite rpily from overgrzing. Sweet ushvel generlly onsists of grsses tht grow slowly in summer ut ten to sty highly nutritious n pltle uring the ry seson (winter). Sweet grss speies inlue Digitri erinth, Pnium mximum n
Pge 2 of 5 TABLE 1: Rumen protozo oserve from impl (Aepyeros melmpus). Speies Present stuy, Vn er Wth n Myurgh (1941), Kleynhns (1977), Dogiel (1925), Keny Dsytrih ruminntium X - X - - Isotrih prostom - - X - - Entoinium uri X - X X X Entoinium nnellum X - X X X Entoinium prvum X - X - - Entoinium simplex - X - - - imple X - X X X mggi X - X - - negletum - - X - - Ostroinium grile X - X X X utum X X X - - eutum X X X - - Epiplstron spinosum - - - X X Totl numer of speies 9 3 11 5 5 Dogiel (1932), Ugn Note: Plese see the full referene list of the rtile, Booyse, D.G. & Dehority, B.A., 2011, Protozo n igestive trt prmeters of the impl, Onerstepoort Journl of Veterinry Reserh 78(1), Art. #327, 5 pges., for more informtion. Urohlo mosmiensis. Sweet grsses reover very slowly from hevy impts like overgrzing. Mixe ushvel is fiel ompose of oth sour n sweet grsses. The vel type in the Rietvlei n Ditholo res is esrie s sourish mixe ushvel, whilst Krooult represents sour ushvel n the vegettion from the Loskop re is esrie s mixe ushvel. In the spring n summer, impl oth fee on grss (Ergrostis) n rowse on trees like Ai kroo n Ai ffr. However, in the winter they re fore to low grze on grss, s little or no rowse is ville. Mesurements After shooting, the nimls were weighe, the igestive trt remove n the ifferent ntomil setions (rumen, smll intestines, eum n olon) were ligte efore seprtion. Eh setion ws weighe n the temperture, ph n length of the orgns were mesure n note. Weights were mesure with n eletroni sle ple of mesuring up to two eiml ples n ph ws mesure with portle, ttery-powere ph meter (Euteh, moel EC-PH- 10/01N, Singpore). Orgn ontent weights were estimte y weighing the orgn full n empty. Smples for fermenttion stuies (200 ml) were ple in fermenttion vessels n gs proution ws mesure every minute y mens of glss syringe (fitte with 20-guge stinless steel neele), inserte through the ruer stopper. Mesurements were tken over perio of 45 min. The ext weight of the smples ws etermine fter omplete fermenttion. Protozo A lrge smple of ingest ws tken from the rumen n the flui squeeze out into 40-mL plsti speimen ottle. For the eum n olon smples, smll liquots were tken t rnom from the ontents n poure into the 40-mL ontiner. These smples were then trnsferre to lrger mrke ontiners n 100 ml 70% methnol ws e to eh. Bk t the lortory, the protozo smples were wshe through sieves s esrie previously (Booyse, Boomker & Dehority 2010). In essene, the entire smple ws wshe through set of sieves with n inner n outer hmer. The smple ws poure into the inner sieve (pore size 110 μm) n wshe with wter. Prtiulte mtter n protozo psse through to the outer sieve, whih h pore size of 37 μm. The outer sieve ws fitte with rining tp to llow the ontents to e rine into ottle. The wshe smple ws then llowe to stn for 30 min n the volume juste k to 40 ml y enting the exess wter. Two rops of Brillint Green stin were e to eh of the 40-mL smples, whih were then llowe to stn for 24 h (Dehority 1984). Three liquots (0.1 ml eh) from eh smple were ple onto seprte mirosope slies n fitte with over slip. Protozo on eh slie were ounte using stnr light mirosope fitte with Pnsoni igitl mer (moel GP220). Conentrtions were etermine y multiplying the men of ll three ounts y 10, thus giving ount per millilitre. Results n isussion Protozo Initil exmintion of the protozo smples revele unexpetely omplete sene of Entoinium speies. Susequent investigtion revele tht these speies were wshe out through the 37-µm pores. The smples were wshe primrily to remove most of the lrger plnt eris, whih interferes with mirosopil oservtion. Beuse ll the smples h een wshe, we were le to enumerte only Isotrihie n the sufmilies Diploiniine n Ophryosoleine in the fmily Ophryosoleie (lrger ophryosolei protozo). However, we were le to shoot n itionl impl, ollet rumen ontents, stin n ount protozo in 0.1-mL smple iretly, without wshing through sieves. Our exmintion revele the presene of numerous Entoinium speies in this lst niml, whih were ientifie n inlue with our previous t in Tle 1.
Pge 3 of 5 TABLE 2: Ourrene n onentrtion of Dsytrih ruminntium n lrger ophryosolei protozo in rumen ontents of the impl. Site Numer Age Sex Dsytrih ruminntium Ophryosolei protozo Ostroinium grile imple mggii eutum Krooul 1 AD F - - - - - - Ourrene (%) - - - - 100 100 - - - Conentrtion (Protozo/mL) - - - - 1200 1600 - - - Rietvlei 2 AD M - - - - - - Ourrene (%) - - - - 100 100 100 50 100 utum Conentrtion (Protozo/mL) - - - - 3940±820 4800±169 1350±240 80 3660±707 - - - - (3360-4520) (4680-4920) (1180-1520) - (3160-4160) Loskop 3 AD M - - - - - - 2 AD F - - - - - - Ourrene (%) - - - 20 100 100 100-80 Conentrtion (Protozo/mL) - - - 200 1280±784 520±440 450±405-750±810 - - - - (400-2320) (80-1120) (80-1040) - (40-1880) Ditholo 24 AD M - - - - - - Ourrene (%) - - - 33 100 92 100 63 100 Conentrtion (Protozo/mL) - - - 1212±1826 6469±5420 1827±1720 945±751 200±143 5371±5183, AD, ult., M, mle; F, femle., Men vlue ± STD., Rnge of vlues. - - - (40-4160) (230-16640) (60-6440) (40-2760) (40-520) (20-17560) Four erlier reports on protozo in the impl re lso inlue in Tle 1 for omprison. To te, 13 speies of protozo hve een oserve in the impl rumen. In South Afri, 11 of these were reporte y Kleynhns in 1977, nine in the present stuy n three y Vn er Wth n Myurgh in 1941. Dogiel reporte totl of five speies from impl in Keny (1925) n Ugn (1932). Tle 2 reflets the ourrene n onentrtion of D. ruminntium n the lrger ophryosoleis (five speies) s groupe y the olletion site. Ostroinium grile ws present in ll 32 nimls, E. mggii ws present in 31 nimls, whilst imple n utum were foun in 30. In ontrst, eutum ws present in only 16 nimls n Dsytrih speies were foun in nine. Thus, the mjority of the lrger ophryosoleis were represente y four speies, nmely O. grile, E. mggii, E. imple n Ep. utum. The totl onentrtion of ll six speies verge out 1.6 x 10 4 per ml in the 24 nimls from Ditholo, 0.32 x 10 4 per ml in the five from Loskop, 1.38 x 10 4 per ml in the two impl from Rietvlei n 0.28 x 10 4 per ml in the single niml from Krooult. The eum n olon ontents were mixe, wshe s esrie ove, n exmine mirosopilly for protozo. The results in Tle 3 show tht only very low onentrtions were present in the ingest from these orgns. Presumly, the protozo woul e igeste s they move through the omsum n smll intestine; however, some ells re oviously le to pss through unhrme. It is possile tht they re emee in lrger foo prtiles n re not expose to the ii n enzymti onitions in those orgns. These onentrtions re onsierly higher thn wht one uthor (B.A.D.) hs previously oserve in omesti ruminnts (unpulishe). A possile reltionship etween the time (i.e. the month when the niml ws hrveste) n the onentrtion of the lrger protozo ws investigte, tking ll 32 nimls into onsiertion. A orreltion oeffiient (r) of 0.65 ws otine (r 2 = 0.42). However, when only the 24 nimls from single lotion (Ditholo) were use, the orreltion oeffiient ws 0.865 (r 2 = 0.75). Figure 1 shows the regression of protozo onentrtion for ll 32 nimls, oring to time of hrvesting. The first smple ws otine in Deemer n lele s 0, followe y Jnury s 1, Ferury s 2, et. This ws se on the ssumption tht the strt of the summer growing seson woul egin in Deemer, when psture n rowse were low, n tht fee woul eome inresingly ville uring the following months. It is lso of interest tht only two gener of the sufmily Diploiniine, n Ostroinium, hve een Conentrtion (protozo/ml) 40000 30000 20000 10000 0 Y = -4143.54 + 2619.63X R-Sq = 42.5 % 0 1 2 3 4 5 6 7 8 9 10 Month Months shown on x-xis run from Deemer (0) to Otoer (10). FIGURE 1: Regression of onentrtion of the lrger protozo in impl rumen ontents oring to the month of olletion.
Pge 4 of 5 TABLE 3: Ourrene n onentrtion of Dsytrih ruminntium n lrger ophryosolei protozo in the eum or olon ontents of impl. Site Numer Age Sex Dsytrih ruminntium Ophryosolei protozo Ostroinium grile imple mggii eutum Krooul 1 AD F - - - - - - Ourrene (%) - - - - 100 100 - - - Conentrtion (Protozo/mL) - - - - 180 50 - - - Rietvlei 2 AD M - - - - - - Ourrene (%) - - - - - - 100 - - Conentrtion (Protozo/mL) - - - - - - 40±0 - - Loskop 3 AD M - - - - - - 2 AD F - - - - - - Ourrene (%) - - - - 60 60 60-60 utum Conentrtion (Protozo/mL) - - - - 50±26 63±29 83±31-163±132 - - - - (30-80) (30-80) (50-110) - (20-280) Ditholo 24 AD M - - - - - - Ourrene (%) - - - 4 75 42 42 8 50 Conentrtion (Protozo/mL) - - - 60 88±84 61±70 38±30 15±7 58±50, AD, ult., M, mle; F, femle., Men vlue ± STD., Rnge of vlues. - - - - (30-360) (10-220) (20-120) (10-20) (10-160) TABLE 4: Physiologil prmeters of the impl igestive trt n gs proution y rumen, eum n olon ontents. Site Orgn Physiologil prmeters Averge niml weight (Kg) Numer Age Sex ph Totl orgn ontents (grms) Dry mtter (%) Averge gs proution in wet orgn ontent (ml/g) Krooul 43 1 AD F - - - - - Rumen - - - - 6.43 1764 12.40 0.18 1.23 Smll intestine - - - - 5.95 901 - - - Ceum - - - - 7.50 200 13.90 0.15 0.64 Colon - - - - 7.13 202 12.80 0.40 0.22 Loskop 77.8±8.61 4 AD M - - - - - (69.19-86.41) 1 AD F - - - - - Rumen - - - - 5.94±0.11 4890±1238 8.54±1.84 0.17±0.14 0.69±0.11 Density - - - - (5.83-6.05) (3651-6128) (6.70-10.38) (0.03-0.31) (0.58-0.08) Smll intestine - - - - 6.99±0.3 950±356 - - - - - - - (6.69-7.29) (594-1306) - - - Ceum - - - - 6.66±0.22 483±364 8.25±1.89 0.09±0.06 0.55±0.05 - - - - (6.44-6.88) (119-846) (4.87-8.25) (0.03-0.16) (0.50-0.80) Colon - - - - 6.74±0.05 360±194 2.96±4.08 0.08±0.06 0.52±0.17 - - - - (6.69-6.79) (166-554) (1.88-4.04) (0.02-0.14) (0.35-0.69) Rietvlei 42.5±3.54 2 AD M - - - - - (39-46.04) - - - - - - - - Rumen - - - - 6.6±0.13 5587±1756 12.89±1.40 0.18±0.10 0.88±0.00 - - - - (6.46-6.73) (3828-7345) (11.49-14.29) (0.09-0.28) (0.87-0.88) Smll intestine - - - - 7.85±1.34 778±219 - - - - - - - (6.51-9.18) (558-997) - - - Ceum - - - - 7.46±0.57 236±11 13.18±2.37 0.20±0.06 0.90±0.03 - - - - (6.88-8.03) (225-247) (1.18-15.54) (0.13-0.26) (0.87-0.93) Colon - - - - 7.75±0.37 291±83 3.78±1.45 0.11±0.02 0.89±0.02 - - - - (7.38-8.12) (208-374) (2.33-5.22) (0.08-0.13) (0.87-0.90) Ditholo 61.33±8.39 24 AD M - - - - -, AD, ult., M, mle; F, femle., Men vlue ± STD., Rnge of vlues. (52.94-69.72) - - - - - - - - Rumen - - - - 5.74±0.32 4007±1169 11.23±2.19 0.38±0.08 0.66±0.13 - - - - (5.24-6.37) (1800-6234) (9.04-13.41) (0.24-0.57) (0.40-0.86) Smll intestine - - - - 7.16±0.71 988±837 - - - - - - - (6.45-7.86) (162-1835) - - - Ceum - - - - 6.81±0.26 212±84 8.71±2.45 0.27±0.21 0.58±0.17 - - - - (6.55-7.07) (127-296) (6.26-11.15) (0.06-0.48) (0.41-0.74) Colon - - - - 6.72±0.51 189±86 7.70±3.28 0.25±0.08 0.45±0.18 - - - - (6.21-7.23) (102-275) (4.44-10.97) (0.16-0.33) (0.27-0.63)
Pge 5 of 5 foun in the impl. Speies of Diploinium, Metinium, Enoploplstron, Elytroplstron n Polyplstron our in mny of the other wil ruminnts in Afri (Dehority & Oenyo 2003; Dogiel 1925, 1932; Kleynhns & Vn Hoven 1975; Vn Hoven 1975, 1978, 1983). Physiologil prmeters Tle 4 presents informtion on the olletion site, ge, sex, orgn ph, weight of the orgn ontents, perentge ry mtter, ensity n gs proution from orgn ontents, inute in vitro (gs proution ws not mesure with smll intestine ingest). Exept for the niml from Krooult n the two from Rietvlei, rumen ph vlues verge elow 6.0. This vlue woul e expete in grzing ruminnts, s emonstrte in the stuy y Dehority n Tirsso (2001), where sheep were fe 1, 6, or 24 times per y. Rumen ry mtter vlues fell within the norml reporte rnge of 10% 13% (Dehority 2003). The im of mesuring gs proution ws to evlute whether this prmeter ws ssoite with protozol onentrtions. When gs proution ws orrelte with numers of lrger protozo, the orreltion oeffiient ws 0.31, initing little or no ssoition. This is not entirely unexpete, euse numer of ftors woul e involve. Proly the most ritil woul e the mount of ville sustrte in the rumen ontents, regrless of the onentrtion of protozo n teri. Gs proution from rumen, eum n olon ontents ws greter in the nimls on the gme frm, suggesting tht more sustrte ws ville in the ingest from these nimls thn from those living in the ushvel. In future stuies it woul e esirle to mesure fermenttion pity s esrie y El-Shzley n Hungte (1965), where itionl sustrte is e. Temperture of orgn ontents rnge from 30 ºC to 41 ºC for the rumen, etween 11 ºC n 28 ºC for the smll intestine, etween 17 ºC n 31 ºC for the eum n etween 13 ºC n 30 ºC for the olon. Length of the orgns rnge from 281 mm to 570 mm for the rumen, 6218 mm to 13 852 mm for the smll intestine, 162 mm to 652 mm for the eum, n 190 mm to 1411 mm for the olon (t not shown). Conlusion The numer of gener n speies of protozo ourring in the impl is limite ompre to other Afrin wil ruminnts n omestite ttle n sheep (Booyse & Dehority in press; Dehority & Oenyo 2003; Dogiel 1932; Kleynhns & Vn Hoven 1976; Vn Hoven 1975, 1978, 1983). Only four speies of Entoinium, two gener of Diploiniine n two speies of hve een oserve. Aknowlegements Authors ontriutions D.G.B. ollete the smples, ws responsile for ll the mesurements n onute the mirosopi stuies. B.A.D. ssiste with ientifition of the speies, ompiltion of the t n writing the mnusript. Referenes Booyse, D.G. & Dehority, B.A., in press, Rumen protozo in n sheep with summry of the worlwie istriution of sheep protozo, Onerstepoort Journl of Veterinry Reserh. PMCi:2628563 Booyse, D.G., Boomker, E.A. & Dehority, B.A., 2010, Protozo in the igestive trt of wil herivores in. I: Wrthogs (Phohoerus ethiopius), Zootx 2492, 63 68. Dehority, B.A., 1984, Evlution of su-smpling n fixtion proeures use for ounting rumen protozo, Applie n Environmentl Miroiology 48, 182 185. PMi:6476828, PMCi:240360 Dehority, B.A., 2003, Rumen Miroiology, Nottinghm University Press, Nottinghm, UK. Dehority, B.A. & Oenyo, A.A., 2003, Influene of iet on the rumen protozol fun of inigenous Afrin wil ruminnts, Journl of Eukryoti Miroiology 50, 220 223. Dehority, B.A. & Tirsso, P.A., 2001, Effet of feeing frequeny on teril n fungl onentrtions, ph n other prmeters in the rumen, Journl of Animl Siene 79, 2908 2912. PMi:11768121 Dogiel, V., 1925, Nouveux infusoires e l fmille es Ophryosoléiés prsites ntilopes Afriines [New infusori of the fmily of ophryosolei prsites in Afrin ntelope], Annles e Prsitologie 2, 116 142. Dogiel, V., 1932, Beshreiung einiger neuer Vertreter er Fmilie Ophryosoleie us friknishen Antilopen nest Revision er Infusorienfun friknisher Wieerkäuer [Desription of some new representtives of the fmily Ophryosoleie from Afrin ntelope together with revision of the infusoril fun of Afrin ruminnts], Arhiv für Protistenkune 77, 92 107. El-Shzley, K. & Hungte, R.E., 1965, Fermenttion pity s mesure of net growth of rumen orgnisms, Applie Miroiology 13, 62 69. PMi:14269247, PMCi:1058191 Kleynhns, C.J., 1977, The rumen protozo of the impl (Aepyeros melmpus) Lihtenstein, n the kuu (Trgelphus strepsieros) Plls, PhD thesis, Deprtment of Zoology, University of Pretori. Kleynhns, C.J. & Vn Hoven, W., 1976, Rumen protozo of the girffe with esription of two new speies, Est Afrin Willife Journl 14, 203 214. Vn er Wth, J.G. & Myurgh, S.J., 1941, Stuies on the limentry trt of merino sheep in. VI. The role of infusori in ruminl igestion with some remrks on ruminnt teri, Onerstepoort Journl of Veterinry Siene n Animl Inustry 17, 61 85. Vn Hoven, W., 1975, Rumen ilites of the Tsessee (Dmlisus luntus luntus) in, Journl of Protozoology 22, 457 462. PMi:811788 Vn Hoven, W., 1978, Development n sesonl hnges in the rumen protozon popultion in young lesok (Dmlisus ors Phillipsi Hrper 1939), South Afrin Journl of Willife Reserh 8, 127 130. Vn Hoven, W., 1983, Rumen ilites with esriptions of two new speies from three Afrin reeuk speies, Journl of Protozoology 30, 688 691.