Articles in PresS. Am J Physiol Endocrinol Metab (March 24, 2015). doi:10.1152/ajpendo.00046.2015
1 2
Formate metabolism in fetal and neonatal sheep. .
3 4
Shannon E Washburn1, Marie A Caudill2, Olga Malysheva2, Amanda J MacFarlane3, Nathalie
5
A Behan3, Brian Harnett4, Luke MacMillan4, Theerawat Pongnopparat4, John T Brosnan4 * and
6
Margaret E. Brosnan4
7 8
1
Department of Veterinary Physiology and Pharmacology and Michael DeBakey Institute,
9
College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, College
10
Station, TX, USA, 2Division of Nutritional Sciences, Cornell University, Ithaca, New York,
11
USA, 3Nutrition Research Division, Health Canada, Ottawa, Canada, 4Department of
12
Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada
13 14
Running Title: Formate metabolism in the fetal lamb.
15 16
*Corresponding author: Dr. John T. Brosnan, Dept. of Biochemistry, Memorial University of
17
Newfoundland, St. John’s, NL, A1B 3X9, CANADA. Email: [email protected]
18 19
Copyright © 2015 by the American Physiological Society.
20 21 22
ABSTRACT
23
By virtue of its role in nucleotide synthesis, as well as the provision of methyl groups for vital
24
methylation reactions, one-carbon metabolism plays a crucial role in growth and development.
25
Formate, a critical, though neglected, component of one-carbon metabolism, occurs
26
extracellularly and may provide insights into cellular events. We examined formate metabolism
27
in chronically cannulated fetal sheep (gestation day 119-121, equivalent to mid-third trimester in
28
humans) and in their mothers, as well as in normal full-term lambs. Plasma formate levels were
29
much higher in fetal lamb plasma and in amniotic fluid (191 ± 62 and 296 ± 154 µM,
30
respectively) than in maternal plasma (33 ± 13 µM). Measurements of folate, vitamin B12 and
31
homocysteine showed that these high formate levels could not be due to vitamin deficiencies.
32
Elevated formate levels were also found in newborn lambs and persisted to about 8 weeks of age.
33
Formate was also found in sheep milk. Potential precursors of one-carbon groups were also
34
measured in fetal and maternal plasma and in amniotic fluid. There were very high
35
concentrations of serine in the fetus (approximately 1.6 mM in plasma and 3.5 mM in the
36
amniotic fluid), compared to maternal plasma (0.19 mM), suggesting increased production of
37
formate; however, we cannot rule out decreased formate utilization.. Dimethylglycine, a choline
38
metabolite, was also 30 times higher in the fetus than in the mother.
39 40
Key-words: one-carbon metabolism, folate, vitamin B12, mitochondria, serine, milk.
41 42
INTRODUCTION
43
Formate is a critical component of folate-mediated, one-carbon metabolism; it plays a key role in
44
the production of one-carbon groups for the synthesis of purines and of thymidine and for the
45
provision of methyl groups for transmethylation reactions (11). These are all processes that are
46
likely to occur at elevated rates in growing tissues, particularly in fetal and neonatal tissues.
47
Purine synthesis is required for the synthesis of nucleotides and nucleic acids. Thymidine
48
synthesis is required for the synthesis of DNA. Methyl groups are required for the synthesis of
49
key molecules, such as phosphatidylcholine and creatine (33) as well as for the methylation of
50
cytosine residues in DNA. This latter function is considered to be crucial for the normal
51
development of fetal and neonatal animals (15). Tetrahydrofolate (THF) is the scaffold on which
52
most one-carbon intermediates are built. The provision of methyl groups occurs via the
53
reduction of 5,10-methylene-THF to 5-methyl-THF by NADPH, catalysed by
54
methylenetetrahydrofolate reductase (MTHFR), followed by the transfer of the methyl group
55
from 5-methyl-THF to homocysteine to form methionine; this is catalysed by methionine
56
synthase, one of only two mammalian enzymes known to require vitamin B12 for the synthesis of
57
their prosthetic groups (21). Methionine is used for the synthesis of S-adenosylmethionine
58
(SAM), the principal biological methylating agent. It has been computed that the human genome
59
contains more than 200 methyltransferases which use SAM to methylate a variety of acceptors,
60
including DNA, RNA and proteins, regulating gene expression, mRNA processing and protein
61
function (28). In addition, methyltransferases are responsible for the synthesis of such key
62
molecules as creatine, phosphatidylcholine and epinephrine (25), and for the detoxification of
63
inorganic arsenic ions (12). Recently, it has been shown that folate metabolism plays a key role
64
in the reduction of NADP+ to NADPH (10).
65 66
Folate is critically important in development, particularly in pregnancy. Low folate status,
67
particularly in the context of the TT genotype of MTHFR, is a risk factor for the development of
68
neural tube defects such as spina bifida and anencephaly (32). Mandatory folic acid fortification
69
of the food supply in the USA, Canada and some other countries has greatly reduced the
70
incidence of neural tube defects (8). The mechanism by which folate supplementation acts is not
71
fully understood.
72 73 74
Formate is primarily produced in mitochondria from serine, glycine, and two intermediates of
75
choline catabolism, sarcosine and dimethylglycine. Mitochondrial 10-formyl-THF can give rise
76
to formate which leaves the mitochondrion and is incorporated into the cytosolic pool of 10-
77
formyl-THF where it may be used for purine synthesis (29). Cytosolic 10-formyl-THF may also
78
be reduced to 5,10-methylene-THF and thence to 5-methyl-THF. Its formyl group may also be
79
oxidized to CO2, providing an exit for excess one-carbon groups from folate metabolism (16).
80
Formate may also arise from a number of other metabolic reactions, such as methanol
81
metabolism, phytanic acid α-oxidation, tryptophan catabolism and cholesterol synthesis (19).
82
Figure 1 shows an outline of one-carbon and formate metabolism. Although intermediates in
83
one-carbon metabolism primarily occur within cells, formate can be found in appreciable
84
quantities in plasma; this may provide insights into cellular one-carbon metabolism.
85
.
86
MATERIALS AND METHODS
87
Animals All aspects of the surgical and experimental protocols were approved by the Texas
88
A&M University Institutional Animal Care and Use Committee. Suffolk ewes aged 2-5 years
89
were obtained from a commercial supplier. Upon arrival at the animal facility, each ewe received
90
an intramuscular injection of Covexin® 8 (Merck Animal Health, Summit, NJ) and an oral bolus
91
of Valbazen® (Zoetis, Kalamazoo, MI). Ewes received progesterone-impregnated vaginal
92
implants (EAZI-BREED™, CIDR®, Zoetis, Kalamazoo, MI). Implants were removed 11 days
93
after placement at which time prostaglandin F2α (20 mg; LUTALYSE®, Zoetis, Kalamazoo, MI)
94
was intramuscularly administered. The following day, ewes were placed with a ram fitted with a
95
marking harness for a period of 24 h. Marked ewes were presumed pregnant until confirmed
96
pregnant ultrasonographically on gestation day (GD) 25 and 92.
97
Ewes were fed TAMU Ewe Ration, a custom ration (Nutrena, Cargill Animal Nutrition,
98
Minneapolis, MN), twice daily. Ration amount (average 2 kg per day) was based on body weight
99
and determined by ARIES® software version 2007 (University of California, Davis). This was a
100
pelleted feed consisting of minimum 12.75% protein, minimum 3.00% crude fat, and maximum
101
25.00% crude fiber. Ewes were allowed ad libitum access to water. Daily feed consumption was
102
monitored; all subjects consumed all of the food offered.
103
Six pregnant ewes were surgically implanted with indwelling cannulas so as to permit
104
simultaneous sampling from the fetal and maternal circulations. ; 5 pregnant ewes were allowed
105
to lamb naturally (no implanted cannulas) in order to measure ewe and lamb plasma and milk
106
formate levels. In addition, 7 non-pregnant ewes were also sampled for comparison.
107
Ewes were allowed to lamb naturally and shortly after birth the lambs were weighed, ear-tagged
108
and their navels were dipped in 7% iodine. They were weighed weekly thereafter and remained
109
with lactating maternal ewes until weaning at 8 weeks age. All lambs were vaccinated against
110
Clostridium perfingens (types C and D) and Clostridium tetani (Bar Vac CD/T, Boehringer
111
Ingelheim Vetmedica, Inc., St. Joseph, MO, USA) at 4-weeks age and again at weaning. Weaned
112
lambs were maintained on a daily ration of sheep feed (5% of body weight) formulated to
113
National Research Council guidelines for growing lambs (Producers Cooperative Association,
114
Bryan, TX, USA), supplemented by alfalfa (1% of body weight).
115
Sample Collection From the cannulated ewes, maternal and fetal samples were collected into
116
lithium heparin tubes via the surgically implanted catheters at GD 117 ± 1 days; amniotic fluid
117
was collected into polystyrene tubes with no additives. From the non-cannulated group, plasma
118
samples were collected by jugular venipuncture into lithium heparin tubes. Samples were
119
collected from the ewes and lambs shortly after lambing occurred, and at 1 week, 2 weeks, 1
120
month and 2 months after lambing. Milk samples were collected from the ewe by hand milking
121
into polystyrene tubes with no additives. Milk samples were obtained shortly after lambing
122
occurred and at 1 week, 2 weeks, 1 month, and 2 months later (time of weaning). Plasma
123
samples were immediately separated via centrifugation, and all samples were stored at -80°C
124
until analysis.
125
Cannulation On day GD 117±1, the pregnant sheep were anesthetized and aseptic surgery was
126
performed.
127
using intravenous administration of diazepam (0.2 mg/kg BW, Abbott Laboratories, North
128
Chicago, IL) and ketamine (4 mg/kg BW, Ketaset®, Fort Dodge, Fort Dodge, IA). The ewes
129
were intubated and ventilated, and anesthesia was maintained with isoflurane (0.5–2.5%,
130
WasoFlo®, Abbott Laboratories) and oxygen. The abdomen was opened at the midline, the
131
uterus was externalized and incised, and a fetal hind limb was exteriorized. An incision was
Methods have been previously described (31, 37). In brief, anesthesia was induced
132
made over the craniolateral aspect of the fetal hind limb, midway between the hock and stifle. A
133
catheter (0.030" inner diameter, 0.050" outer diameter polyvinyl chloride) was advanced from
134
the fetal cranial tibial artery into the abdominal aorta to the level of the diaphragm; the procedure
135
was then repeated on the other hind limb. The fetus was returned to the uterus, and the uterus and
136
maternal midiline were closed. The maternal femoral vessels were isolated, cannulated and
137
catheters (0.050" inner diameter, 0.090" outer diameter polyvinyl chloride) were advanced from
138
the maternal femoral artery and vein to the level of the diaphragm of the abdominal aorta and
139
vena cava, respectively. Fetal and maternal catheters were passed through the abdominal wall
140
where they were stored in a cloth pouch.
141 142
Analyses. Formate was analysed by the GC-MS method of Lamarre et al. (17). Total plasma
143
homocysteine (tHcy) was measured as described by Vester and Rasmussen (35). Total plasma
144
and amniotic folate were measured using the Lactobacillus casei microbiological assay (14).
145
Total plasma and amniotic vitamin B12 were analyzed using the Advia Centaur XP Immunoassay
146
(Siemens Canada), as described by the manufacturer. Amino acids were derivatized with o-
147
phthaldehyde and assayed by HPLC, as described (38). Choline and its derivatives,
148
dimethylglycine and betaine , were quantified by LC-MS/MS (13, 36) with modifications based
149
on available instrumentation (41, 42)
150
Expression and analysis of data. Data were expressed as means ± standard deviation, with the
151
number of observations in parentheses. Statistical analysis was carried out with Graph Pad 3
152
statistical package. Data were analyzed by means of a t-test, a One-way ANOVA or a Repeated
153
Measures ANOVA, followed by Dunnet’s Multiple Comparison Post-hoc Test, as indicated in
154
figures and tables.
155
RESULTS
156
Figure 2 shows plasma formate levels in maternal and fetal plasma and in amniotic fluid, taken
157
from six pregnant ewes between gestation days 119 and 121 (full term is 147 ± 2 days),
158
corresponding to mid third trimester in humans. Very high levels of formate were evident in fetal
159
plasma (191 ± 62 µM) and in amniotic fluid (296 ± 154 µM). Both of these were significantly
160
higher than the formate concentration in maternal plasma (33 ± 13 µM). We then compared the
161
maternal formate concentrations with those of a separate group of non-pregnant ewes; the
162
formate concentration in the pregnant ewes was significantly higher (p
1 2
Formate metabolism in fetal and neonatal sheep. .
3 4
Shannon E Washburn1, Marie A Caudill2, Olga Malysheva2, Amanda J MacFarlane3, Nathalie
5
A Behan3, Brian Harnett4, Luke MacMillan4, Theerawat Pongnopparat4, John T Brosnan4 * and
6
Margaret E. Brosnan4
7 8
1
Department of Veterinary Physiology and Pharmacology and Michael DeBakey Institute,
9
College of Veterinary Medicine and Biomedical Sciences, Texas A & M University, College
10
Station, TX, USA, 2Division of Nutritional Sciences, Cornell University, Ithaca, New York,
11
USA, 3Nutrition Research Division, Health Canada, Ottawa, Canada, 4Department of
12
Biochemistry, Memorial University of Newfoundland, St. John’s, NL, Canada
13 14
Running Title: Formate metabolism in the fetal lamb.
15 16
*Corresponding author: Dr. John T. Brosnan, Dept. of Biochemistry, Memorial University of
17
Newfoundland, St. John’s, NL, A1B 3X9, CANADA. Email: [email protected]
18 19
Copyright © 2015 by the American Physiological Society.
20 21 22
ABSTRACT
23
By virtue of its role in nucleotide synthesis, as well as the provision of methyl groups for vital
24
methylation reactions, one-carbon metabolism plays a crucial role in growth and development.
25
Formate, a critical, though neglected, component of one-carbon metabolism, occurs
26
extracellularly and may provide insights into cellular events. We examined formate metabolism
27
in chronically cannulated fetal sheep (gestation day 119-121, equivalent to mid-third trimester in
28
humans) and in their mothers, as well as in normal full-term lambs. Plasma formate levels were
29
much higher in fetal lamb plasma and in amniotic fluid (191 ± 62 and 296 ± 154 µM,
30
respectively) than in maternal plasma (33 ± 13 µM). Measurements of folate, vitamin B12 and
31
homocysteine showed that these high formate levels could not be due to vitamin deficiencies.
32
Elevated formate levels were also found in newborn lambs and persisted to about 8 weeks of age.
33
Formate was also found in sheep milk. Potential precursors of one-carbon groups were also
34
measured in fetal and maternal plasma and in amniotic fluid. There were very high
35
concentrations of serine in the fetus (approximately 1.6 mM in plasma and 3.5 mM in the
36
amniotic fluid), compared to maternal plasma (0.19 mM), suggesting increased production of
37
formate; however, we cannot rule out decreased formate utilization.. Dimethylglycine, a choline
38
metabolite, was also 30 times higher in the fetus than in the mother.
39 40
Key-words: one-carbon metabolism, folate, vitamin B12, mitochondria, serine, milk.
41 42
INTRODUCTION
43
Formate is a critical component of folate-mediated, one-carbon metabolism; it plays a key role in
44
the production of one-carbon groups for the synthesis of purines and of thymidine and for the
45
provision of methyl groups for transmethylation reactions (11). These are all processes that are
46
likely to occur at elevated rates in growing tissues, particularly in fetal and neonatal tissues.
47
Purine synthesis is required for the synthesis of nucleotides and nucleic acids. Thymidine
48
synthesis is required for the synthesis of DNA. Methyl groups are required for the synthesis of
49
key molecules, such as phosphatidylcholine and creatine (33) as well as for the methylation of
50
cytosine residues in DNA. This latter function is considered to be crucial for the normal
51
development of fetal and neonatal animals (15). Tetrahydrofolate (THF) is the scaffold on which
52
most one-carbon intermediates are built. The provision of methyl groups occurs via the
53
reduction of 5,10-methylene-THF to 5-methyl-THF by NADPH, catalysed by
54
methylenetetrahydrofolate reductase (MTHFR), followed by the transfer of the methyl group
55
from 5-methyl-THF to homocysteine to form methionine; this is catalysed by methionine
56
synthase, one of only two mammalian enzymes known to require vitamin B12 for the synthesis of
57
their prosthetic groups (21). Methionine is used for the synthesis of S-adenosylmethionine
58
(SAM), the principal biological methylating agent. It has been computed that the human genome
59
contains more than 200 methyltransferases which use SAM to methylate a variety of acceptors,
60
including DNA, RNA and proteins, regulating gene expression, mRNA processing and protein
61
function (28). In addition, methyltransferases are responsible for the synthesis of such key
62
molecules as creatine, phosphatidylcholine and epinephrine (25), and for the detoxification of
63
inorganic arsenic ions (12). Recently, it has been shown that folate metabolism plays a key role
64
in the reduction of NADP+ to NADPH (10).
65 66
Folate is critically important in development, particularly in pregnancy. Low folate status,
67
particularly in the context of the TT genotype of MTHFR, is a risk factor for the development of
68
neural tube defects such as spina bifida and anencephaly (32). Mandatory folic acid fortification
69
of the food supply in the USA, Canada and some other countries has greatly reduced the
70
incidence of neural tube defects (8). The mechanism by which folate supplementation acts is not
71
fully understood.
72 73 74
Formate is primarily produced in mitochondria from serine, glycine, and two intermediates of
75
choline catabolism, sarcosine and dimethylglycine. Mitochondrial 10-formyl-THF can give rise
76
to formate which leaves the mitochondrion and is incorporated into the cytosolic pool of 10-
77
formyl-THF where it may be used for purine synthesis (29). Cytosolic 10-formyl-THF may also
78
be reduced to 5,10-methylene-THF and thence to 5-methyl-THF. Its formyl group may also be
79
oxidized to CO2, providing an exit for excess one-carbon groups from folate metabolism (16).
80
Formate may also arise from a number of other metabolic reactions, such as methanol
81
metabolism, phytanic acid α-oxidation, tryptophan catabolism and cholesterol synthesis (19).
82
Figure 1 shows an outline of one-carbon and formate metabolism. Although intermediates in
83
one-carbon metabolism primarily occur within cells, formate can be found in appreciable
84
quantities in plasma; this may provide insights into cellular one-carbon metabolism.
85
.
86
MATERIALS AND METHODS
87
Animals All aspects of the surgical and experimental protocols were approved by the Texas
88
A&M University Institutional Animal Care and Use Committee. Suffolk ewes aged 2-5 years
89
were obtained from a commercial supplier. Upon arrival at the animal facility, each ewe received
90
an intramuscular injection of Covexin® 8 (Merck Animal Health, Summit, NJ) and an oral bolus
91
of Valbazen® (Zoetis, Kalamazoo, MI). Ewes received progesterone-impregnated vaginal
92
implants (EAZI-BREED™, CIDR®, Zoetis, Kalamazoo, MI). Implants were removed 11 days
93
after placement at which time prostaglandin F2α (20 mg; LUTALYSE®, Zoetis, Kalamazoo, MI)
94
was intramuscularly administered. The following day, ewes were placed with a ram fitted with a
95
marking harness for a period of 24 h. Marked ewes were presumed pregnant until confirmed
96
pregnant ultrasonographically on gestation day (GD) 25 and 92.
97
Ewes were fed TAMU Ewe Ration, a custom ration (Nutrena, Cargill Animal Nutrition,
98
Minneapolis, MN), twice daily. Ration amount (average 2 kg per day) was based on body weight
99
and determined by ARIES® software version 2007 (University of California, Davis). This was a
100
pelleted feed consisting of minimum 12.75% protein, minimum 3.00% crude fat, and maximum
101
25.00% crude fiber. Ewes were allowed ad libitum access to water. Daily feed consumption was
102
monitored; all subjects consumed all of the food offered.
103
Six pregnant ewes were surgically implanted with indwelling cannulas so as to permit
104
simultaneous sampling from the fetal and maternal circulations. ; 5 pregnant ewes were allowed
105
to lamb naturally (no implanted cannulas) in order to measure ewe and lamb plasma and milk
106
formate levels. In addition, 7 non-pregnant ewes were also sampled for comparison.
107
Ewes were allowed to lamb naturally and shortly after birth the lambs were weighed, ear-tagged
108
and their navels were dipped in 7% iodine. They were weighed weekly thereafter and remained
109
with lactating maternal ewes until weaning at 8 weeks age. All lambs were vaccinated against
110
Clostridium perfingens (types C and D) and Clostridium tetani (Bar Vac CD/T, Boehringer
111
Ingelheim Vetmedica, Inc., St. Joseph, MO, USA) at 4-weeks age and again at weaning. Weaned
112
lambs were maintained on a daily ration of sheep feed (5% of body weight) formulated to
113
National Research Council guidelines for growing lambs (Producers Cooperative Association,
114
Bryan, TX, USA), supplemented by alfalfa (1% of body weight).
115
Sample Collection From the cannulated ewes, maternal and fetal samples were collected into
116
lithium heparin tubes via the surgically implanted catheters at GD 117 ± 1 days; amniotic fluid
117
was collected into polystyrene tubes with no additives. From the non-cannulated group, plasma
118
samples were collected by jugular venipuncture into lithium heparin tubes. Samples were
119
collected from the ewes and lambs shortly after lambing occurred, and at 1 week, 2 weeks, 1
120
month and 2 months after lambing. Milk samples were collected from the ewe by hand milking
121
into polystyrene tubes with no additives. Milk samples were obtained shortly after lambing
122
occurred and at 1 week, 2 weeks, 1 month, and 2 months later (time of weaning). Plasma
123
samples were immediately separated via centrifugation, and all samples were stored at -80°C
124
until analysis.
125
Cannulation On day GD 117±1, the pregnant sheep were anesthetized and aseptic surgery was
126
performed.
127
using intravenous administration of diazepam (0.2 mg/kg BW, Abbott Laboratories, North
128
Chicago, IL) and ketamine (4 mg/kg BW, Ketaset®, Fort Dodge, Fort Dodge, IA). The ewes
129
were intubated and ventilated, and anesthesia was maintained with isoflurane (0.5–2.5%,
130
WasoFlo®, Abbott Laboratories) and oxygen. The abdomen was opened at the midline, the
131
uterus was externalized and incised, and a fetal hind limb was exteriorized. An incision was
Methods have been previously described (31, 37). In brief, anesthesia was induced
132
made over the craniolateral aspect of the fetal hind limb, midway between the hock and stifle. A
133
catheter (0.030" inner diameter, 0.050" outer diameter polyvinyl chloride) was advanced from
134
the fetal cranial tibial artery into the abdominal aorta to the level of the diaphragm; the procedure
135
was then repeated on the other hind limb. The fetus was returned to the uterus, and the uterus and
136
maternal midiline were closed. The maternal femoral vessels were isolated, cannulated and
137
catheters (0.050" inner diameter, 0.090" outer diameter polyvinyl chloride) were advanced from
138
the maternal femoral artery and vein to the level of the diaphragm of the abdominal aorta and
139
vena cava, respectively. Fetal and maternal catheters were passed through the abdominal wall
140
where they were stored in a cloth pouch.
141 142
Analyses. Formate was analysed by the GC-MS method of Lamarre et al. (17). Total plasma
143
homocysteine (tHcy) was measured as described by Vester and Rasmussen (35). Total plasma
144
and amniotic folate were measured using the Lactobacillus casei microbiological assay (14).
145
Total plasma and amniotic vitamin B12 were analyzed using the Advia Centaur XP Immunoassay
146
(Siemens Canada), as described by the manufacturer. Amino acids were derivatized with o-
147
phthaldehyde and assayed by HPLC, as described (38). Choline and its derivatives,
148
dimethylglycine and betaine , were quantified by LC-MS/MS (13, 36) with modifications based
149
on available instrumentation (41, 42)
150
Expression and analysis of data. Data were expressed as means ± standard deviation, with the
151
number of observations in parentheses. Statistical analysis was carried out with Graph Pad 3
152
statistical package. Data were analyzed by means of a t-test, a One-way ANOVA or a Repeated
153
Measures ANOVA, followed by Dunnet’s Multiple Comparison Post-hoc Test, as indicated in
154
figures and tables.
155
RESULTS
156
Figure 2 shows plasma formate levels in maternal and fetal plasma and in amniotic fluid, taken
157
from six pregnant ewes between gestation days 119 and 121 (full term is 147 ± 2 days),
158
corresponding to mid third trimester in humans. Very high levels of formate were evident in fetal
159
plasma (191 ± 62 µM) and in amniotic fluid (296 ± 154 µM). Both of these were significantly
160
higher than the formate concentration in maternal plasma (33 ± 13 µM). We then compared the
161
maternal formate concentrations with those of a separate group of non-pregnant ewes; the
162
formate concentration in the pregnant ewes was significantly higher (p
