BIOLOGY OF REPRODUCTION 66, 1797–1804 (2002)
Effect of Elevated Systemic Concentrations of Ammonia and Urea on the Metabolite and Ionic Composition of Oviductal Fluid in Cattle1 D.A. Kenny,3,4 P.G. Humpherson,5 H.J. Leese,5 D.G. Morris,3 A.D. Tomos,6 M.G. Diskin,3 and J.M. Sreenan2,3 Teagasc Research Centre,3 Athenry, Co. Galway, Ireland Department of Animal Science and Production,4 Faculty of Agriculture, National University of Ireland Dublin, Dublin 4, Ireland Department of Biology,5 University of York, York YO10 5YW, United Kingdom School of Biological Sciences,6 University of Wales Bangor, Bangor, Gwynedd, LL57 2UW, Wales, United Kingdom ABSTRACT 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 5 25). Heifers were randomly assigned to 1 of 3 treatments consisting of jugular vein infusion with either ammonium chloride (n 5 8), urea (n 5 8), or saline (n 5 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
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 elSupported by Greenvale Animal Feeds, Thurles, Co. Tipperary, Ireland. Correspondence: Joseph Sreenan, Teagasc Research Centre, Athenry, Co. Galway, Ireland. FAX: 353 91 845847; e-mail:
[email protected]
2
Received: 15 November 2001. First decision: 28 November 2001. Accepted: 28 December 2001. Q 2002 by the Society for the Study of Reproduction, Inc. ISSN: 0006-3363. http://www.biolreprod.org
MATERIALS AND METHODS
Animals
INTRODUCTION
1
evated 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.
Twenty-five nulliparous, cross-bred beef heifers with a mean 6 SEM body weight and body condition score [17] of 384 6 6 kg and 3.22 6 0.06 units, respectively, were used in this study. Estrous cycles were synchronized using 2 injections of a synthetic prostaglandin (PG) F2a analogue (500 mg 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 PGF2a injections. Only animals that were recorded in standing estrus following the second PGF2a 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 5 Day 0) in a 2 3 3 factorial study as follows: saline on Day 2 (n 5 5) or Day 8 (n 5 4); urea on Day 2 (n 5
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4) or Day 8 (n 5 4); and ammonium chloride on Day 2 (n 5 4) or Day 8 (n 5 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 mmol21 of NH4Cl kg21 min21 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 mmol21 of urea kg21 min21 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 mmol21 of urea kg21 min21 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 1.5ml tubes, centrifuged at 15 000 3 g for 7 min, snap-frozen in liquid nitrogen (21968C), and stored at 2808 C until analysis.
assay. Plasma samples for ammonia and urea analysis were frozen in liquid nitrogen immediately after harvesting and stored at 2808C 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 NAD1. 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 NAD1 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 NAD1, 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 mIU/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 mIU/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 508C. 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 ml of sample in a 200-ml microcentrifuge tube. Five microliters of this mixture were manually injected onto the column. Quantification of K1, Mg21, and Ca21 were based on the ratio of peak heights to that of the internal standard. For Na1 determination, 2 ml of sample were diluted with 498 ml 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 Na1 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 intraand 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.
Blood Sampling Blood was sampled for determination of plasma concentrations of ammonia, urea, glucose, insulin, and progesterone. Samples were taken at 230, 215, 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 3 g at 48C for 15 min. Plasma was separated, and samples for determination of insulin, glucose, and progesterone concentration were stored at 2208C until
Statistical Analysis Results are presented as least square mean 6 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
Infusion Treatment and Volume of Oviductal Fluid Recovered
No day of sampling 3 treatment interaction (P . 0.05) or day of sampling 3 treatment 3 oviduct interaction (P
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BLOOD AMMONIA AND UREA AND OVIDUCT FLUID IN CATTLE TABLE 1. Effect of infusion treatment and day of estrous cycle on plasma concentrations of lactate, K1, Na1, Mg21, and Ca21.a Treatment Control Lactate (mmol/L) K1 (mM) Na1 (mM) Mg21 (mM) Ca21 (mM)
1.60 1.87 244.68 0.33 0.70
6 6 6 6 6
0.459 0.775 46.501 0.100 0.200
Urea 0.658 2.19 180.61 0.28 0.60
6 6 6 6 6
0.484 0.566 33.959 0.073 0.146
Day NH4Cl 0.579 2.68 148.11 0.40 0.85
6 6 6 6 6
0.484 0.490 31.766 0.063 0.127
2 0.603 2.61 194.86 0.34 0.78
6 6 6 6 6
8 0.382 0.422 26.547 0.055 0.109
1.29 1.88 187.40 0.33 0.65
6 6 6 6 6
0.395 0.582 34.890 0.075 0.150
No effect of treatment 3 day, treatment, and day on any variable measured (P . 0.05). Values are the least square mean 6 SEM of plasma samples taken during oviductal fluid collection.
a
. 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 6 61.326 ml vs. 333.20 6 60.062 ml, P 5 0.05) and also tended to be higher on Day 2 than on Day 8 (499.72 6 59.007 ml vs. 338.249 6 62.953 ml, P 5 0.07).
Infusion Treatment and Plasma Metabolite and Ionic Concentration
No treatment 3 day of sampling interaction for plasma concentrations of urea, ammonia, glucose, or insulin was observed (P . 0.05), but an interaction of infusion treatment 3 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). An infusion treatment 3 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 3 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 3 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 3 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).
Infusion Treatment and Plasma Concentration of Progesterone
No treatment 3 day of sampling 3 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 3 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. 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, K1, Na1, or Mg21 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). Ovi-
FIG. 1. Effect of infusion treatment on mean plasma concentrations of insulin (A), glucose (B), ammonia (C), and urea (D).
ductal 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.
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).
a
14.01 416.54 2.12 4.99 3.26 179.85 0.33 0.51
0.350 119.770 0.468 1.806 0.559 21.085 0.050 0.66
6 6 6 6 6 6 6 6
0.350 174.050 0.509 1.806 0.661 24.948 0.060 0.078
6 6 6 6 6 6 6 6
5.91 451.94 2.95 10.02 3.48 185.00 0.40 0.80
8.27 1187.08 2.83 7.98 3.84 162.36 0.35 0.68
6 6 6 6 6 6 6 6
0.369 118.059 0.493 1.904 0.559 21.085 0.050 0.066
NH4Cl 9.55 815.38 2.17 6.36 3.78 147.47 0.36 0.72
6 6 6 6 6 6 6 6
2 0.291 96.170 0.389 1.502 0.441 16.632 0.040 0.052
Day 9.25 555.19 3.10 8.98 3.26 203.997 0.35 0.61
Pooled least square mean 6 SEM of ipsilateral and contralateral oviducts. There was no treatment by day of sampling interaction observed.
Urea (mmol/L) Ammonia (mmol/L) Glucose (mmol/L) Lactate (mmol/L) K1 (mM) Na1 (mM) Mg21 (mM) Ca21 (mM)
Urea
Control
Treatment
TABLE 2. Effect of infusion treatment and day of estrous cycle on the biochemical composition of oviduct fluid.a
6 6 6 6 6 6 6 6
8 0.291 105.610 0.412 1.502 0.527 19.879 0.047 0.062
P , 0.05
P , 0.001 P , 0.001
Treatment
P , 0.05
P 5 0.08
Day
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BLOOD AMMONIA AND UREA AND OVIDUCT FLUID IN CATTLE TABLE 3. Relationship between concentration of plasma and oviductal fluid metabolites.a Oviductal fluid Plasma Urea Ammonia Glucose Lactate K1 Mg21 Ca21 Na21
Urea
Ammonia
Glucose
Lactate
K1
Mg21
Ca21
Na21
0.97 (P , 0.001) 0.09 (P . 0.10) 0.06 (P . 0.10) 0.05 (P . 0.10) 0.002 (P . 0.10) 0.04 (P . 0.10) 0.03 (P . 0.10) 0.02 (P . 0.10)
0.05 (P . 0.10) 0.56 (P , 0.001) 0.004 (P . 0.10) 0.08 (P . 0.10) 0.08 (P . 0.10) 0.05 (P . 0.10) 0.07 (P . 0.10) 0.01 (P . 0.10)
0.05 (P . 010) 0.10 (P . 0.10) 0.24 (P , 0.05) 0.11 (P , 0.10) 0.04 (P . 0.10) 0.20 (P , 0.05) 0.24 (P , 0.05) 0.003 (P . 0.10)
0.10 (P . 0.10) 0.06 (P . 0.10) 0.01 (P . 0.10) 0.63 (P , 0.001) 0.12 (P . 0.10) 0.02 (P . 0.10) 0.07 (P . 0.10) 0.03 (P . 0.10)
0.02 (P . 0.10) 0.02 (P . 0.10) 0.10 (P . 0.10) 0.01 (P . 0.10) 0.04 (P . 0.10) 0.10 (P . 0.10) 0.06 (P . 0.10) 0.04 (P . 0.10)
0.09 (P . 0.10) 0.0001 (P . 0.10) 0.01 (P . 0.10) 0.11 (P . 0.10) 0.03 (P . 0.10) 0.24 (P , 0.05) 0.12 (P . 0.13) 0.20 (P 5 0.06)
0.47 (P , 0.001) 0.01 (P . 0.10) 0.003 (P . 0.10) 0.09 (P . 0.10) 0.04 (P . 0.10) 0.22 (P , 0.05) 0.19 (P 5 0.06) 0.25 (P , 0.05)
0.01 (P . 0.10) 0.003 (P . 0.10) 0.001 (P . 0.10) 0.0001 (P . 0.10) 0.02 (P . 0.10) 0.08 (P . 0.10) 0.01 (P . 0.10) 0.02 (P . 0.10)
a The square of the correlation coefficient (R2) with appropriate levels of significance is presented. Values in bold are the relationships that are statistically significant.
DISCUSSION
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 mmol/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 ipsior 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 ammo-
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nium 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 mmol/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 Dglucose 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
BLOOD AMMONIA AND UREA AND OVIDUCT FLUID IN CATTLE
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 chlo-
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ride 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 mmol/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 mmol/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.
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