Influence of dietary protein source and exogenous progesterone on liver mixed function oxidase
activity in ovariectomized ewes
by Kathryn Jo Wiley
A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in
Animal Science
Montana State University
© Copyright by Kathryn Jo Wiley (1990)
Abstract:
Nineteen ovo-hysterectomized Rambouillet ewes from 3 genetic lines resulting in different
reproductive prolificacy (high-H, control-C, low-L) were assigned to one of two treatments: Urea (U)
and Blood Meal (BM) . Diets were isocaloric and isonitrogenous and ewes were fed at 1.5 X
maintenance for nonpregnant, non-lactating dry ewes. Liver biopsies were taken surgically before the
trial (pre-trt) and 19d after trial had begun (post-trt) and analyzed for microsomal protein and
cytochrome P-450. Progesterone was infused through jugular catheters and blood samples drawn over a
period of 12 h. Blood samples were analyzed for progesterone and blood urea nitrogen (BUN). Initial
and final live weights, dry matter and protein intake, and pre and post microsomal protein content were
not different (P>.10). Pre-treatment cytochrome P-450 concentrations were not different (P>.10)
between treatment groups. However, post-treatment cytochrome P-450 concentrations were different
between U and BM (P<.05) with U group having greater concentration of cytochrome P-450 (1.3 vs .73
nmol mg-1 protein). Blood urea nitrogen concentration tended to be higher in U group (P=. 14) with
13.2 mg dl-1 in U and 8.2 mg dl-1 in SM. High line ewes tended to have greater P-450 concentrations
than other lines (P>.10), which resulted in a significant change (P<.10) in P-450 concentrations in H
compared to L and C. Quadratic regression on the progesterone indicated no difference between
treatments for progesterone disappearance rates. These data suggest that protein quality influenced
P-450 concentration. The P-450 concentrations appeared to be higher in ewes fed U that had been
selected for high reproductive rates. Progesterone disappearance rates could possibly have been
confounded since progesterone injection and sampling sites were the same. 
INFLUENCE OF DIETARY PROTEIN SOURCE AND EXOGENOUS
PROGESTERONE ON LIVER MIXED FUNCTION OXIDASE 
ACTIVITY IN OVARIECTOMIZED EWES
by
Kathryn Jo Wiley
A thesis submitted in partial fulfillment 
of the requirements for the degree
of ^
Master of Science
in
Animal Science
MONTANA STATE UNIVERSITY 
Bozeman, Montana
February 1990
/(W ?
APPROVAL
of a thesis submitted by
Kathryn Jo Wiley
This thesis has been read by each member of the thesis 
committee and has been found to be satisfactory regarding 
content, English usage, format, citations, bibliographic 
style, and consistency, and is ready for submission to the 
College of Graduate Studies.
Date Chairperson, Graduate Committee
Approved for the Major Department
- i- v - cX Cl
Date Head, Major Department
Approved for the College of Graduate Studies
Date Graduate Dean
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the 
requirements for a master's degree at Montana State 
University, I agree that the Library shall make it available 
to borrowers under rules of the Library. Brief quotations 
from this thesis are allowable without special permission, 
provided that accurate acknowledgment of source is made.
Permission for extensive quotation from or reproduction 
of this thesis may be granted by my major professor, or in 
his/her absence, by the Dean of Libraries when, in the opinion 
of either, the proposed use of the material is for scholarly 
purposes. Any copying or use of the material in this thesis 
for financial gain shall not be allowed without my written 
permission.
iv
ACKNOWLEDGEMENTS
I am very grateful to all the people involved within the 
completion of this project. Firstly, I want to thank Drs. Sam 
Rogers and Jack Robbins, who cared enough about the project 
to suggest the names of other biochemists to assist me with 
the P-450 lab protocol. Secondly, I want to thank Drs. Al 
Jesaitis and Ed Dratz, for the use of their lab facilities, 
equipment, and supplies in the determination of the microsomal 
protein and P-450 analysis. I want to include Dr. Mark Quinn, 
with his help with the microsomal protein analysis and Dr. 
Jesaitis1 lab technician Dan Siemsen, for his continuous 
guidance, encouragement and interest in this project. Thirdly, 
I want to thank Drs. Rodney Kott and Mark Petersen, Ruth 
Kemalyan, Bob Padula, Alan Danielson, Connie Clark and Scott 
Wiley for their assistance with the surgeries and/or window 
bleeds. I also would like to include Drs. Mike Tess and Mike 
McInerney, for their help with the progesterone analysis. I 
especially want to thank my major professor, Dr. Verl Thomas 
for his guidance, concern and encouragement throughout this 
study, and Byron Hould, manager for Ft. Ellis, whose 
cooperativity was greatly appreciated. Lastly, a special thank 
you to my husband', Scott Wiley, for sharing his expertise and 
knowledge in this field of study. His commitment and 
encouragement have enabled me to complete1 this project.
VTABLE OF CONTENTS
Page
LIST OF TABLES ............. . ....................  vi
LIST OF FIGURES ...................... ...... . vii
ABSTRACT ..................... ....................  viii
1. INTRODUCTION ................................. I
2. LITERATURE R E V I E W .........    3
Cytochrome P-450 . . . . . .................... . 3
Induction of P-450 ............,............  6
Nutritional Induction of P-450 .........   7
Nutrition and Reproduction  ..............  9
Progesterone . ................    11
Effects of Nutrition on Plasma Progesterone 12
Effect of Type of Protein on Reproduction . 14
3. MATERIALS AND METHODS .................   17
Animals ..............   17
Facilities .................................. 18
Treatments ............................... . . 18
Surgical Procedure .........................  18
Microsomal Preparation and P-450 Analysis . 20
Progesterone . .......................    22
Statistical Analysis  ................... . . 23
4. RESULTS AND DISCUSSION ...................   24
Progesterone Disappearance Rates ...........  32
5. CONCLUSION .................................   37
LITERATURE CITED ............................  38
vi
LIST OF TABLES
Table Page
1. Ingredient and nutrient composition of
experimental diets ..............................  19
2. Influence of protein supplement on ewe body
weight and change, blood urea nitrogen and 
microsomal protein concentration ...........   24
3. P-450 concentrations pre and post-treatment by
 ^ewe .................     26
4. Analysis of variance for linear and quadratic
regression on progesterone ...................... . 35
5. Mean progesterone concentrations ug/ml by time
with standard deviations ........................  36
vii
LIST OF FIGURES
Figure Page
1. Least square means of P-450 as influenced
by protein supplement ...................... 27
2. Least square means of change in P-450 as
influenced by protein .....................  29
3. Least square means of P-450 as influenced
by line .... . ...............................  3 0
4. Least square means of change in P-450 as
influenced by line ....... ....... .........  31
5. Least square means of post p-450 as
influenced by protein and line ..........  33
6. Least square means of change in p-450 as 
influenced by protein and line 34
viii
ABSTRACT
Nineteen ovo-hysterectomized Rambouillet ewes from 3 genetic 
lines resulting in different reproductive prolificacy (high- 
H , control-C, low-L) were assigned to one of two treatments: 
Urea (U) and Blood Meal (BM) . Diets were isocaloric and 
isonitrogenous and ewes were fed at 1.5 X maintenance for non­
pregnant, non-lactating dry ewes. Liver biopsies were taken 
surgically before the trial (pre-trt) and 19 d after trial had 
begun (post-trt) and analyzed for microsomal protein and 
cytochrome P-450. Progesterone was infused through jugular 
catheters and blood samples drawn over a period of 12 h. 
Blood samples were analyzed for progesterone and blood urea 
nitrogen (BUN). Initial and final live weights, dry matter 
and protein intake, and pre and post microsomal protein 
content were not different (P>.10). Pre-treatment cytochrome 
P-450 concentrations were not different (P>.10) between 
treatment groups. However, post-treatment cytochrome P-450 
concentrations were different between U and BM (P<.05) with 
U group having greater concentration of cytochrome P-450 (1.3 
vs .73 nmol mg" protein). Blood urea nitrogen concentration 
tended to be higher in U group (P=. 14) with 13.2 mg dl"1 in U 
and 8.2 mg dl"1 in SM. High line ewes tended to have greater 
P-450 concentrations than other lines (P>.10), which resulted 
in a significant change (P<.10) in P-450 concentrations in H 
compared to L and C . Quadratic regression on the progesterone 
indicated no difference between treatments for progesterone 
disappearance rates. These data suggest that protein quality 
influenced P-450 concentration. The P-450 concentrations 
appeared to be higher in ewes fed U that had been selected for 
high reproductive rates. Progesterone disappearance rates 
could possibly have been confounded since progesterone 
injection and sampling sites were the same.
ICHAPTER I 
INTRODUCTION
Cytochrome systems are complex enzyme systems found in 
most forms of living organisms. Since the early 1960's the 
majority of research has been to determine their location in 
different tissues and mode of action. Cytochrome systems play 
a key role in the nutritional/reproductive axis in ruminants., 
Cytochromes can be manipulated nutritionally and in turn, 
affect animal reproduction.
It has been shown that nutrition increases the 
concentration of the cytochrome systems in liver cells. In 
turn, these systems affect reproduction by influencing the 
concentrations of circulating hormones related to 
reproduction. (Thomford and Dziuk, 1986; Smith, 1988; Thomford 
and Dziuk, 1988). An example of this is the flushing effect 
in sheep. "Flushing" has long been used as a management tool 
to increase ovulation rate by subjecting the ewes to a high 
plane of nutrition three to four weeks prior to breeding. 
Smith (1988) indicated that quantity or quality of dietary 
protein fed may affect these cytochrome enzyme systems. 
However, increased plane of nutrition associated with flushing 
if continued after conception has occurred has a carryover
2effect (Smith et al. , 1983) which increases early embryo 
mortality in sheep (Williams and Gumming, 1982 Parr et al., 
1982; Parr et al., 1987). However, the mechanism describing 
this phenomena has not been determined. Diskin et al., (1988) 
states that, "Embryo mortality is a major cause of economic 
loss in the sheep industry."
The objective of this study was to determine the 
influence of supplemental protein source on cytochrome systems 
and secondly, to determine whether clearance rate of 
progesterone from the blood increases with increased enzyme 
activity. These data may lead to a better understanding of 
how nutrition impacts reproduction in sheep.
3CHAPTER 2 
LITERATURE REVIEW 
Cytochrome P-450
Cytochromes P-450 are found in the testis (Menard et al. , 
1973), adrenal glands (DuBois et al., 1981; Kramer et al., 
1982 Ohashi et al., 1983), liver (Conney and Klutch, 1963;
Omura and Sato 1964a; Omura and Sato 1964b; Kaminsky et al., 
1981; Thomford and Dziuk, 1986; Thomford and Dziuk, 1988), 
kidney, lung, colon, heart, brain, spleen, small intestine, 
muscle (Kaminsky et al., 1981), and rumen papillae (Smith et 
al., 1983 ; Watkins et al., 1987). P-450 has also been found
in ovaries and associated structures (Funkenstein et al., 
1984; Rodgers et al., 1986; Tfzeciak et al., 1986; Waterman 
et al., 1986;) such as the inner mitochondrial membrane of the 
corpus luteum (Rodgers et al., 1986; Rodgers et al., 1988), 
in bovine ovarian follicles (Rodgers et al., 1986), and in 
granulosa cells of developing follicles (Funkenstein et al., 
1984). These systems are located in the microsomes or 
mitochondria of cells, depending on the type of reactions they 
catalyze.
Hepatic cytochrome P-450, also referred to as mixed 
function oxidases (MFC's), are a complex enzyme system
4involving the heme protein, P-450, which catalyzes the 
oxidation of a wide variety of drugs, chemical toxins, 
environmental pollutants, fatty acids and steroids (Kaminsky 
et al., 1981) and are either non-specific for substrate or 
have overlapping substrate specificities (Kaminsky et al.. , 
1981).
Mixed function oxidases occur in a variety of forms 
called isoenzymes or isozymes, depending on the type of 
reactions they are involved in and these isozymes can be 
expressed constitutively, while others are expressed as a 
result of hormonal or chemical stimuli (Eisen, 1986).
Cytochrome P-450 may be classified into two separate 
functional groups, the first being involved in the metabolism 
of steroid hormones in the body, by incorporation of 
cholesterol into the cells of steroid synthesizing organs. 
One of the P-450 isozymes (P-450 SCC) catalyzes the rate - 
limiting step in the synthesis of progesterone, estrogen, and 
testosterone, and is considered an endogenous substance. 
These isozymes are carefully regulated by protein hormones 
released by the pituitary, but have the capability of 
induction (Waterman et al., 1986).
The second is involved with the catabolism of xenobiotics 
and other exogenous substances foreign to normal bodily 
syntheses. These isozymes can be induced in vivo, by exposing 
them to xenobiotics or steroids not directly produced by the 
body. This exposure can cause an increase in concentration of
5the enzyme and/or activity. Substances such as steroid 
hormones synthesized by the gonads and adrenal glands are 
known to be catabolized by the liver in rats (Thomford and 
Dziuk, 1986). These compounds are converted into substances 
excreted by the kidney in the urine or are converted in the 
liver and excreted from the body in the bile (Williams, 197 3) . 
The mechanism by which these enzymes hydroxylate testosterone 
and progesterone in the liver is unclear at this time. Thomas 
et al. , (1987) reported that hepatic MFO1s exhibited a 
preference for endogenous steroid hormones over drugs and 
xenobiotics due to the Km values for progesterone, 
testosterone and estradiol 17(3 which have been shown to be 10 
times lower than those of some drug oxidations.
There are volumes of literature available describing the 
mechanism of steroid biosynthesis by P-450 in the gonads and 
adrenal glands, but very little is known about their mode of 
action in the liver. It is interesting that species, sex and 
age influence enzyme concentrations and xenobiotic metabolism 
(Smith et al., 1983; Stegman et al., 1988). Interpolating 
data from one species to another has been a major stumbling 
block for researchers attempting to incorporate current 
knowledge into applicable fields such as animal production 
(Watkins et al., 1987).
6Induction of P-450
Cytochrome systems can be induced which results in 
increased enzyme concentration and/or activity. This section 
will discuss how the second functional group of P-450 can be 
induced. Jefcoate (1986) discussed in great detail four 
different mechanisms which rapidly affect the activity of P- 
450. One mechanism is the control of substrate; by increasing 
the amount of substrate enzyme systems respond by increasing 
activity, and increased enzyme activity is paralleled by 
accelerated drug metabolism. (Conney and Klutch, 1963).
Most data from studies on rats report increased liver 
size when P-450 enzyme systems are induced (Clinton et al., 
1977; Truex et al., 197 7; Edes et al., 1979; Eisen, 1986). 
Smith (1988) found increased liver size in ewes when these 
enzyme systems were induced with phenobarbital. A possible 
explanation for this response may be that the liver releases 
by-products from the reactions it catalyzes. These products 
exhibit activity involved with inflammatory responses 
(Waterman et al., 1986).
Earlier research (Conney and Klutch, 1963; Omura and 
Sato, 1964a; Omura and Sato, 1964b; Shetty et al., 1972) 
involving induction of liver microsomal enzymes found 
phenobarbital (PB) to be a potent inducer, and research was 
directed toward investigating the biochemical pathways
7associated with measuring the enzyme systems. Numerous studies 
have found increased liver MFO activity in swine, rats, and 
sheep treated with phenobarbital (Conney and Klutch, 1963; 
Kaminsky et al., 1981; Thomford and Dziuk, 1986).
Shetty et al. (1972) investigated the mode of action of 
PB. Sheep were injected with PB over time, then antipyrine 
was given. Antipyrine was known at that time to be 
metabolized by hepatic enzymes. Their data indicated a 
significant increase in antipyrine plasma clearance rate in 
those sheep induced with PB. They interpreted the data to mean 
that xenobiotics actually induced the liver P-450 system. 
Conney and Klutch, (1963) found that rate of metabolism of 
testosterone and androstene 3,17 dione in rats was faster in 
those treated with PB. They hypothesized that treatment with 
PB stimulated the activity of the enzyme systems located in 
the liver microsomes involved in hydroxylation of testosterone 
and androstene 3,17 dione. They also noted that pregnenolone 
16a carbonitrile is a potent inducer of drug metabolizing 
enzymes (Lu et al., 1972).
Nutritional Induction of P-450
Several studies have reported that quality or quantity 
of protein may play a key role with induction (Thomford and 
Dziuk, 1986; Smith, 1988; Thomford and Dziuk, 1988). It's 
been documented that an amino acid deficiency or toxicity may 
induce MFC activity. Clinton et al. (1977) conducted an
8experiment to determine the influence of dietary protein level 
on P-450 concentrations. Growing and adult female rats were 
fed either 7.5%, 15%, or 45% protein diets supplemented with 
methionine. An increase in P-450 concentration as percent 
protein fed increased was observed. However, the adult rats 
responded to a lesser degree than younger rats due to the 
age-dependent decline in activity. (Clinton et al., 1977).
Edes et al. (1979) reported sulphur containing amino 
acids influenced MFO activity, and feeding a diet deficient 
in sulphur amino acids resulted in decreased MFO activity in 
rats. It appears that a three-way interaction has been 
observed. As nutritional protein levels increase, MFO 
activities in the liver increase, thereby increasing 
degradation of drugs and other exogenous substances (Clinton 
et al., 1977). Level of protein appears to have more effect 
on MFC's than energy level (Clinton et al 1977). This is 
supported by Truex et al. (1977) whp found rats fed protein 
deficient diets had reduced MFO activity and drug metabolism 
rates. Thomford and Dziuk (1988) fed ovariectomized ewes a 
high (14.8%) and low (4.7%) crude protein diet. They found 
that feeding the high CP diet increased P-450 concentrations 
by 20% within 10 days of feeding and no significant difference 
in microsomal protein was found between treatment groups.
9Nutrition and Reproduction
Studies on effects of plane of nutrition on reproduction 
have been done as far back as the early 1900's, and research 
has produced extensive results with sheep (El-Sheikh et al., 
1954) .
Sheep producers in Australia have found lupin grain an 
excellent source of protein and energy for ruminants. Many 
studies have investigated the effects of feeding lupin grain 
on ewe reproduction. Brien et al. (1981) found that ewes 
grazing pasture and fed lupin had significantly lower plasma 
progesterone concentrations than those grazing pasture without 
lupin supplementation. He concluded that high plane of 
nutrition at mating lowers plasma progesterone concentrations 
and suggested this may be the cause of early embryo mortality. 
Coop (1966) conducted an experiment to determine the influence 
of different planes of nutrition on bodyweight prior to and 
at breeding. Ewes were allocated into three groups, (HE, MM, 
LH) and all were of the same body condition at the time of the 
study. The (HE) group was fed to gain body condition fpr 40 
d prior to flushing, then diets were switched for them to lose 
body condition 3 weeks prior to breeding until the end of the 
breeding season. The MM group was fed to maintain body 
condition throughout the study, and the LH group were fed for 
40 d to lose body condition until the diets were switched 3 
weeks prior to breeding to gain body condition throughout the
10
breeding season.' Ewes in the HL group had higher first 
service conception rates, more lambed the first 17.d of the 
lambing season and fewer open ewes; however, the percentage 
of lambs born per ewe lambing was lower than the other 
treatment groups. The LH group had the lowest first service 
conception, the longest lambing season, but the highest 
percentage of lambs born per ewes lambing. The MM ewes were 
intermediate in response to the HL and LH groups.
Coop (1966) conducted a study involving time of breeding 
cycle and level of nutrition, without consideration of body 
condition. Ewes were placed in three different nqtritional 
planes, (HH) and fed a high plane of nutrition 3 weeks prior 
and during the breeding season, (HL) ewes fed a high plane of 
nutrition prior to breeding, then switched to low during the 
breeding season, and (LL) ewes fed below maintenance diet 
prior to and during the breeding season. The results were 
basically the same as the study described previously; the ewes 
fed a high level of nutrition before and during mating (HH) 
showed an increase in number of lambs born per ewe mated, 
more open ewes, lower first service conception rates and fewer 
lambs born first 17 days of lambing season. Ewes fed the low 
level of nutrition (LL) before and during gating had the 
lowest fertility, fewest lambs born, and more open ewes th^n 
(HH) . Ewes fed a high plane of nutrition (HL) prior to mating 
and switched to low plane of nutrition at mating showed higher 
fertility, less open ewes, but fewer lambs born per ewe mated.
11
Coop (19 66) concluded that ewes of.higher live weight 
at mating have improved conception rates, a lower incidence 
of barrenness and respond to flushing more than thinner ewes. 
He also stated, "special time relationship between flushing 
and mating is much less important than was previously 
thought".
Foote et al. (1959) conducted a three year study- 
involving the effects of feeding levels on ovulation rate and 
embryo survival. . His data indicated a possible "carryover" 
effect of the flushing response such that whatever was 
stimulated as a result of flushing remained stimulated ipto 
the breeding season and early pregnancy. It's been postulated 
that .the "carryover" effect may be influenping utppine 
environment (Edey, 1969) or possibly the peripheral 
concentrations of progesterone, the hormone essential for 
maintenance of pregnancy (Edey, 1969; Bassett et al., 1969; 
Bindon, 1975; Gumming et al., 1975; Tepperman, 1981; Brien et 
al., 1981; Williams and Gumming, 1982; Dial and Dziuk, 1983; 
Parr et al., 1987; Smith 1988; Rodgers et al., 1988).
Progesterone
Progesterone, a steroid hormone, is synthesized by the 
corpus luteum located on the ovary and adrenal glands, and is 
essential for the maintenance of pregnancy. This hormone can 
be catabolizpd at a faster rate than normal due to greater MFO 
activity (Thomas et al., 1987). Clearance rates of
12
progesterone influence serum concentration which affects 
maintenance of pregnancy.
Bedford et al. (1972) conducted a study to determine 
metabolic clearance rate of progesterone in sheep and found 
that progesterone was removed rapidly. Little §t al. (1966) 
determined that progesterone in ovariectomized human females 
and in males is rapidly removed by both hepatic and, extra 
hepatic tissues, and found no difference between single- 
injection of labelled progesterone and continuous infusion. 
It has been determined that greater than 75% of the 
progesterone is removed by the liver (Bedford et al., 1972). 
Little (1966) also mentioned that disappearance rate of 
progesterone from other studies were inconclusive sincg they 
were not able to determine whether the disappearance was due 
to metabolism or rather the distribution of the hormone into 
a large volume. Bedford et al. (1972) noted that there is no 
evidence of a 1 progesterone-conserving mechanism1 in sheep but 
in pregnant guinea pigs. There appears to be a mechanism 
associated with an increased concentration of a protein with 
high affinity for progesterone.
Effects of Nutrition on Plasma Progesterone
Parr et al. (1987) found that overfeeding during early 
pregnancy reduced serum progesterone concentration thereby 
reducing pregnancy rate; in sheep. Plasma progesterone 
concentrations sufficient for proper embryo implantation and
13
support for early pregnancy (up to 50 days) lie between 2 
-3 ng/ml, afterward a steady increase occurs until parturition 
(Bassett et al., 1969; Bindon, 197 5; Brien et al., 1981, Parr 
et al. , 1987). Parr et al. (1982) fed, ewes either 25% or 
100% maintenance diets from the day of mating until d 11 or 
12. Plasma progesterone concentrations were higher in ew$s 
underfed than those fed the 100% maintenance diet. Parr et 
al. (1987) fed three groups of ewes diets containing either
a low [25% of maintenance (M)], a medium (100% M) or a high 
(200% M) level of nutrients. Ewes were then either implanted 
or not implanted (control) with 34 0 mg progesterone. Ewes fed 
the high ration had pregnancy rates significantly lower 
compared to the medipm and low rations. Progesterone 
supplementation did not affect the low and medium groups, but 
increased the pregnancy rate in the high ration group by 28%. 
Progesterone concentration was less than 1.5 ng/ml for control 
ewes in the high ration group which is below the threshhold 
to maintain pregnancy (Bassett et al., 1969; Bindon, 1975;
Brien et al., 1981; Parr et al., 1987) . Mean progesterone 
concentration increased to 2.8 ng/ml when progesterone 
implants were used in the H group. They postulated that a 
higher plane of nutrition increased blood flow to the liver 
and progesterone catabolism (Bedford et al., 1972).
Gumming et al. (1975) imposed three planes of nutrition 
from the second to the sixteenth day post mating: 25%,(1/4M); 
100%(M); and 200% (2M) of maintenance. Ewes fed 1/4M ration
14
had elevated progesterone concentrations compared to the Othqr 
groups. No explanation for the rise in the underfed ewes was 
given. This research was similar to Williams and Gumming 
(1982) who fed three groups of ewes at either 1/4 maintenance, 
maintenance, 2X maintenance. They found that plasma
progesterone concentrations were consistently higher in 1/4M 
ewes than with the M or 2M ewes and they concluded that there 
is an inverse relationship between concentration of 
progesterone and nutrition in ewes. Brien et al. (1981)
investigated the effects of lupin grain supplements on 
progesterone concentrations and early embryo mortality. TjlQy 
concluded that a high plane of nutrition (with lupin 
supplementation) at mating lowers plasma progesterone an4 this 
may be a factor in early embryonic death.
Effect of Type of Protein on Reproduction 
Smith (1988) in his discussion on protein intake and 
flushing response indicated that differences in ruminal 
degradation rate of protein may influence ewe reproduction. 
In dairy cattle, Ferguson et al. (1988) reported that cows 
fed diets containing greater percentage of crude protein had 
lower conception rates and days open, and feeding rumen 
protected protein improved conception rates and decreased 
number of services/conception and days open. They
hypothesized that feeding excess ruminally degradable protein 
lead to higher concentrations of ammonia, urea or other
15
nitrogenous compounds in the blood and uterine fluids that are 
toxic to spermatozoa, ova or embryos. Feeding a high crude 
protein ruminally degradable feed increased blood urea 
nitrogen (BUN) over cows fed rumen-protected protein. He 
concluded BUN concentrations greater than 20 mg/dl may 
predispose cows to infertility. Thompson et al. (1973) found 
that ewes fed purified diets containing urea as the major N 
source required several services before conception occurred. 
However, in their study they found no detrimental influence 
on reproductive efficiency due to feeding urea to ewes, cows, 
rams, and bulls.
Several authors (Menard and Purvis, 1973; Thomas et al., 
1986; Smith, 1988; Thomford and Dziuk, 1988) reported that 
excess protein or merely overfeeding influences enzymatic 
activity which in turn affects hormonal levels associated with 
early embryo mortality.
Menard and Purvis (1973) indicated that enhanced 
production of steroids stimulated P-450 concentration in the 
liver. He postulated that due to the increase in microsomal 
enzymes, estradiol metabolism increased, thereby causing more 
FSH circulation, and increased ovulation rates. Thomas et al. 
(1987) made the same inference. When mixed function oxidase 
activity is increased, steroid hormones are catabolized at a 
faster rate, which in turn influences a greater negative 
feedback on the pituitary and hypothalmus (Thomford and Dziuk, 
1988) . Smith (1988) mentions that in looking at the mechanism
16
underlying the flushing response, estradiol 17)6 depresses 
ovulation rate in the ewe and that it appears to lower 
Follicle Stimulating Hormone (FSH) levels as well.. FSH is a 
protein hormone released by the pituitary gland and promotes 
follicular growth and development in the ovaries. Therefore, 
when removed from peripheral blood at a faster rate due to a 
higher MFO activity in the liver, estradiol 17)6 will reduce 
the negative feedback control on the hypothalmus and 
pituitary, thereby increasing LH and FSH release from the 
pituitary, and thereby causing more follicles on the ovary to 
develop (Thomas, et al., 1987).
In summary, nutrition can affect reproduction^.by 
influencing enzymatic activity in the liver. Flushing and 
associated high plane of nutrition has a three week carryover 
effect on reproduction. Since flushing is practiced before 
and during the breeding season, this "carryover effect" may 
be of major importance to early pregnant ewes. It has been 
shown that overfeeding induces liver MFO activity which in 
turn catabolizes progesterone at a faster rate. Thus early 
pregnant ewes are subject to losing embryos from their first 
service due to reduced progesterone concentrations which is 
insufficient to maintain pregnancy.
The purpose of this study is to determine what type of 
excess protein fed will stimulate MFO activity and to 
determine whether progesterone disappearance rate increases 
as MFO activity increases.
17
CHAPTER 3
MATERIALS AND METHODS 
Animals
The experiment was conducted at the Fort Ellis 
Agricultural Experiment Station near Bozeman, Montana.
Rambouillet ewes from three genetic lines developed by 
Burfening et al (1989) were used. Ewes were either selected 
for high (H) or tow (L) reproduction based on their dam's 
reproductive rate beginning in 1969. By 1972, the remaining 
foundation ewes from L and H were random bred to form the 
control (C) line.
Nineteen Rambouillet ewes were available from a previous 
study which consisted of H line (n=4), L line (n=9) and C line 
(n=6)'. These ewes had undergone ovo-hysterectomies. The use 
of ovariectomized ewes should minimize changes in steroids due 
to estrus cycles which could affect mixed function oxidase 
levels (Thomford and Dziuk, 1988).
The trial began June I, 1989 when liver biopsies were 
taken to determine baseline values for P-450 apd a repeat 
biopsy was taken on June 27, 1989. Following the second 
biopsy ewes were fed their experimental diets until July 11, 
1989 when a 12 h window bleed for progesterone was conducted 
prior to the study the ewes were maintained on pasture.
18
Facilities
Ewes were penned individually in 1.46 m2 pens. Ewes were 
in close proximity of each other and bedded with wood chips. 
They were protected from inclement weather and wind to 
minimize stress. Ewes were fed twice daily and clean water 
was available at all times. Surgeries were performed in the 
surgical facilities located at the Fort Ellis Agricultural 
Experiment Station.
Treatments
Following the initial liver biopsies, nineteen ewes were 
randomly assigned to one of two dietary protein supplement 
groups: blood meal (BM) or urea (U) . Pelleted diets were
formulated to be isocaloric and isonitrogenous (Table I) . 
Quantity fed was calculated at 1.5 times maintenance protein 
requirement for non-pregnant dry ewes (NRC, 1985). This level 
of dietary protein was used to ensure a stimulation of P-450 
concentration (Thomford and Dziuk, 1988).
Diets were fed twice daily at approximately 083 0 and 17 00 
h . Ewe weights were recorded prior to and at the end of the 
trial.
Surgical Procedure
On June I, 1989, liver biopsies were taken surgically 
from 18 ewes. One ewe reacted to the biotal and was removed 
from the project. Repeat biopsies were taken on June 27, 
1989. Food and water were withheld twelve hours prior to the
19
Table I. INGREDIENT AND NUTRIENT COMPOSITION OF 
EXPERIMENTAL DIETS
Protein Supplement
Ingredient (%) UREA BLOOD MEAL
Straw .81.1 83.9
Molasses 5.9 7.8
Wheat Millrun 8.0
Urea . 9
Blood Meal 4.0
Gypsum . 3 . 2
Dicalcium phosphate . I . 5
Bentonite 3.2 3.2
Salt-Iodized .2 .2
Trace mineralized salt .2 . 2
Dry matter and nutrient cone.
Dry matter intake, kg 1.86 1.82
Crude protein, % 
Metabolizable energy, 
Meal kg"1
7.70 7.30
3.73 3.98
surgeries. Liver biopsy samples were obtained from eighteen 
and sixteen ewes respectively during the initial and repeat 
surgery.
Ewes were shaved on the right side, from the ninth rib 
to 10 cm caudal to the end of the last rib. Twelve ml of 4% 
Biotal (Bioceutics) were given I . V. in the carotid artery with 
an 18 x 1-1/2 inch needle. When the ewe became prone, she was 
placed on a surgery table, and halothane immediately
20
administered. Lying on her left side, the shaved area was 
surgically scrubbed and a sterile surgery drape was used. An 
incision was made starting approximately 5 to 10 cm caudal 
and 5 to 10 cm lateral to the sternum and then continuing for 
10 cm parallel to the end of the ribs with an electro-surgical 
unit. Both muscle layers and peritoneum were opened, and a 
"thumbnail" liver biopsy was taken by sliding two fingers and 
thumb into the incision moving toward the ribs and pinching 
off I to 5 g liver. Wisniewski et al. (1986) found uniformity 
of hepatic xenobiotic metabolizing enzymes throughout the 
livers of cattle, goats and sheep. Therefore, we were not 
concerned about sampling from the same lobe each time. Liver 
samples were immediately placed on ice and kept at 0° C until 
microsomal preparations were performed. Peritoneum and each 
muscle layer, were sutured and the skin surgically clamped. 
Twelve ml of penicillin were administered intramuscularly. 
Each ewe was then placed in an indoor pen adjacent to the 
surgery room and kept under surveillance for 24 h. Hay and 
water were available. Following the twenty-four hour 
surveillance period, ewes were placed back in their pens and 
fed their respective diets.
Microsomal Preparation and P-450 Analysis 
Liver samples were taken, weighed and recorded, and 
rinsed in cold .!SM KCL. Each sample was placed in a glass 
tube marked with the ewe's identification number, and stored
21
on ice. Liver enzyme assays were completed on the same day 
of the surgeries in a cold room kept at 0-4 ° C.
Two to five g of sample were minced with scissors, and 
rinsed three times in approximately 10 v . I SM cold KCL to 
remove all blood. Minced samples were then placed in a 15 ml 
teflon-glass homogenizer and 10% w/v of Tris Buffer (.05M 
Trizma/1.SM KCL, pH 7.4) was added. Samples were homogenized 
for approximately 60 seconds, and centrifuged at 12,000 x g 
for 20 minutes at 4° C.
Centrifuge tubes were weighed, marked and recorded. 
After the centrifugation was completed, supernatant was 
poured off into the ultra-centrifuge tubes. Supernatant was 
centrifuged at 100,000 x g for 90 minutes to obtain a 
microsomal pellet. After completion of the last spin, the 
supernatant was poured off and a reddish-brown gel-like pellet 
was obtained. Pellet weight was recorded.
Pellets were resuspended to approximately 20% w/v with 
Tris-Buffer and microsomal protein analysis conducted using 
Pierce BCA protein assay reagent9. Resuspended pellets were 
diluted to approximately 5 mg protein ml'1 A baseline was 
recorded on the sample, then the sample was reduced with 
sodium dithionite, and another baseline recorded. Carbon 
monoxide was bubbled in, and difference spectra was recorded 
from 400-500 nm.
9Pierce Chemical Company, 3747 Meridian Road, Rockford, IL 
61105
22
An extinction coefficient of 91 nM"1 cm'1 was used to 
calculate cytochrome P-450 concentration.
Progesterone
Five ewes from each protein supplement group were 
randomly selected for use in a window bleed. On July 11, 1989 
indwelling jugular catheters were placed in each of the ten 
ewes. One ewe pulled her catheter out shortly after insertion 
and was removed from the window bleed. A 10 ml blood sample 
was taken at 0930 for baseline progesterone and blood urea 
nitrogen (BUN) analyses. At 0945, 25 mg progesterone was 
injected through the catheters followed by 5 ml saline. At 
1000 the first blood sample was taken, in increments of 15 
min for the first 2 h, then every 30 min for the next 2 h, 
then every 60 min for the remaining 8 h. Blood samples were 
refrigerated immediately until centrifugation. Blood samples 
were centrifuged for 30 min at 12,000 x g. Upon completion 
of centrifugation, serum was poured into serum tubes and 
frozen at -20° C.
Serum samples were sent to Dr. Dennis Hallford, Endocrine 
Laboratory at New Mexico State University, Las Cruces, New 
Mexico for progesterone analyses. Validation for the assay was 
done according to Hallford et al. (1982). The Progesterone - 
11 BSA antiserum (GDN 337) was kindly supplied by Dr. G.D. 
Niswender, Department of Physiology and Biophysics, Colorado 
State University, Fort Collins, Colorado. Serum samples were
23.
analyzed for BUN using an Ames Pacer semi-automated analyzer13.
Statistical Analysis
Microsomal Protein, P-450, BUN and weight data were 
analyzed using a completely randomized design with least- 
squares analysis of variance using the General Linear Models 
Procedure of SAS (1987). The statistical model included the 
fixed effects of protein supplement (BM.vs U), Line (H,L,C) 
and their two-way interaction. Means were separated using 
least significant difference. Progesterone data were analyzed 
SAS (1987). Model included the fixed effects of. treatment and 
by least squares analysis of variance by the GLM procedure of 
linear and inverse quadratic regressions of time. Regressions 
were initially evaluated within time to determine differences 
in disappearance pattern. Regression values were not 
different (P>.10) and therefore regression values within 
treatment were combined. Linear regression was not different 
(P>.10) from zero.
bMiles Laboratory, Inc., Elkhart, IN.
CHAPTER 4
RESULTS AND DISCUSSION
Initial and final live weights were not different (P>.10) 
between protein supplement groups (Table 2) . Mean initial and
Table 2. INFLUENCE OF PROTEIN SUPPLEMENT ON EWE BODY WEIGHT 
AND CHANGE, BLOOD UREA NITROGEN AND MICROSOMAL PROTEIN 
CONCENTRATION
BM
Protein Suoolement 
. U SEa P
Weight, kg 
Initial 60.7 59.7 5.68 0.78
Final 61.7 61.0 6.24 0.84
Change . 99 •1.19 1.19 0.79
Blood Urea N, mg dl'1 8.2 13.2 1.87 0.12
Microsomal Protein,
(mg g"1)
Pre-treatment 18.12 17; 82 0.96 0.83
Post-treatment 13.51 14.58 0.49 0.14
Change -4.74 -3.15 1.32 0.45
Standard error of least square means
final weights were 60.7 and 61.7 kg for BM and 59.7 and 60.9 
kg for U, respectively. Weight change during the trial for 
BM and U were similar (P>.10) ; .99 and 1.19 kg, respectively.
25
Microsomal‘protein concentrations pre and post-treatment 
for BM and U were 18.12 and 17.82 mg g"1 liver (pre) and 13.51 
and 14.58 mg g'1 liver (post), respectively, with no difference 
(P>.10) between treatments (Table 2). Pre and post treatment 
microsomal protein concentrations lie in the range found by 
Thomas et al. (1987). No differences (P>.40) in change in 
microsomal protein concentrations were detected. Associated 
enzymes and P-450 involved with hydroxylation of drugs are 
found in the microsomes of the liver. It has been shown that 
phenobarbital and other xenobiotics have the ability to induce 
the formation of microsomal enzymes and P-450. (Mayes, 1988). 
Our data are in agreement with that of Thomford and Dziuk 
(1988) who reported that increased protein intake had no 
effect on microsomal protein concentrations on liver of ewes 
but did elevate cytochrome P-450 concentrations. Average 
post-treatment concentration of microsomal protein in our 
study (14.08 mg g"1) was higher than their reported values 
(7.7 mg g"1) .
Calculated pre and post-enzyme concentrations for 
individual ewes are reported in Table 3. Range of values for 
enzyme concentrations agree with other published values 
(Thomford and Dziuk, 1986; Thomas et al., 1987; Wisniewski 
et al., 1987; Thomford and Dziuk, 1988).
Pre-treatment enzyme concentrations were not different 
(P>. 10; .69 BM vs .92 U nmol mg'1 protein). However, post­
enzyme concentrations were different (P<.05) with U fed ewes
26
Table 3. P-450 CONCENTRATIONS PRE AND POST-TREATMENT BY EWEa
Ewe ID
Protein
Supplement
P-450 Concentration 
Pre-treatment Post-treatment
2002 BM .9080902 1.3182560
2008 BM .3389306 . 9478077
2011 BM .9313917
2017 BM .8713542 . 5030785
2019 UREA . 7764586 .6662652
2028 BM .5347362 .5347362
2029 UREA 1.0720560 .7760003
2048 BM .8038958 1.0273220
2054 UREA .4947130
2422 BM .6138049 .7697201
2429 UREA 1.0141120 2.0480870
2473 UREA .7211695 1.7380780
2645 UREA 1.0317550 I.0695950
2648 UREA 1.1571450 1.6214110
2650 BM .7709571 . 6323807
2655 UREA .7148759 1.0664330
2679 UREA .9354623 1.3469060
2705 BM . 8995252
dP-450 concentrations in nmol mg"1 microsomal protein
having greater concentrations of cytochrome P-450 than the BM 
group (Figure I; 1.3 vs .73 nmol mg 1 protein) . These data 
suggest a relationship between dietary protein source and 
cytochrome P-450 concentration. Dry matter and protein intakes 
between treatment groups were similar (128.8 g'1, BM versus 
138.4 g d protein, U) . Therefore, differences in cytochrome
N
M
O
L
 
P-
45
0 
MG
 
M
I
C
R
O
S
O
M
A
L
 
P
R
O
T
E
I
N
figure i .  Least Square Means of P-450
as Influenced by Protein Supplement
0.7
PRE-TREATMENT POST-TREATMENT
DO
28
P-450 should be related to protein source and not protein 
intake. Ewes fed U tended (P> .10) to have higher 
concentrations of BUN (Table 2) than those fed BM with 8.2 mg 
dl'1 in the BM group and 13.2 mg dl"1 for U. BUN values are 
indicative of N catabolism in the body. In our situation, 
excess N was probably available from urea and converted to 
ammonia in the rumen. Ammonia was absorbed into the portal 
blood system and was filtered in the liver. Thus the liver 
may have increased cytochrome P-450 activity to clear excess 
ammonia to prevent ammonia buildup in the body (Conney and 
Klutch, 1963; Jefcoate, 1986; Rodwell, 1988). Change in P- 
450 concentration (Figure 2) therefore tended (P=.15) to be 
greater for those fed U compared to BM (.04 vs .38 nmol mg’1).
Pre and post-treatment cytochrome P-450 concentrations 
were not different (P>.10) between lines (Figure 3). Pre­
treatment least square means of enzyme concentration in nmol 
mg"1 microsomal protein for H , L, and C lines were .74, .87, 
.81, respectively. Although not significantly different 
(P>. 10) H line ewes tended to have greater P-450 
concentrations than the other lines. This resulted in a 
significant change (P<.10) in P-450 concentration in H line 
compared to C or L line ewes (Figure 4).
It has been documented that the H ewes are superior to 
the L ewes in litter size, but were significantly lower in 
first service conception (Pc.OS) although no fertility 
differences were found over all services (Schoenian 1988).
N
M
O
L
 
P-
45
0 
MG
' 
M
I
C
R
O
S
O
M
A
L
 
PR
O
T
E
I
N
figure 2 . Least Square Means of Change in
P-450 as Influenced by Protein
PROTEIN SUPPLEMENT
I
N
M
O
L
 
P-
45
0 
MG
''
 
M
I
C
R
O
S
O
M
A
L
 
P
R
O
T
E
I
N
FIGURE 3. Least Square Means of P-450
as Influenced by Line
PRE-TREATMENT POST-TREATMENT
N
M
O
L
 
P-
45
0 
MG
 
M
I
C
R
O
S
O
M
A
L
 
P
RO
TE
IN
0.59
figure 4 . Least Square Means of Change in
P-450 as Influenced by Line
- 0.05
32
In Schoenian1s master's thesis she stated, "Embryo survival 
was lowest in the H line". We suggest lower progesterone 
concentrations may be the underlying cause of this due to the 
increased mixed funtion oxidase activity in the liver 
catabolizing progesterone at a faster rate (Menard and Purvis, 
1973 ; Thomas et al. , 1986; Smith, 1988; Thomford and Dziuk, 
1988). Although it was not the primary objective of our work 
to determine differences in P-450 concentrations between 
reproductive lines, these data suggest selection for 
reproduction may be influencing liver enzyme systems.
A significant treatment by line interaction was detected 
(P<.05) with respect to P-450 concentration and change. (H) 
line ewes fed U had greater (P<.05) concentrations of P-450 
than all other treatment combinations (Figure 5). High line 
ewes fed BM and U had values of .77 and 1.9 respectively, (C) 
BM and U .63 and 1.3, respectively and (L) were .8 and .72 
nmol/mg microsomal protein respectively. Therefore, P-450 
concentration change was greatest (P<.05) in the (H) line'fed 
U (Figure 6).
However, these results must be treated with caution since 
the number of ewes from H line was small, (n = 3) with 2 H 
line ewes in the U group and I in the BM group.
Progesterone Disappearance Rates
Quadratic regression was significant (P<.01; Table 4) for 
best fit of the data points. No difference (P>.10), however
N
M
O
L
 
P-
45
0 
MG
'1
 
M
I
C
R
O
S
O
M
A
L
 
P
R
O
T
E
I
N
figure 5 . Least Square Means of Post
P-450 as Influenced by Protein and Line
2.5
N
M
O
L
 
P-
45
0 
MG
"1
 
M
I
C
R
O
S
O
M
A
L
 
P
R
O
T
E
I
N
figure e. Least Square Means of Change in
P-450 as Influenced by Protein and Line
1.5
0.5
- 0.5
- 0.14
- 0.2
HIGH LINE, BM I 
CONTROL, UREAl
HIGH LINE, UREv 
LOW LINE, BM
CONTROL, BM 
LOW LINE, UREA
#
W
f t
-1
I35
Table 4. ANALYSIS OF VARIANCE FOR LINEAR AND QUADRATIC 
REGRESSION ON PROGESTERONE3
Source DF
Mean
Square F Value P
Treatment I 2 . 2 0.90 0.34
Ewe (Trt) 5 11.2 4.56 0.0007
Linear I 0.5 0.21 0.65
Quadratic
a-TJ 2 n o .  T l ______
2 1070.2 433.5 0.0001
was detected between protein supplement treatments. Least 
square treatment means were: U, I.69±.20 and BM 2.021.17. It 
is apparent when looking at progesterone concentrations in 
Table 5 that time had more effect on progesterone 
disappearance than did treatment. Treatment means and 
standard deviations within time were similar. It appears from 
the data that more frequent sampling should have occurred 
immediately following progesterone injection, progesterone 
could have been infused in the side opposite the sampling 
site, and perhaps I mg progesterone could have been infused.
36
Table 5. MEAN PROGESTERONE CONCENTRATIONS UG/ML BY TIME WITH 
STANDARD DEVIATIONS
TREATMENT
TIME (min) BM STD DEV UREA STD DEV
0 19.12 6.93 20.66 8.7
15 4.11 2.01 2.80 0.94
30 2.20 0.90 2.92 1.27
45 1.27 0.58 1.51 0.29
60 1.13 0.26 1.41 0.26
75 0.91 0.28 1.08 0.2 5
90 0.91 0.2 0 1.28 0.64
>90 0.42 0.10 0.77 0.56
37
CHAPTER 5 
CONCLUSION
Progesterone disappearance rates were probably not 
meaningful since progesterone injection and sampling sites 
were the same, and blood samples were probably not taken 
frequently enough (recommend 5 incremental blood samples for 
first 30 minutes, followed by 15 minute samples drawn for next 
3 h) . This study demonstrated that protein quality influenced 
cytochrome P-450 concentrations. The inducer of the 
cytochrome system in our experiment appeared to be ruminal 
ammonial concentrations and not amino acids. It was 
interesting that the cytochrome P-450 system seemed to be more 
induced in ewes fed U that had been selected for high 
reproductive rate. This information presents a number of 
questions involving the relationship to genetic selection and 
response to environment. Perhaps genetics is simply selecting 
for more enzymes, or their sensitivity to the environment 
and/or substrate. A few questions I pose for further research 
are; Have the H line ewes been selected for synthesizing more 
of these enzymes? Are their enzyme systems more sensitive to 
different substrate? Have the L line ewes been selected for 
little response to different substrates?
LITERATURE CITED
39
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