through the gastrointestinal tract of sheep

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1 Net flux of folates and vitamin through the gastrointestinal tract of sheep C. L. Girard 1 and D. Rémond 3 1 Agriculture et Agroalimentaire Canada, Centre de recherche et développement sur le bovin laitier et le porc, C. P. 90, Lennoxville, Québec, Canada J1M 1Z3; and 3 Unité Nutrition et Métabolisme Protéique, INRA, St-Genès-Champanelle, France. Contribution no. 790, received 17 June 2002, accepted 30 December Girard, C. L. and Rémond, D Net flux of folates and vitamin through the gastrointestinal tract of sheep. Can. J. Anim. Sci. 83: In Trial 1, three wethers were equipped with ruminal and caecal cannulas, catheters in the right ruminal and caecal veins and a mesenteric artery, as well as blood flow probes around the right ruminal and caecal arteries. Blood samples were collected every 15 min from 30 min before to 60 min after the injection of 9 µmol of folic acid and vitamin per kilogram body weight (BW) in the rumen and after injection of half this dose in the caecum. There was a net release of these vitamins from the rumen shortly after the injection (P < 0.05). In Trial 2, four wethers were equipped with a ruminal cannula, catheters in the right ruminal, mesenteric and portal veins and a mesenteric artery and blood flow probes around the right ruminal and the mesenteric arteries and the portal vein. Blood samples were taken 30 and 60 min before, and 30, 60, 120, 180, 240 and 300 min after the intraruminal injection of 0.32 g of folic acid and 0.98 g of vitamin. In Trial 3, blood samples were taken from two wethers previously used in Trial 2, before and 10, 40, 70 and 100 min after the intraruminal injection of 0.32 g of folic acid. These intraruminal injections led to a net release of the vitamins from the mesenteric and portal-drained viscera (P 0.17) but also from the other gastrointestinal tissues, probably mostly from the duodenum. Therefore, in sheep, the main site of absorption for folic acid is the proximal intestine whereas the absorption of vitamin appears in a more distal part of the small intestine. Key words: Sheep, folic acid, vitamin, absorption Girard, C. L. et Rémond, D Flux net de folates et de vitamine en provenance du tractus gastrointestinal du mouton. Can. J. Anim. Sci. 83: Trois moutons équipés de canules dans le rumen et le caecum, de cathéters dans les veines ruminale droite et caecale et une artère mésentérique et de sondes débitmétriques ultrasoniques autour des artères ruminale droite et caecale ont été utilisés lors de l Essai 1. Des échantillons de sang ont été prélevés à toutes les 15 min, de 30 min avant jusqu à 60 min après l injection de 9 µmol d acide folique et de vitamin par kg de PV dans le rumen et de la moitié de cette dose dans le caecum. Il y a eu transfert net de ces vitamines vers la circulation sanguine à travers les parois du rumen rapidement après l injection (P < 0.05). Lors de l Essai 2, 4 moutons ont été équipés d une canule dans le rumen, de cathéters dans les veines ruminale droite, mésentérique et porte et dans une artère mésentérique et de sondes débitmétriques ultrasoniques autour des artères ruminale droite et mésentérique et de la veine porte. Des échantillons de sang ont été prélevés 60 et 30 min avant et 30, 60, 120, 180, 240 et 300 min après l injection intraruminale de 0.32 g d acide ptéroylmonoglutamique et 0.98 g de vitamine. Dans l essai 3, des échantillons de sang ont été prélevés chez 2 des moutons de l essai 2, avant et 10, 40, 70 et 100 min après l injection intraruminale de 0.32 g d acide folique. Ces injections intraruminales d acide folique et de vitamine ont entraîné une libération nette de ces vitamines par les tissus drainés par les veines mésentérique et porte (P 0.17) mais surtout par les autres tissus gastrointestinaux, probablement principalement le duodénum. Chez le mouton, le principal site d absorption de l acide folique est l intestin grêle proximal alors que l absorption de la vitamine se produit dans une section plus distale de l intestin grêle. Production of folates and vitamin by the ruminal microflora is generally considered sufficient to avoid deficiency symptoms in ruminants (McDowell 1989). However, serum folates of ewes decrease from mating to mid-gestation and are greater for prolific than non-prolific breeds (Girard et al. 1996). In dairy cows, serum concentration of vitamin is low in early lactation (Girard and Matte 1999). Indeed, studies on dairy cows indicate that under some circumstances supplements of folic acid and vitamin improve production performance (Girard et al. 1995; Girard and Matte 1997, 1998). These results suggest that ruminal production is not sufficient to maximize the production traits and, under specific conditions, ruminants could benefit from vitamin supplementation. Nevertheless, little effort has been made to understand the mechanisms of vitamin absorption in ruminants. Rérat et al. (1958a) did not Mots clés: Mouton, acide folique, vitamine, absorption 273 succeed in demonstrating vitamin absorption through the rumen wall of fed sheep, but when a vitamin solution was infused into an empty rumen, vitamin reached blood circulation across the rumen wall. Moreover, the rumen wall could retain the vitamin and release it later into the blood or the rumen cavity (Rérat et al. 1958b). Similarly, Smith and Marston (1970) failed to detect vitamin absorption from the rumen of sheep, but they also observed an accumulation of vitamin in the rumen wall. To our knowledge, there is no information in the literature on the fate of folic acid in the gastrointestinal tract of sheep. The present paper reports a series of trials designed to elucidate the fate of vitamin and folic acid across the Abbreviations: BW, body weight

2 274 CANADIAN JOURNAL OF ANIMAL SCIENCE gastrointestinal tract of sheep. In order to measure the net flux of vitamins through the different parts of the gastrointestinal tract, concentrated solutions of these two vitamins were injected into the rumen or caecum of sheep. MATERIALS AND METHODS Trial 1 Three mature Texel wethers (45 ± 5 kg BW) were used. They were fitted with ruminal and caecal cannulae, indwelling catheters in the right ruminal and caecal veins and a mesenteric artery, and blood flow probes (Transonic Systems Inc., Ithaca, NY) around the right ruminal and caecal arteries. The catheter in the right ruminal vein and the probe around the ruminal artery were implanted as described by Rémond et al. (1993b). Like for probes placed around the ruminal artery, probes (Type 3R) used on caecal artery were wrapped in silicone strips. Surgical procedures and post-surgical care were conducted in accordance with French national legislation on the care and use of laboratory animals. Whethers were housed in individual pens (1 1.5 m) in a room under continuous lighting with controlled temperature (19 to 23 C). Sheep were given a diet (Table 1) containing 700 g of chopped hay and 300 g of concentrate distributed in equal portions at 0030 and These amounts ensured rapid and complete consumption of each meal. The sheep had free access to water and a salt block. The experiment began about 8 wk after surgery. The last 4 h of a feeding cycle were chosen for the vitamin injections because during this period ruminal blood flow and physicochemical conditions in the rumen are nearly stable (Rémond et al. 1993a). A solution (95 ± 10 ml), at ph 7, containing 9 µmol of pteroylmonoglutamic acid (folic acid) and cyanocobalamin (vitamin ) per kilogram BW was injected in the rumen. It represented ± g of pteroylmonoglutamic acid and ± g of cyanocobalamin. The amount of pteroylmonoglutamic acid was determined from a production study conducted on cows (Girard and Matte 1998) and adapted to sheep on a BW basis; the same amount of cyanocobalamin was given on a molar basis. Half of these doses and volumes were injected into the caecum. Ruminal and caecal injections were performed simultaneously at Blood samples (13 ml per catheter) were collected at 15-min intervals from 30 min before to 60 min after the injection. Blood flow was recorded continuously from 15 min before the first blood sampling to 15 min after the last sampling. Each sheep received the treatment twice, with at least 1 wk between experiment days. Data were pooled as a mean of the 2 d. Trial 2 Four mature Texel wethers (64 ± 3 kg BW) were surgically prepared with a ruminal cannula, indwelling catheters and blood flow probes. The catheters were implanted in the right ruminal vein, the cranial mesenteric vein, the portal vein and a mesenteric artery. Blood flow probes were placed around the right ruminal artery, the cranial mesenteric artery and the portal vein. The catheters, equipment and surgical procedures have been described previously (Rémond et al. 2000). Surgical procedures and post-surgical care were conducted in Table 1. Composition of diet in Trial 1 Item % as fed Ingredient composition Chopped orchardgrass hay 70.0 Ground corn 16.9 Soybean meal 10.9 Molasses 1.7 Fat-soluble vitamin and mineral premix 0.5 Chemical composition Dry matter (%) 84.9 Crude protein (% of DM) 14.0 Metabolizable energy (MJ kg 1 DM) 10.6 accordance with the French national legislation on the care and use of laboratory animals. The sheep were housed as described in Trial 1. They were offered a diet containing 800 g of chopped orchardgrass hay and 350 g of extruded peas, providing 274 g of N d 1 and 10.6 MJ of ME d 1. Feed was delivered in 12 equal portions at 2 h-intervals from an automatic feeder. The sheep were given free access to water and a salt block. The experiment started approximately 5 wk after surgery. On experiment days, 1 h 45 min after the meal at 0800, the sheep received an intraruminal injection of 0.32 g of pteroylmonoglutamic acid and 0.98 g of cyanocobalamin diluted in 400 ml of water. Blood samples (8 ml) were simultaneously withdrawn through the four catheters 30 and 60 min before and 30, 60, 120, 180, 240 and 300 min after the injection. Blood flow was recorded as described in Trial 1. Trial 3 This trial was performed to get a clearer picture of pteroylmonoglutamate absorption within the first 2 h following ruminal injection. It was conducted on two sheep from Trial 2. Environmental conditions were similar to Trial 2. On experiment days, the sheep received an intraruminal injection of 0.32 g pteroylmonoglutamic acid diluted in 400 ml of water. Blood samples (8 ml) were simultaneously withdrawn through the four catheters 110 and 50 min before and 10, 40, 70 and 100 min after the injection. Laboratory Analysis Immediately after collection, blood was transferred from the syringes to EDTA-treated tubes (2.7 ml) for haemoglobin determination and to tubes without additives for vitamin determinations. Packed cell volume was measured in duplicate on fresh blood by microcentrifugation. Haemoglobin was measured on fresh blood as described by Rémond et al. (1993a). Blood was left to clot in the dark at room temperature for 2 h. Serum was collected after centrifugation at 3800 g for 10 min at 4 C and frozen at 20 C for vitamin determination. Folates and vitamin were determined in duplicate by radioassay with a commercial kit designed for human plasma or serum [Quantaphase Folate II and Quantaphase, Bio-Rad Laboratories (Canada) Ltd, Mississauga, ON], validated for sheep serum by conducting recovery and parallelism tests (Girard et al. 1996, 1999). Inter-assay coefficients of variation were 3.36 and 4.31% for folates and vitamin in sheep serum, respectively.

3 GIRARD AND RÉMOND ABSORPTION OF FOLIC ACID AND VITAMIN IN SHEEP 275 Statistical Analysis The net flux of folic acid or vitamin across rumen and caecal walls, mesenteric and portal-drained viscera was calculated according to the following equation: Net flux = ([X] v PF v ) ([X] a PF a ) where [X] v and [X] a are the concentrations of the studied vitamins in plasma from venous (v) and arterial (a) blood and PF v and PF a are the plasma flow in vein and artery, respectively. For each sampling time, the plasma flow in the ruminal, caecal and mesenteric arteries or portal vein was obtained by calculating the mean whole blood flow recorded by the flow probe from 5 min before to 5 min after blood sampling, corrected for the packed cell volume. The plasma flow in the right ruminal, caecal or mesenteric veins or in the arteries irrigating portal-drained tissues was calculated from the blood flow from the corresponding artery or vein corrected with haemoglobin concentration ratio between vein and artery to correct fluxes for water movement across vessels. For Trials 2 and 3, net flux from the other gastrointestinal tissues was calculated as: Net flux from the portal-drained viscera [(2 net flux from the right rumen) + net flux from the mesenteric tissues]. This value represents an evaluation of the net release (or uptake) by the omasum, abomasum, duodenum, spleen, pancreas, omental fat, and large intestine (Getty 1975; Barone 1996). For the vitamin flux, a negative value indicates net tissue removal whereas a positive value denotes net release. For each trial, mean values of samples collected before the injection were used as the baseline level (T0). Data were analyzed using the repeated statement of the Mixed procedure of SAS software with a spatial power adjustment for the variance-covariance matrix (SAS Institute, Inc. 1999). Only the time effect was included in the model as there were no comparisons to be done between animals. When the P value for the effect of time was smaller than 0.20, the adjusted mean for each time was compared to the mean at T0 with a Dunnett-type correction. Based on similar studies (Guerino et al. 1991; Reynolds et al. 1991), the cut-off values for degree of significance used throughout the manuscript were as follows: means were considered significantly different at P 0.10 and to tend to differ at 0.10 P Morevoer, this choice of cut-off values was reinforced by the fact that with absorption curves like those obtained in the present trials, the high variability among animals is mainly due to the fact that the events, as peak of absorption, were generally not perfectly synchronized among animals. RESULTS Trial 1 Plasma flow rates in the right ruminal artery and the caecal artery were ± 4.7 and 41.2 ± 4.4 ml min 1, respectively. Concentrations of folates (P = 0.02) and vitamin (P = 0.05) in arterial plasma increased after ruminal and caecal injections of the vitamins (Table 2). The net flux of folates across the rumen wall changed with time (P = 0.05; Table 2); it was greater than before the injection during the 60 min-period following the ruminal injection (P 0.09). The net flux of vitamin across the rumen wall also changed with time (P = 0.02; Table 2). However, only the net flux observed 60 min after the intraruminal injection differed significantly from the preinjection flux (P = 0.006). To evaluate the flux from the entire rumen, the net flux recorded across the right rumen vein can be multiplied by two (Rémond et al. 1993b). Thus, during the 60-min period following the injection, a net release of 11.8 µg of folates and 15.7 µg of vitamin was observed, roughly representing and 0.003% of the dose of folic acid and vitamin, respectively. The net flux of folates and vitamin across the caecum wall tended to change with time (P = 0.2; Table 2). For the two vitamins, only the net flux observed 15 min after the caecal injection of vitamins (P = 0.07) differed from net flux before the caecal injection. Trial 2 Plasma flow rates were ± 17.2, ± 3.5 and ± 5.8 ml min 1 in the portal vein, the right ruminal artery and the mesenteric artery, respectively. Concentrations of folates in arterial plasma increased throughout the experimental period (P = ; Table 3). The net flux of folates through the portal-drained viscera tended to vary with time (P = 0.17; Table 3); it tended to differ from the pre-injection flux at 120 (P = 0.12), 180 (P = 0.12) and 240 (P = 0.02) min after the injection. However, the net flux of folates through the rumen wall (P = 0.99) and the mesenteric tissues (P = 0.58) did not change with time. Therefore, most of the folic acid reaching blood circulation, 2.0% of the infused dose, was transferred across gastrointestinal tissues other than rumen and small intestine. The net flux of folates through these parts of the gastrointestinal tract tended to change with time (P = 0.19; Table 3) and to be greater than before the injection from 120 to 240 min post-injection (P = 0.10; 0.17 and 0.03, respectively). Concentrations of vitamin in arterial plasma increased during the experimental period (P = ; Table 4). Net flux of vitamin through the rumen wall did not change following the ruminal injection of vitamins (P = 0.22; Table 4). The net flux across mesenteric (P = 0.04) and portal-drained (P = 0.11) tissues varied during the experimental period (Table 4). The net flux across portal-drained tissues differed from the pre-injection level from 120 to 300 min post-injection (P = 0.12, 0.01, 0.11 and 0.07, respectively); approximately 0.09% of the injected dose reached portal circulation. The net flux across mesenteric tissues differed from the pre-injection level 240 and 300 min post-injection (P = 0.05 and P = 0.06). Net flux from gastrointestinal tissues other than rumen and small intestine did not change during the studied period (P = 0.36).

4 276 CANADIAN JOURNAL OF ANIMAL SCIENCE Table 2. Arterial concentrations and net flux of folates and vitamin across the wall of the right rumen and the caecum before and after an injection of vitamins in rumen or caecum (Trial 1; n = 3) z Time post-injection (min) P Folates Arterial concentrations (ng ml 1 ) 1.09 ± ± ± ± ± Net flux across the right rumen 9.19 ± ± ± ± ± wall (ng min 1 ) Net flux across the caecum 1.09 ± ± ± ± ± wall (ng min 1 ) Vitamin Arterial concentrations (ng ml 1 ) 6.52 ± ± ± ± ± Net flux across the right rumen 8.34 ± ± ± ± ± wall (ng min 1 ) Net flux across the caecum wall (ng min 1 ) 2.45 ± ± ± ± ± Table 3. Arterial concentrations and net flux of folates from the rumen, portal-drained, mesenteric and the other gastrointestinal tissues before and after an intraruminal injection of vitamins (Trial 2; n = 4) z Trial 3 Plasma flows were ± 77.6, ± 8.9 and ± 42.3 ml min 1 in the portal vein, the right ruminal artery and the mesenteric artery, respectively. Arterial concentrations of folates increased during the experimental period (P = 0.004: Table 5). The net portal flux of folates changed with time (P = 0.08: Table 5); it was greater than the pre-injection flux at 40 min (P = 0.16) and 70 min (P = 0.05) following the ruminal injection of folic acid. The net flux of folates through the rumen wall tended to change with time; it was significantly higher than the basal level only at 10 min post-injection (P = 0.11; Table 5) but was lower 70 min (P = 0.10) and 100 min (P = 0.06) after the injection in the rumen. One hundred minutes after the ruminal injection of folic acid, the net flux through mesenteric tissues was lower than before the injection (P = 0.09: Table 5). The net flux of folates through the remaining part of the gastrointestinal tract, omasum, abomasum, duodenum and large intestine, tended to vary with time (P = 0.11); it was higher than before the injection at 70 min (P = 0.06) and 100 min (P = 0.15) post-injection (Table 5). DISCUSSION In the present study, about 2.7% of the folic acid injected into the rumen reached the portal vein of the sheep, and this Time post-infusion (min) P Arterial concentrations (ng ml 1 ) 1.55 ± ± ± ± ± ± ± Rumen net flux (µg min 1 ) ± ± ± ± ± ± ± Portal-drained viscera net flux (µg min 1 ) 0.27 ± ± ± ± ± ± ± Mesenteric net flux (µg min 1 ) 0.18 ± ± ± ± ± ± ± Other gastrointestinal tissues net flux (µg min 1 ) 0.40 ± ± ± ± ± ± ± absorption took place within the 4 h following the injection (Trial 2). Similarly, in dairy cows, 98% of the amount of folates released from the portal-drained viscera during the 24 h following ingestion of the vitamin are transferred during the first 6 h, and this release accounted for 2.5 to 4.3% of the dose of folic acid ingested (Girard et al. 2001). It is noteworthy that although portal recovery was low, the arterial concentration of folates was about eight times greater 5 h after than before the vitamin supply for both species. A net release of folic acid was observed across the rumen wall soon after intraruminal injection. Similar observations were made in dairy cows, in which a net release of folic acid across the rumen wall was seen during the first 20 min following an infusion of this vitamin into the rumen (Girard et al. 2001). These observations suggest that rumen absorption of folic acid is significant only when high rumen concentrations are generated. Thus although the rumen wall possesses the capacity to absorb folic acid, the net transfer across its epithelium seems low. In monogastric animals, folic acid is efficiently absorbed in the distal duodenum and jejunum by both a saturable active process and by passive diffusion (Le Grusse and Watier 1993; Combs 1998). In the present study there was no significant positive net flux of folates from the mesenteric tissues (Trial 2). However, the cranial mesenteric vein,

5 GIRARD AND RÉMOND ABSORPTION OF FOLIC ACID AND VITAMIN IN SHEEP 277 Table 4. Arterial concentrations and net flux of vitamin from the rumen, portal-drained, mesenteric and the other gastrointestinal tissues before and after intraruminal injection of vitamins (Trial 2; n = 4) z at the sampling site, does not take into account the duodenum and probably part of the proximal jejunum (Neutze et al 1994). When CrEDTA (liquid phase marker) is injected into the rumen of sheep, it appears at the duodenum in less than 1 h, and the greatest concentrations in duodenal samples are observed between 3 and 4 h postinjection (L. Bernard, personal communication). Therefore, in the present study it was not possible to conclude from which segment of the gut between the rumen and the jejunum (omasum, abomasum and duodenum) folate absorption took place. As for the rumen, this vitamin could be transferred across the omasum wall and contribute to its absorption when high luminal concentrations are generated. Nevertheless it seems reasonable to hypothesize that in the present study most of the absorption occurred in the duodenum and the first part of the jejunum. In dairy cows, during the 24 h following ingestion of a vitamin supplement, 0.27% of the dose ingested was released from the portal-drained viscera according to a biphasic pattern, 49% of this amount appeared 4 to 10 h after the meal whereas 35% appeared 20 to 24 h after ingestion of the supplement (Girard et al. 2001). Calculations show that 0.13% of the dose ingested was released from the portaldrained viscera within the first 10 h following the supplemented meal. In the present study, the portal recovery of vitamin within the period studied (5 h) was 0.09% of the dose. However, 5 h postinjection, portal net release of vitamin was still high and the portal recovery was probably underestimated because the absorption process was not complete. In monogastric animals, vitamin in low doses (1 2 µg) is absorbed efficiently in the terminal ileum by an Time post-infusion (min) P Arterial concentrations (ng ml 1 ) 1.86 ± ± ± ± ± ± ± Rumen net flux (ng min 1 ) ± ± ± ± ± ± ± Portal-drained viscera net flux (ng min 1 ) ± ± ± ± ± ± ± Mesenteric net flux (ng min 1 ) ± ± ± ± ± ± ± Other gastrointestinal tissues net flux (ng min 1 ) ± ± ± ± ± ± ± Table 5. Arterial concentrations and net flux of folates from the rumen, portal-drained, mesenteric and the other gastrointestinal tissues before and after intraruminal injection of folic acid (Trial 3; n = 2) z Time post-injection (min) P Arterial concentrations (ng ml 1 ) 1.16 ± ± ± ± ± Rumen net flux (µg min 1 ) ± ± ± ± ± Portal-drained viscera net flux (µg min 1 ) ± ± ± ± ± Mesenteric net flux (µg min 1 ) ± ± ± ± ± Other gastrointestinal tissues net flux (µg min 1 ) ± ± ± ± ± active saturable transport involving binding proteins as intrinsic factor and transcobalamin (Combs 1998; Quadros et al. 1999). At high doses, vitamin is absorbed by diffusion through the small intestine but the efficiency of this process is very low, around 1% of the amount ingested (Le Grusse and Watier 1993; Combs 1998). After perfusing the rumen and the small intestine of fed sheep with blood from other sheep and measuring changes in concentrations of vitamin in blood after 2 h of perfusion, Rérat et al. (1958a) did not demonstrate absorption of vitamin through the rumen wall, but showed absorption in the distal small intestine. Similarly, from the distribution of a radioactive dose of vitamin, Smith and Marston (1970) concluded that in sheep, the site of absorption of vitamin is the small intestine although they measured an important accumulation of vitamin in the rumen wall. However, using measurements of blood enrichment in the vitamin, Rérat et al. (1958b) showed some absorption of vitamin through the rumen wall when the vitamin solution was placed in the empty rumen of sheep. In dairy cows, there is a positive net flux of vitamin through the rumen wall from 15 to 60 min after a ruminal infusion of vitamins but the amount transferred through the rumen wall is small, % of the amount infused (Girard et al. 2001). In sheep, a net release of vitamin from the rumen was detected 60 min post-injection (Trial 1), but the proportion of the amount injected that was transferred to blood circulation was higher than in cows (0.002%). Thus, in agreement with Rérat et al. (1958b), this study showed that the ruminal wall of sheep has the capacity to absorb vitamin when high concentrations of this vitamin are generated in the liquid phase of

6 278 CANADIAN JOURNAL OF ANIMAL SCIENCE the rumen. In Trial 2 significant net release of vitamin in the mesenteric vein was observed from 4 to 5 h after the injection. At these times, the release from mesenteric tissues accounted for 43 to 49% of the portal net appearance. Therefore, in sheep, the main site of absorption appeared to be the distal small intestine. Results from Trial 1 showed a trend towards a release of vitamins from the caecum. As synthesis of B-vitamins by the microflora has been demonstrated in the caecum of other species (McDowell 1989), absorption of these vitamins across the caecum wall could be of physiological significance, even in the absence of dietary supplements of vitamins. CONCLUSION Data from the present experiment demonstrate that, in spite of extensive losses in the gastrointestinal tract, significant amounts of folic acid and vitamin from an exogenous source were released by the portal-drained viscera. Understanding the fate of these supplements will help in the design of further studies on metabolism of these two vitamins in the ruminant. Results from the present experiment demonstrate that, as for monogastrics, the main site of absorption of folic acid is the proximal intestine whereas the absorption of vitamin appears more distal in the small intestine. IMPLICATIONS In sheep, the main site of absorption of supplements of folic acid and vitamin is the small intestine. Therefore, if dietary supplements of these vitamins are used to improve animal performance, the amounts given should be sufficient to insure that significant amounts of these vitamins would reach the small intestine in spite of destruction or utilization by ruminal microorganisms. ACKNOWLEDGEMENTS The authors are grateful to L. Bernard, J. P. Chaise, E. Delval and Chrystiane Plante for technical assistance. Barone, R Anatomie comparée des mammifères domestiques. Vol. 5. Angiologie. Editions Vigot, Paris, France. Combs, G. F., Jr The vitamins. Fundamental aspects in nutrition and health. 2nd ed. Academic Press, San Diego, CA. 618 pp. Getty, R The anatomy of the domestic animals. Vol. 1. 5th ed. W.B. Saunders Co., Philadelphia, PA. Girard, C. L. and Matte, J. J Parenteral supplements of vitamin and milk performance of dairy cows. J. Dairy Sci. 80 (Suppl. 1): 240 (Abstr.). Girard, C. L. and Matte, J. J Dietary supplements of folic acid during lactation: Effects on the performance of dairy cows. J. Dairy Sci. 81: Girard, C. L. and Matte, J. J Changes in serum concentrations of folates, pyridoxal, pyridoxal-5-phosphate and vitamin during lactation of dairy cows fed dietary supplements of folic acid. Can. J. Anim. Sci. 79: Girard, C. L., Castonguay, F., Fahmy, M. H. and Matte, J. J Serum and milk folates during the first two gestations and lactations in Romanov, Finnsheep, and Suffolk ewes. J. Anim. Sci. 74: Girard, C. L., Castonguay, F. and Matte, J. 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