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Biology of Reproduction 66, 1797-1804 (2002)
© 2002 Society for the Study of Reproduction, Inc.

Regular Article

Effect of Elevated Systemic Concentrations of Ammonia and Urea on the Metabolite and Ionic Composition of Oviductal Fluid in Cattle1

D.A. Kennya,b, P.G. Humphersonc, H.J. Leesec, D.G. Morrisa, A.D. Tomosd, M.G. Diskina, and J.M. Sreenan2,,a

a Teagasc Research Centre, Athenry, Co. Galway, Ireland b Department of Animal Science and Production, Faculty of Agriculture, National University of Ireland Dublin, Dublin 4, Ireland c Department of Biology, University of York, York YO10 5YW, United Kingdom d School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd, LL57 2UW, Wales, United Kingdom


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High dietary protein leads to elevated systemic concentrations of ammonia and urea, and these, in turn, have been associated with reduced fertility in cattle. The effect of elevating systemic concentrations of ammonia and urea on the concentrations of electrolytes and nonelectrolytes in bovine oviductal fluid were studied using estrus-synchronized, nulliparous heifers (n = 25). Heifers were randomly assigned to 1 of 3 treatments consisting of jugular vein infusion with either ammonium chloride (n = 8), urea (n = 8), or saline (n = 9). Oviducts were catheterized, and fluid was recovered over a 3-h period on either Day 2 or 8 of the estrous cycle. No difference (P > 0.05) was found in the concentrations of any electrolyte or nonelectrolyte between oviducts ipsi- or contralateral to the corpus luteum. Plasma and oviductal concentrations of urea were increased by infusion with urea (P < 0.001) and ammonium chloride (P < 0.05) but not by saline (P > 0.05). Plasma and oviductal concentrations of ammonia were elevated by infusion with ammonium chloride (P < 0.001) but not by infusion with urea or saline (P > 0.05). No effect (P > 0.05) of treatment was found on oviductal or plasma concentrations of glucose, lactate, magnesium, potassium, or sodium or on plasma concentrations of insulin or progesterone. The concentration of calcium in oviductal fluid was reduced by urea infusion and was negatively associated with systemic and oviductal concentrations of urea. Oviductal concentrations of sodium were higher on Day 8 than on Day 2 (P < 0.05). No effect of sample day was found on any of the other electrolytes or nonelectrolytes measured (P > 0.05). Elevated systemic concentrations of ammonia and urea are unlikely to reduce embryo survival through disruptions in the oviductal environment.

embryo, female reproductive tract, fallopian tubes, fertilization, oviduct


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
High dietary protein leads to elevated systemic concentrations of ammonia and urea, and these, in turn, have been associated with reduced fertility in cattle [1–3]. Whereas evidence relating to the extent of the reduced fertility is equivocal, a number of possible mechanisms by which elevated systemic ammonia or urea may reduce cow fertility have been proposed. Reports have suggested that elevated systemic concentrations of ammonia and/or urea may compromise early embryo development in the oviduct [4–8]. The importance of oviductal fluid as an environment that facilitates gamete transport and maturation, fertilization, and early embryo development has been recently documented [9]. Because it provides the nutritional environment for the early embryo, the metabolite and ionic composition of oviductal fluid is clearly important. However, to our knowledge, no data are available regarding the effect of elevated systemic concentrations of either ammonia or urea on the composition of oviductal fluid. Furthermore, most attempts at collecting oviductal fluid in the cow have involved chronic catheterization of ligated oviducts, exteriorization of the cannulae, and fluid collection over a period of several days or weeks [10–16]. This approach, involving tubal ligation and long, exteriorized cannulae, likely leads to artifacts and/or losses in constituents. To collect oviductal fluid in as physiological a manner as possible, we have used a simple technique, involving gentle insertion and tying of a catheter in the ampulla of the oviduct. Clear tubal fluid emerges in the catheter and may be collected over a 3-h period without recourse to tubal ligation.

The objectives of the present study were to establish the relationship between systemic and oviductal concentrations of ammonia and urea and to examine the effects of elevated systemic concentrations of ammonia and urea on the concentration of other constituents in oviductal fluid to gain a better understanding of the possible role played by these catabolites in protein-mediated infertility in cattle.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Twenty-five nulliparous, cross-bred beef heifers with a mean ± SEM body weight and body condition score [17] of 384 ± 6 kg and 3.22 ± 0.06 units, respectively, were used in this study. Estrous cycles were synchronized using 2 injections of a synthetic prostaglandin (PG) F2{alpha} analogue (500 µg of cloprostenol; Estrumate; Coopers Animal Health, Berkhamsted, U.K.) administered i.m. 11 days apart. Observations for estrous activity were carried out at 0800, 1200, 1600, and 2100 h, starting 24 h after each of the PGF2{alpha} injections. Only animals that were recorded in standing estrus following the second PGF2{alpha} injection were used for oviductal fluid recovery.

Experimental Design

To elevate systemic concentrations of ammonia and urea, heifers were randomly allocated to 1 of 3 infusion treatments: saline (control), urea, or ammonium chloride. Treatments were carried out on either Day 2 or Day 8 of the estrous cycle (estrus = Day 0) in a 2 x 3 factorial study as follows: saline on Day 2 (n = 5) or Day 8 (n = 4); urea on Day 2 (n = 4) or Day 8 (n = 4); and ammonium chloride on Day 2 (n = 4) or Day 8 (n = 4).

Infusion Protocol

At 24 h before the scheduled collection of oviductal fluid, heifers were sedated with 0.1 mg/kg of xylazine (Chanazine, 2% [w/v]; Chanelle Pharmaceuticals Manufacturing Ltd., Loughrea, Co. Galway, Ireland). Cannulae were then inserted approximately 10 cm into the left and the right jugular veins; filled with sterile, heparinized saline (55 IU/ml); and checked regularly to ensure patency. Heifers were then housed in tie stalls, where they remained before recovery of oviductal fluid.

The infusion protocol and the desired concentrations of experimental solutions were validated in a preliminary experiment. The concentration of ammonium chloride used was adapted from that reported by Fernandez et al. [18]. Experimental solutions were infused into the left jugular vein using a peristaltic pump (Watson Marlow Ltd., Falmouth, Cornwall, U.K.), and infusion was initiated 4 h before the commencement of and throughout oviductal fluid recovery.

Animals on the ammonia treatment were infused with a solution of ammonium chloride (NH4Cl; Merck, Darmstadt, Germany) and saline (0.9% NaCl) solution at a rate of 15 µmol-1 of NH4Cl kg-1 min-1 over a period of 420 min to elevate systemic concentrations of ammonia. Animals on the urea treatment were first infused with a loading dose of a urea-saline solution at a rate of 2100 µmol-1 of urea kg-1 min-1 over a period of 25 min to rapidly increase basal systemic concentrations of urea. Systemic concentrations of urea were then maintained by the subsequent, continuous infusion of a urea-saline solution at a rate of 9.7 µmol-1 of urea kg-1 min-1 over a period of 395 min. Control animals were infused with physiological saline (0.9% NaCl) at the same pump rate as those on the ammonia and urea treatments.

Oviductal Fluid Recovery

Heifers were fasted from food for 24 h and from water for 12 h before oviductal fluid recovery, which was carried out during midventral laparotomy under license in accordance with the European Community Directive 86-609-EC. Anesthesia was induced using thiopentone sodium (5 g i.v.; Rhone Merieux, Harlow, U.K.) and maintained over the collection period by closed-circuit anesthesia with halothane (May and Baker Ltd., Dagenham, U.K.) and oxygen. Oviduct catheters (length, 35 cm) were prepared from polyethylene tubing (Portex Ltd., Hythe, Kent, U.K.) with an internal diameter of 2.16 mm and an external diameter of 3.25 mm and were sterilized before use. Following laparotomy, the ovaries and oviducts were exteriorized, and a catheter was inserted through the tubal ostium of each oviduct to a distance of approximately 3 cm. The catheters were retained within the oviduct using two ligatures of absorbable suture material, with care being taken not to occlude the lumen. The ovaries and catheterized oviducts were returned to the peritoneal cavity with the distal ends of each catheter exteriorized and covered with sterile surgical gauze. Oviductal fluid was allowed to accumulate in the catheter for a period of 3 h. At the end of the collection period, the catheters were removed, and the volume of fluid was recorded. The fluid was then transferred to 1.5-ml tubes, centrifuged at 15 000 x g for 7 min, snap-frozen in liquid nitrogen (-196°C), and stored at -80° C until analysis.

Blood Sampling

Blood was sampled for determination of plasma concentrations of ammonia, urea, glucose, insulin, and progesterone. Samples were taken at -30, -15, 0 (start of infusion), 30, 60, 120, 180, 240, 300, 360, and 420 min. During the 3-h period of oviductal fluid collection, additional blood samples were taken for determination of plasma concentrations of lactate, calcium, potassium, magnesium, and sodium. The anticoagulants used were EDTA for insulin and progesterone samples, lithium fluoride for glucose and lactate samples, and lithium heparin for ammonia, urea, calcium, potassium, magnesium, and sodium samples. At each sampling time, 25 ml of blood were withdrawn from the right jugular vein cannula. Heparinized saline was used to ensure patency of the cannula and to prevent coagulation of blood between samplings. At each sample, 5 ml of blood were withdrawn from the cannula and discarded to prevent contamination from the anticoagulant solution. A 20-ml blood sample was then taken with a sterile syringe and divided between the respective blood-sampling vials.

Immediately after sampling, blood was centrifuged at 1250 x g at 4°C for 15 min. Plasma was separated, and samples for determination of insulin, glucose, and progesterone concentration were stored at -20°C until assay. Plasma samples for ammonia and urea analysis were frozen in liquid nitrogen immediately after harvesting and stored at -80°C until assay. To allow a direct comparison, aliquots from the plasma samples collected during oviductal fluid collection were treated in a manner similar to the oviductal fluid samples, centrifuged immediately after collection, and frozen in liquid nitrogen.

Metabolite and Ion Assays

Plasma and oviductal fluid concentrations of urea and ammonia were determined using an autoanalyzer (Cobas Mira; Roche Products Ltd., Welwyn Garden City, Hertfordshire, U.K.). The method was based on two reactions: first, the hydrolysis of urea with urease; and second, the reaction of the resulting ammonia with 2-oxoglutarate in the presence of glutamate dehydrogenase and NADH to form L-glutamate and NAD+. The amount of NADH oxidized is directly proportional to the amount of urea present. Plasma and oviductal fluid concentrations of ammonia were determined using reaction 2 of the above method, with the amount of NAD+ formed being proportional to the amount of ammonia present. Test kits for determination of urea were supplied by Roche (ref. no. 0736856) and for ammonia by Sigma (catalog no. 171-UV; Poole, Dorset, England, U.K.).

Plasma and oviductal concentrations of glucose and lactate were measured using an autoanalyzer (Cobas Mira). Glucose concentration was determined by the glucose oxidase-peroxidase test combination, purchased from Boehringer Mannheim (ref. no. 124036; Lewes, East Sussex, England, U.K.). Lactate was assayed by monitoring the formation of NADH from NAD+, catalyzed by lactate dehydrogenase in the presence of hydrazine sulfate [19].

Plasma insulin was measured by a 125I-labeled insulin double-antibody RIA as previously described [20]. The sensitivity of the assay was 2.8 µIU/ml. The intra- and interassay coefficients of variation were 0.16 and 0.05 and 0.14 and 0.15 for pools of plasma that contained 4.86 and 21.02 µIU/ml of insulin, respectively.

Cation concentrations were measured using a Dionex 2000i ion chromatograph (Camberley, Surrey, U.K.) fitted with a CS12 cation-exchange column and a self-regenerating cation suppressor operated in auto-regeneration mode. The eluant was 20 mM methanesulfonic acid, and the column was operated at 50°C. The system was coupled to a Shimadzu CR5A plotting integrator (Milton Keynes, U.K.). Ten microliters of 1 mM CsCl were mixed with 2.5 µl of sample in a 200-µl microcentrifuge tube. Five microliters of this mixture were manually injected onto the column. Quantification of K+, Mg2+, and Ca2+ were based on the ratio of peak heights to that of the internal standard. For Na+ determination, 2 µl of sample were diluted with 498 µl of eluant, and quantification was based on external standards. Two runs at different dilutions were necessary because of the large differences between the concentrations of Na+ and those of the other cations.

Progesterone Assay

Plasma progesterone was measured without previous extraction and using a 125I-labeled progesterone double-antibody RIA as previously described [20]. The sensitivity of the assay was 0.14 ng/ml, and the intra- and interassay coefficients of variation were 0.12 and 0.14 and 0.11 and 0.12 for plasma controls with progesterone concentrations of 0.23 and 10.17 ng/ml, respectively.

Statistical Analysis

Results are presented as least square mean ± SEM. Plasma and oviduct metabolite and ion concentrations as well as volumes of oviductal fluid were analyzed using factorial structured ANOVA (PROC GLM; Statistical Analysis Systems Institute, Cary, NC) [21], regression, and correlations to determine the relationship and effects of infusion treatment on the volume of oviductal fluid, systemic and oviductal fluid metabolite and ionic concentrations, and systemic concentrations of progesterone. Repeated measures were used to analyze metabolite and progesterone data over time (PROC GLM) [21].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Infusion Treatment and Volume of Oviductal Fluid Recovered

No day of sampling x treatment interaction (P > 0.05) or day of sampling x treatment x oviduct interaction (P > 0.05) on the volume of oviductal fluid recovered was observed. No effect of treatment on the volume of oviductal fluid recovered was observed (P > 0.05). The volume of fluid recovered over the 3-h collection period tended to be higher from the oviduct ipsilateral to the corpus luteum (504.77 ± 61.326 µl vs. 333.20 ± 60.062 µl, P = 0.05) and also tended to be higher on Day 2 than on Day 8 (499.72 ± 59.007 µl vs. 338.249 ± 62.953 µl, P = 0.07).

Infusion Treatment and Plasma Metabolite and Ionic Concentration

No treatment x day of sampling interaction for plasma concentrations of urea, ammonia, glucose, or insulin was observed (P > 0.05), but an interaction of infusion treatment x time of sampling was observed for plasma urea. The effect of infusion treatment on plasma concentrations of urea, ammonia, glucose, and insulin are presented in Figure 1. In animals infused with urea, plasma concentrations of urea were higher during the infusion period than during the preinfusion period (P < 0.001). Plasma concentrations of urea were higher (P < 0.001) in animals infused with urea at all sample times during the infusion period than in animals infused with either saline or ammonium chloride. Plasma urea was not affected by infusion of either saline or ammonium chloride (P > 0.05).



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  		FIG. 1. Effect of infusion treatment on mean plasma concentrations of insulin (A), glucose (B), ammonia (C), and urea (D)

An infusion treatment x time of sampling was observed for plasma ammonia. In animals infused with ammonium chloride, plasma concentrations of ammonia were higher during the infusion period than during the preinfusion period (P < 0.001). Plasma concentrations of ammonia were higher (P < 0.001) in animals infused with ammonia at all sample times during the infusion period than in animals infused with either saline or ammonium chloride. Plasma concentration of ammonia was not affected by the infusion of either saline or urea (P > 0.05).

No treatment x day of sampling interaction was observed for glucose (P > 0.05). Plasma concentration of glucose was not affected by infusion treatment or by day of sampling (P > 0.05). Glucose was affected by sample time (P < 0.01) and increased in all treatments over time (Fig. 1).

No treatment x day of sampling interaction was observed for insulin (P > 0.05). Plasma concentrations of insulin were not affected by treatment, day of sampling, or time of sampling (P > 0.05).

No treatment x day of sampling interaction was observed for plasma concentrations of lactate, potassium, sodium, magnesium, and calcium (P > 0.05). No effect of treatment (P > 0.05) or of day of sampling (P > 0.05) was observed on plasma concentrations of lactate, potassium, sodium, and magnesium (Table 1).


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  		TABLE 1. Effect of infusion treatment and day of estrous cycle on plasma concentrations of lactate, K+, Na+, Mg2+, and Ca2+.a

Infusion Treatment and Plasma Concentration of Progesterone

No treatment x day of sampling x time of sampling interaction was observed for progesterone (P > 0.05). Plasma progesterone concentration was affected by day of sampling (P < 0.05) but not by treatment or by time of sampling (P > 0.05).

Infusion Treatment and Oviductal Fluid Metabolite and Ionic Concentration

The effect of infusion treatment on oviductal fluid metabolite concentrations is presented in Table 2. No treatment x day of sampling interaction was observed for any variable measured (P > 0.05). Oviductal fluid sodium concentrations were affected by day of sampling and were higher on Day 8 than on Day 2 (P < 0.05). No effect of day of sampling (P > 0.05) was observed on any other parameter of oviductal fluid composition measured. No difference was observed, within treatment, in oviductal fluid metabolite or ion concentrations between the oviduct ipsilateral or contralateral to the corpus luteum (P > 0.05), so these data were pooled.


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  		TABLE 2. Effect of infusion treatment and day of estrous cycle on the biochemical composition of oviduct fluid.a

The concentration of urea in oviductal fluid was significantly higher in the animals infused with urea than in the animals infused with either saline or ammonium chloride (P < 0.001). Oviductal fluid concentrations of urea were higher in animals infused with ammonium chloride than in those infused with saline (P < 0.01).

The concentration of ammonia in oviductal fluid was higher in animals infused with ammonium chloride than in animals infused with either saline or urea (P < 0.01), but the concentration of oviductal ammonia was not different between animals infused with either saline or urea (P > 0.05). Oviductal fluid concentrations of glucose, lactate, K+, Na+, or Mg2+ were not affected by infusion treatment (P > 0.05). The concentration of calcium in oviductal fluid was lower in animals infused with urea than in those infused with saline or ammonium chloride. Oviduct calcium was not different between animals infused with either saline or ammonium chloride (P > 0.05).

Relationship Between Plasma and Oviductal Fluid Concentration of Metabolites and Ions

The relationship between plasma and oviductal fluid metabolite concentration is presented in Table 3. The plasma and oviductal fluid concentrations of urea, ammonia, glucose, lactate, and magnesium were all positively related. No association was found between plasma and oviductal fluid concentrations of calcium, potassium, or sodium. Plasma concentration of calcium was positively associated with oviductal fluid concentrations of glucose (P < 0.05). Oviductal fluid calcium concentration was positively associated (P < 0.05) with plasma concentrations of magnesium and sodium but negatively associated (P < 0.001) with plasma concentrations of urea.


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  		TABLE 3. Relationship between concentration of plasma and oviductal fluid metabolites.a

Relationship Between Plasma Progesterone and Oviductal Fluid Concentration of Metabolites and Ions

No relationship was observed between the plasma concentration of progesterone measured on either Day 2 or Day 8 and the plasma or oviductal fluid concentrations of any metabolite or ion measured (P > 0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous reports implicating elevated systemic ammonia and/or urea with reduced fertility in cattle fed high-protein diets have postulated that one or both of these metabolites may act to compromise oocyte competence [5, 22] or to alter the oviductal-utero environment [2, 4, 5, 7, 23] and, in this way, to compromise early embryo development. The results of the present study are, to our knowledge, the first to report the relationship between elevated systemic concentrations of ammonia and urea and the composition of oviductal fluid in cattle. Furthermore, the infusion protocol employed enabled examination of the discrete effects of ammonia and urea on the metabolite and ionic composition of oviductal fluid. The infusion of ammonium chloride and of urea into the systemic circulation significantly elevated the oviductal concentrations of ammonia and urea, respectively. Whereas the oviductal concentration of urea was strongly reflective of the systemic concentrations, the relationship between systemic and oviductal concentrations of ammonia, although still highly significant, was somewhat weaker, with ammonia being higher in the oviduct than in the blood plasma. Despite generating systemic and oviductal concentrations of ammonia in excess of 820 and 1180 µmol/L, respectively, and of urea of up to 14 mmol/L, with the exception of calcium, we found no effect of elevating systemic ammonia or urea on any of the other components of oviductal fluid measured. Furthermore, with the exception of sodium, we found no difference between Days 2 and 8 of the estrous cycle in the concentrations of any of the constituents of oviductal fluid measured, nor was any interaction found between treatment and day of collection on the concentration of any constituent measured.

Previous studies that attempted to establish the composition of oviductal fluid in cattle have involved aspiration of fluid from the reproductive tracts of animals postmortem [24]; use of chronic, indwelling catheters and oviduct ligation over several days [10–16]; or culture of bovine oviductal epithelial cells in vitro [25]. However, oviductal fluid or cells collected postmortem are likely to suffer from rapid degenerative effects on the oviductal epithelium, cell death, and alterations in the transport of molecules between cells, all of which could lead to anomalies in the concentrations of some constituents. Methods involving tubal ligation change the directional flow of fluid, whereas long catheters exteriorized over prolonged periods of time are also likely to lead to artifacts in both volume and composition of the fluid collected [26]. Furthermore, with this long-term approach, the oviduct and/or uterus may be predisposed to infectious agents and inflammatory reactions. The technique used in the present study, in which we recovered oviductal fluid in vivo over a short period of time, involved minimal disturbance of normal physiology and allowed for the evaluation of treatment effects in as physiological a manner as possible.

No relationship was found between systemic concentrations of ammonia or urea and the volume of oviductal fluid collected. A tendency was observed for the volume recovered to be higher on Day 2 than on Day 8, which is consistent with previous reports for a number of species, including cattle [10, 11, 13, 14, 27, 28]. Variations in the volume of oviductal fluid between various phases of the estrous cycle are due to endocrinological regulation of the secretory activity of oviductal epithelium, which increases under the influence of estradiol and declines as progesterone increases [27, 29]. The frequency and amplitude of muscular contractions along the oviductal wall in the cow is also increased at estrus and over the subsequent 3 days [30]. Furthermore, the directional flow of oviductal fluid is oriented toward the peritoneum during this period and reverses around the time of entry by the ovum or embryo into the uterus [29]. A strong tendency was observed for the recovery of fluid to be higher from the ipsilateral rather than the contralateral oviduct, which is also consistent with previous reports [11] and is probably a consequence of the greater muscular activity of the ipsilateral oviduct [30].

No difference was observed in the concentrations of metabolites or ions measured in the oviductal fluid ipsilateral or contralateral to the corpus luteum, and this allowed us to average the data from both oviducts for the remaining analyses. To our knowledge, no equivalent data have been published that compare the concentrations of ions and metabolites between opposite oviducts; however, a recent study [24] reported no difference in the amino acid concentration of bovine oviductal fluid harvested from the ipsi- or contralateral oviduct.

Oviductal and plasma concentrations of urea in the present study were significantly elevated following infusion of urea and, to a lesser extent, following infusion of ammonium chloride into the systemic circulation of heifers, with concentrations reaching 14 mmol/L in blood plasma and oviductal fluid in the urea-infused animals. Additionally, no difference was observed in oviduct or systemic concentrations of urea between Days 2 and 8 of the estrous cycle, which is consistent with a previous report [23] that showed no relationship between uterine or systemic concentrations of urea and stage of the estrous cycle in dairy cows.

Oviductal and systemic concentrations of ammonia were significantly increased following the infusion of ammonium chloride. This agrees with other reports [18, 31] that showed significant increases in systemic ammonia in steers that had been infused with NH4Cl (12 µmol/kg). In the present study, the absence of any difference between Days 2 and 8 of the estrous cycle in either oviductal or systemic concentrations of ammonia is also consistent with recent reports from this laboratory that showed no change in systemic concentrations of ammonia with day of the estrous cycle in heifers consuming high-protein pasture [20] or fed a high-rumen degradable protein (RDP) diet indoors [32].

To our knowledge, the present study is the first to quantify the concentration of ammonia or urea in the oviductal fluid of cattle and to demonstrate the relationship between systemic and oviductal concentrations of ammonia and urea. Urea is a small molecule, which readily diffuses across membranes and equilibrates within the body. The strong, positive relationship between systemic and oviductal concentrations of urea reported here agrees with a previous report in rabbits [33] and is consistent with other studies reporting the relationship between systemic and uterine fluid concentrations of urea in dairy cows [23] and ewes [4]. As with urea, a positive association was found between systemic and oviductal concentrations of ammonia, but ammonia was significantly higher in oviductal fluid than in blood. A recent report by Hammon et al. [34] indicates relatively high concentrations of ammonia in follicular fluid compared to blood. Consistent with this, Visek [35], in a review of the literature, reported that concentrations of ammonia may vary by up to 10-fold or greater between different compartments of the body.

In the present study, oviductal and systemic concentrations of glucose were not affected by ammonia or urea infusion, nor were they related to systemic concentrations of ammonia or urea. Hyperglycemia has been associated with elevated systemic ammonia in cattle [18, 31, 36]. Fernandez et al. [31] suggested that elevated systemic glucose induced by hyperammonemia might be mediated through decreased glucose utilization by peripheral tissues or, perhaps, through increased glycogenolysis. In the present study, systemic concentrations of glucose did not differ with stage of the estrous cycle, which is consistent with reports in Holstein cows [23] and beef heifers [20].

The glucose concentration of oviductal fluid was lower than that of plasma across all treatments in the present study, and this agrees with previous reports [29]. Oocytes and early embryos up to the 8-cell stage have a preference for pyruvate before switching to glucose at the blastocyst stage [37, 38]. In vitro studies have demonstrated that D-glucose supplementation of bovine embryo culture is not required until Day 3 or 4, at which stage supplementation improves development [39]. Furthermore, glucose concentrations in the range found in blood plasma (5–6 mM) are generally inhibitory to embryo development [40], and this may be due to elevated glucose interfering with cell signaling or altering the Krebs cycle [38]. Consequently, the selective permeability of the oviductal epithelium may maintain hypoglycemic conditions within the oviduct to aid early embryo survival and development [41, 42].

Systemic concentrations of lactate have been reported to be elevated [18, 36] or reduced [31] in animals experiencing hyperammonemia. In the present study, lactate was variable within treatment and not affected by cycle stage. Despite generating systemic concentrations of ammonia in excess of 820 mmol/L, we could not establish any relationship between concentrations of ammonia and lactate in either oviductal fluid or plasma. The higher lactate concentration recorded across treatments in oviductal fluid compared to plasma was probably the result of glucose metabolism within the oviduct [29].

Literature concerning the relationship between dietary protein and insulin is contradictory, with systemic insulin concentrations reported to be elevated [43], reduced [44], or unaltered [20] in cattle consuming diets with a high CP (crude protein) concentration. Reductions in systemic insulin have been reported to be associated with high systemic concentrations of ammonia in a number of studies [31, 44], and these reductions have been suggested to be mediated through inhibitory effects of ammonia on the beta cell or through the release of catecholamines from the pancreas [44]. Systemic insulin was variable within all three treatments in the present study; however, despite recording systemic concentrations of ammonia previously reported to be toxic to cattle [36], we could not establish any relationship between the two metabolites.

To our knowledge, the present study is the first to examine the relationship between systemic concentrations of ammonia and urea and the ionic concentration of oviductal fluid. High-protein diets and elevations in systemic ammonia and urea have been previously associated, alternatively, with changes in the ionic composition of uterine fluid [23] and uterine pH [2]. We found no effect of either ammonia or urea infusion on oviductal concentrations of magnesium, sodium, or potassium. The oviductal concentration of calcium was, however, reduced by infusion with urea but not by infusion with ammonium chloride. Oviductal calcium was also negatively related to systemic and oviductal concentrations of urea. In contrast, Jordan et al. [23] found no difference in the concentration of calcium in uterine fluid or in plasma between animals consuming either 12% or 23% CP diets, despite a 3.5-fold difference in plasma concentrations of urea. In their study, however, the systemic concentrations of urea generated were only approximately half those generated in the present study. Lapointe and Sirard [45] reported that the binding of certain oviductal proteins to spermatozoan membranes in vitro was dependent on the concentration of calcium in the medium, and they suggested that this binding may be important for subsequent sperm survival. In contrast, Walker et al. [46] reported that varying the concentration of calcium in culture media from 1.3 to 5 mM had no effect on the in vitro development of ovine embryos. Whereas gamete intracellular calcium is essential for sperm capacitation and fertilization, the role of oviductal calcium and the significance of the reduction induced by urea infusion shown in the present study are not clear.

No difference was found in either oviductal or systemic concentrations of calcium, magnesium, or potassium between Day 2 and Day 8 of the estrous cycle in the current study. The concentration of sodium was, however, higher on Day 8 than on Day 2. Grippo et al. [14], using chronic catheterization, failed to observe any relationship between oviductal concentrations of sodium and stage of the estrous cycle. Paisley and Mickelsen [11], also using exteriorized chronic catheterization, reported lower concentrations of sodium in the ipsilateral oviduct, but this was confined only to the day of ovulation.

The passage of ions between the oviduct and the systemic circulation is important for the movement of water, which is, by far, the largest component of oviductal fluid [9]. In the present study, plasma and oviductal fluid were similar with respect to their concentrations of calcium, magnesium, and sodium. Oviductal concentrations of sodium have been shown previously to be isotonic between the oviduct and blood [11, 14], but the concentrations of calcium and magnesium have been reported to vary between the oviduct and the systemic circulation [14]. In the present study, a higher concentration of potassium was found in oviductal fluid than in plasma, which is consistent with observations in other species [47], including cattle [14].

Butler [48] recently reported an inverse relationship between systemic urea and progesterone and postulated that high dietary CP or systemic urea may impair fertility by interfering with the normal inductive effects of progesterone on the microenvironment of the uterus, thereby leading to suboptimal conditions for the maintenance of embryo growth and development [49]. Despite generating very high systemic concentrations of urea, albeit over a relatively short period, we found no evidence to suggest any reduction in plasma progesterone concentrations. Furthermore, we failed to discover an association between systemic concentrations of progesterone and any oviductal or systemic constituent measured.

Elevated systemic concentrations of urea have frequently been cited as a probable causative agent in protein-mediated infertility in cattle [1, 2, 49]. Elrod and Butler [2] as well as Butler et al. [50] reported threshold systemic urea concentrations of 5.7 and 6.8 mmol/L, above which a 30% and 20% reduction in pregnancy rate for Holstein heifers and cows, respectively, was observed. However, Gath et al. [6], in an embryo-transfer study, reported that despite generating systemic urea concentrations of more than 7 mmol/L, neither fertilization rate nor subsequent embryo survival rate in recipient heifers was compromised. Similarly, two recent studies at this laboratory [20], despite generating systemic urea concentrations of up to 25 mmol/L, failed to establish any detrimental effect on embryo survival rate. Consistent with these findings, the results of the present study show that even with systemic and oviductal concentrations of urea peaking at more than 14 mmol/L, with the exception of calcium, no relationship is observed between urea and any oviductal or systemic metabolite or ion measured. Similarly, Berardinelli et al. [51] reported that feeding excess RDP to ewes generated significant increases in systemic concentrations of urea but did not affect either oviductal or uterine protein secretion in the early postovulatory period.

High systemic ammonia has also been suggested by a number of authors to be the underlying cause of infertility in animals fed high-protein diets [3, 4], but to our knowledge, no direct evidence supports this. In the past, one of the difficulties in partitioning the possible effects of ammonia and urea on cattle fertility was the relationship between systemic ammonia and urea. It is possible to increase systemic urea without increasing ammonia, but it is more difficult to increase ammonia without also elevating urea. Furthermore, systemic ammonia, by its nature, is quickly elevated following feeding and peaks within a short time before returning to basal levels. Infusing ammonium chloride circumvented this problem and allowed us to elevate, and to maintain the elevation in, systemic concentrations of ammonia independent of urea, thus enabling us to study changes in plasma and oviductal constituents in the presence of subtoxic [35] concentrations of ammonia. The mean peak systemic concentration of 820 µmol/L recorded in the ammonium chloride-infused animals is significantly greater than that previously reported to be associated with impaired embryo survival in cattle [3] and sheep [4], yet systemic ammonia was not related, with the exception of oviductal ammonia and urea, to any of the oviductal constituents measured. Hammon et al. [52] reported that the effects from in vitro exposure of bovine oocytes and early embryos to ammonia are dependent on the timing and duration of exposure and on the concentration of ammonia used. The high oviductal concentrations of ammonia recorded in the control animals of the present study suggest that bovine gametes, as part of their normal environment, are accustomed to relatively high concentrations of ammonia. This hypothesis is strengthened by the findings of a recent study [33], which reported physiological follicular ammonia concentrations of up to 366 µmol/L during the in vivo development of bovine oocytes.

In summary, the present study is the first, to our knowledge, to quantify the in vivo concentrations of ions and metabolites in bovine oviductal fluid without recourse to chronic catheterization or in vitro epithelial cell cultures. Furthermore, the present results show for the first time, again to our knowledge, the relationship between elevated systemic concentrations of ammonia and urea and the biochemical composition of oviductal fluid in cattle. With the exception of calcium, artificially high systemic or oviductal concentrations of either ammonia or urea were not related to the concentration of any constituent of oviductal fluid measured. In light of these results, it can be concluded that elevations in systemic concentrations of ammonia or urea per se, particularly of the magnitude observed under normal feeding conditions, are unlikely to impair embryo survival in cattle as a consequence of disruptions to the oviductal environment. The present findings also support those of two recent reports from this laboratory, which failed to find a relationship between systemic concentrations of ammonia or urea and embryo survival [20, 32] or development in heifers [20]. Notwithstanding this, however, interactions, if they exist, between negative energy balance and high systemic concentrations of ammonia and/or urea may act to compromise embryo survival in the high-yielding dairy cow.


   ACKNOWLEDGMENTS
 
The assistance of Prof. W.R. Butler in devising the experimental urea solutions is gratefully acknowledged. The authors also gratefully acknowledge the technical assistance of Dr. J. Gorham; Messrs. P. Joyce, W. Connolly, J. Nally, G. Morris, and J. Larkin; and Mrs. A. Glynn. The authors also wish to thank the farm staff at the Belclare Research Centre for their care of the animals.


   FOOTNOTES
 
First decision: 28 November 2001.

1 Supported by Greenvale Animal Feeds, Thurles, Co. Tipperary, Ireland. Back

2 Correspondence: Joseph Sreenan, Teagasc Research Centre, Athenry, Co. Galway, Ireland. FAX: 353 91 845847; jsreenan{at}athenry.teagasc.ie Back

Accepted: December 28, 2001.

Received: November 15, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Canfield RW, Sniffen CJ, Butler WR. Effects of excess degradable protein on postpartum reproduction and energy balance in dairy cattle. J Dairy Sci 1990; 73:2342-2349[Abstract]
  2. Elrod CC, Butler WR. Reduction of fertility and alteration of uterine pH in heifers fed excess ruminally degradable protein. J Anim Sci 1993; 71:694-701[Abstract]
  3. Sinclair KD, Kuran M, Gebbie FE, Webb R, McEvoy TG. Nitrogen metabolism and fertility in cattle: II. Development of oocytes recovered from heifers offered diets differing in their rate of nitrogen release in the rumen. J Anim Sci 2000; 78:2670-2680[Abstract/Free Full Text]
  4. McEvoy T, Robinson J, Aitken R, Findlay P, Robertson I. Dietary excesses of urea influence the viability and metabolism of preimplantation sheep embryos and may affect fetal growth among survivors. Anim Reprod Sci 1997; 47:71-90[CrossRef][Medline]
  5. O'Callaghan D, Boland MP. Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants. Anim Sci 1999; 68:299-314
  6. Gath VP, Lonergan P, Boland MP, O'Callaghan D. Effect of diet type on establishment of pregnancy and embryo development in heifers. Theriogenology 1999; 51:224 (abstract)
  7. Berardinelli JG, Weng J, Burfening PJ, Adair R. Effect of excess degradable intake protein on early embryonic development, ovarian steroids, and blood urea nitrogen on days 2, 3, 4, and 5 of the estrous cycle in mature ewes. J Anim Sci 2001; 79:193-199[Abstract/Free Full Text]
  8. Fahey J, Boland MP, O'Callaghan D. Effects of dietary urea on embryo development in superovulated donor ewes and on embryo survival following transfer in recipient ewes. Anim Sci 2001; 72:395-400
  9. Leese HJ, Tay JI, Reischl J, Downing SJ. Formation of fallopian tubal fluid: role of a neglected epithelium. Reproduction 2001; 121:339-346[Abstract]
  10. Stanke DF, Sikes JD, DeYoung DW, Tumbleson ME. Proteins and amino acids in bovine oviductal fluid. J Reprod Fertil 1974; 38:493-496[Medline]
  11. Paisley LG, Mickelsen WD. Continuous collection and analysis of bovine oviduct fluid: preliminary results. Theriogenology 1979; 11::375-384[Medline]
  12. Kavanaugh JF, Killian GJ. Bovine oviductal cannulations. J Invest Surg 1988; 1:201-208[Medline]
  13. Gerena RL, Killian GJ. Electrophoretic characterization of proteins in oviduct fluid of cows during the estrous cycle. J Exp Zool 1990; 256::113-120[CrossRef][Medline]
  14. Grippo AA, Henault MA, Anderson SH, Killian GJ. Cation concentrations in fluid from the oviduct ampulla and isthmus of cows during the estrous cycle. J Dairy Sci 1992; 75:58-65[Abstract]
  15. Guérin P, Gallois E, Croteau S, Revol N, Maurin F, Guillaud J, Menezo Y. Collection and amino acid composition of oviduct and follicular secretions in domestic animals. Rev Med Vet 1995; 146:805-814
  16. Greve T, Schmidt M, Host Hansen T. Collection of oviduct fluid in heifers. Dtsch Tierarztl Wochenschr 1996; 103:291-293
  17. Lowman BG, Scott NA, Somerville SH. Condition Scoring of Cattle. Rev. Ed. Bulletin, East of Scotland College of Agriculture; 1976, No 6
  18. Fernandez JM, Croom WJ Jr, Johnson AD, Jaquette RD, Edens FW. Subclinical ammonia toxicity in steers: effects on blood metabolite and regulatory hormone concentrations. J Anim Sci 1988; 66:3259-3266
  19. Gardner DK, Leese HJ. Assessment of embryo metabolism and viability. In: Trounson AO, Gardner DK (eds.), Handbook of In Vitro Fertilization, 2nd ed. London: CRC Press; 2000: 347–371
  20. Kenny DA, Boland MP, Diskin MG, Sreenan JM. Effect of pasture crude protein and fermentable energy supplementation on blood metabolite and progesterone concentrations and on embryo survival in heifers. Anim Sci 2001; 73:501-512
  21. Statistical Analysis Systems Institute. SAS/STAT User's Guide, Release 8.1. Cary, NC: SAS Institute; 2001
  22. De Wit AAC, Cesar MLF, Kruip TAM. Effect of urea during in vitro maturation on nuclear maturation and embryo development of bovine cumulus-oocyte-complexes. J Dairy Sci 2001; 84:1800-1804[Abstract]
  23. Jordan ER, Chapman TE, Holtan DW, Swanson LV. Relationship of dietary crude protein to composition of uterine secretions and blood in high-producing postpartum dairy cows. J Dairy Sci 1983; 66:1854-1862
  24. Elhassan YM, Wu G, Leanez AC, Tasca RJ, Watson AJ, Westhusin ME. Amino acid concentrations in fluids from the bovine oviduct and uterus and in KSOM-based culture media. Theriogenology 2001;1907–1918
  25. Cox CI, Leese HJ. Retention of functional characteristics by bovine oviduct and uterine epithelia in vitro. Anim Reprod Sci 1997; 46:169-178[CrossRef][Medline]
  26. Black DL, Kumar A, Crowley LV, Duby RT, Spilman CH. Composition of oviduct fluid kept at two temperatures during collection from sheep. J Reprod Fertil 1970; 22:597-600[Medline]
  27. Killian GJ, Chapman DA, Kavanaugh JF, Deaver DR, Wiggin HB. Changes in phospholipids, cholesterol and protein content of oviduct fluid of cows during the estrous cycle. J Reprod Fertil 1989; 86:419-426[Abstract]
  28. Ehrenwald E, Foote RH, Parks JE. Bovine oviductal fluid components and their potential role in sperm cholesterol efflux. Mol Reprod Dev 1990; 25:195-204[CrossRef][Medline]
  29. Menezo Y, Guerin P. The mammalian oviduct: biochemistry and physiology. Eur J Obstet Gynecol Reprod Biol 1997; 73:99-104[CrossRef][Medline]
  30. Bennett WA, Watts TL, Blair WD, Waldhalm SJ, Fuquay JW. Patterns of oviductal motility in the cow during the estrous cycle. J Reprod Fertil 1988; 83:537-543[Abstract]
  31. Fernandez JM, Croom WJ Jr, Tate LP Jr, Johnson AD. Subclinical ammonia toxicity in steers: effects on hepatic and portal drained visceral flux of metabolites and regulatory hormones. J Anim Sci 1990; 68:1726-1742[Abstract]
  32. Kenny DA, Boland MP, Diskin MG, Sreenan JM. Effect of rumen degradable protein with or without fermentable carbohydrate supplementation on blood metabolites and embryo survival in cattle. Anim Sci 2002; 74:(in press)
  33. Leese HJ, Aldridge S, Jeffries KS. The movement of amino acids into rabbit oviductal fluid. J Reprod Fertil 1979; 56:623-626[Abstract]
  34. Hammon DS, Wang S, Holyoak GR. Ammonia concentration in bovine follicular fluid and its effect during in vitro maturation on subsequent embryo development. Anim Reprod Sci 2000; 58:1-8[CrossRef][Medline]
  35. Visek WJ. Ammonia: its effects on biological systems, metabolic hormones and reproduction. J Dairy Sci 1984; 67:481-498
  36. Symonds HW, Mather DL, Collis KA. The maximum capacity of the liver of the adult dairy cow to metabolize ammonia. Br J Nutr 1981; 46:481-486[CrossRef][Medline]
  37. Leese HJ. Metabolism of the preimplantation mammalian embryo. Oxf Rev Reprod Biol 1991; 13:35-72[Medline]
  38. Boland MP, Lonergan P, O'Callaghan D. Effect of nutrition on endocrine parameters, ovarian physiology, and oocyte and embryo development. Theriogenology 2001; 55:1323-1340[CrossRef][Medline]
  39. Kim JH, Funahashi H, Niwa K, Okuda K. Glucose requirement at different developmental stages of in vitro fertilized bovine embryos cultured in semidefined medium. Theriogenology 1993; 39:875-886
  40. Thompson JG. Defining the requirements for bovine embryo culture. Theriogenology 1996; 45:27-40[CrossRef]
  41. Edwards LJ, Batt PA, Gandolfi F, Gardner DK. Modifications made to culture medium by bovine oviduct epithelial cells: changes to carbohydrates stimulate bovine embryo development. Mol Reprod Dev 1997; 46:146-154[CrossRef][Medline]
  42. Nichol R, Hunter RH, Gardner DK, Partridge R, Leese HJ, Cooke GM. Concentrations of energy substrates in oviduct fluid in unilaterally ovariectomized pigs. Res Vet Sci 1998; 63:263-264
  43. Blauweikel R, Kincaid RL. Effect of crude protein and solubility on performance and blood constituents of dairy cows. J Dairy Sci 1986; 69:2091-2098
  44. Sinclair KD, Sinclair LA, Robinson JJ. Nitrogen metabolism and fertility in cattle: I. Adaptive changes in intake and metabolism to diets differing in their rate of energy and nitrogen release in the rumen. J Anim Sci 2000; 78:2659-2669[Abstract/Free Full Text]
  45. Lapointe S, Sirard MA. Importance of calcium for the binding of oviductal fluid proteins to the membranes of bovine spermatozoa. Mol Reprod Dev 1996; 44:234-240[Medline]
  46. Walker SK, Seamark RF, Quinn P, Warnes GM, Ashman RJ, Smith DH, Ancell P. Culture of pronuclear embryos of sheep in a simple medium. In: Proceedings of the 11th International Congress of Animal Reproduction and Artificial Insemination; 1988; Dublin, Ireland. Abstract 483
  47. Leese HJ. The formation and function of oviduct fluid. J Reprod Fertil 1988; 82:843-856[CrossRef][Medline]
  48. Butler WR. Nutritional effects on resumption of ovarian cyclicity and conception rate in postpartum dairy cows. In: Fertility in the High-Producing Dairy Cow. BSAS Occasional Publication No. 26. 2001; 1:133-145
  49. Butler WR. Review: effect of protein nutrition on ovarian and uterine physiology in dairy cattle. J Dairy Sci 1998; 81:2533-2539[Abstract]
  50. Butler WR, Calaman JJ, Beam SW. Plasma and milk urea nitrogen in relation to pregnancy rate in lactating dairy cattle. J Anim Sci 1996; 74:858-865[Abstract]
  51. Berardinelli JG, Weng J, Burfening PJ, Adair R. Oviductal and uterine protein on days 2 and 4 of the estrous cycle of ewes fed excess rumen degradable protein. Biol Reprod 1997; 56:(suppl 1):135 (abstract 212).
  52. Hammon DS, Wang S, Holyoak GR. Effects of ammonia during different stages of culture on development of in vitro produced bovine embryos. Anim Reprod Sci 2000; 59:23-30[Medline]



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