THE USE OF ALTRENOGEST TO CONTROL REPRODUCTIVE

FUNCTION IN BEEF CATTLE

A Dissertation

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Submitted to the Graduate Faculty of
Louisiana State University and
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Agricultural and Mechanical College
In partial fulfillment of the
Requirements for the degree of
Doctor of Philosophy
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in
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The Interdepartmental Program in Animal and Dairy Sciences

by
Clarence Edward Ferguson
B.S., McNeese State University, 1996
M.S. Stephen F. Austin State University, 1999
M.N.A.S., Southwest Missouri State University, 2000

December 2004
 UMI Number: 3151828

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UMI Microform 3151828

Copyright 2005 by ProQuest Information and Learning Company.
All rights reserved. This microform edition is protected against
unauthorized copying under Title 17, United States Code.

ProQuest Information and Learning Company

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 ACKNOWLEDGEMENTS
The author would like to convey his sincere gratitude to his major professor, Dr.
Robert A. Godke for his direction, patience, and commitment to train him as a research
scientist. The author believes that the training he received in Dr. Godke’s Program has
been and will continue to be an invaluable experience that will aid him in overcoming
obstacles later in life. Appreciation is extended to the members of his graduate
committee, Dr. John Lynn, Dr. Kathy Williams, Dr. Earl Pope for their advice and
guidance. A special thanks is extended to Dr. John Lynn for all of his advice, patience
and friendship which will always be remembered. Also, the authors would like to
acknowledge the assistance provided by the Louisiana State University School of
Veterinary Medicine professors, Dr. Dale Paccamonti, Dr. Sara Lyle and Dr. Bruce Elits

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for their advice and instruction in developing experimental approaches. The author
would also like to thank the crew at the Center for Reproductive Biology at St. Gaberial
Research Station for all their assistance and loan of experimental animals which made
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many of these experiments possible. The author would also like to thank Dr. Matthew
Wheeler for his critical review of this dissertation which was greatly appreciated.
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Furthermore, the author would like to thank all of the graduate students and
students; Toyna Davidson, Oscar Perez, Luke Lenard, Glen Gentry, Kyle Hebert, Allison
Morisan, Andrea Huval, Trey Champman, as well as post-doctoral researcher, Masimo
Maurakami, for their assistance and invaluable friendships.
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The author would like to thank his father Ronnie Ferguson, mother Dixie
Ferguson and grandmother Velma Jordan, who unselfishly sacrificed to allow their son
to succeed in life and his sister Rene Ferguson for her encouragement to complete his
goal swiftly. Also, a special thank you is extended to Bennie Deaton whose love and
support throughout his life has always been appreciated.
Most importantly, the author would like to give special appreciation to a very
special person who shared his many trials and accomplishments during the completion
of this dissertation. No only has she improved the quality of his life she provided a
brighter future and a greater appreciation for live but she more importantly taught him
the meaning unconditional love. Thank you Tonya Davidson.

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 TABLE OF CONTENTS
ACKNOWLEDGEMENTS ................................................................................................. ii

LIST OF TABLES.............................................................................................................. v

LIST OF FIGURES...........................................................................................................vii

ABSTRACT .......................................................................................................................ix

CHAPTER 1. INTRODUCTION ........................................................................................ 1

CHAPTER 2. LITERATURE REVIEW .............................................................................. 4

Role of Oral Progestins in Control of Reproduction .............................................. 4
Effects of P4 on the Oviducts during Early Pregnancy ........................................ 14
Effects of P4 on Embryo Transport into the Uterus.............................................. 17
Effects of P4 on Uterine Growth and Proliferation ............................................... 19

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Effects of P4 on the Synthesis and Secretion of Uterine Proteins
and Growth Factors............................................................................................. 21
P4 Effects on the Immune System Function during Pregnancy........................... 24
Effects of P4 on Embryo-Uterine Synchrony during Early Pregnancy ................. 25
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Effects of P4 on Embryo Developmental Rates ................................................... 29
Effects of P4 on Embryo Metabolism ................................................................... 32
Effects of P4 on Post-Implantation Development................................................. 33
Embryonic P4 Synthesis during Early Pregnancy................................................ 34
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P4 Levels in Pregnant and Nonpregnant Female ................................................ 35
Maintenance of Pregnancy in Ovariectomized and CL Ablated Females ........... 40

CHAPTER 3. THE USE OF ALTRENOGEST TO SYNCHRONIZE ESTRUS IN

YEARLING BEEF HEIFERS .................................................................... 43
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Introduction.......................................................................................................... 43
Materials and Methods ........................................................................................ 45
Results ................................................................................................................ 48
Discussion........................................................................................................... 55

CHAPTER 4. EFFECTS OF ADMINISTRATION OF P4 OR ALTRENOGEST ON

DAYS 3 TO 5 POST-MATING ON PREGNANCY RATES ...................... 60
Introduction ......................................................................................................... 60
Materials and Methods........................................................................................ 62
Results ................................................................................................................ 70
Discussion........................................................................................................... 75

CHAPTER 5. EFFECT OF PROGESTERONE ON BOVINE EMBRYO

DEVELOPMENT IN VITRO ..................................................................... 81
Introduction….. ................................................................................................... 81
Materials and Methods ....................................................................................... 83
Results ............................................................................................................... 87
Discussion .......................................................................................................... 98

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CHAPTER 6. AN ATTEMPT TO DETECT PROGESTERONE RECEPTORS IN
EARLY STAGES OF IN VITRO PRODUCED BOVINE EMBRYOS ...... 102
Introduction.................................................................................................... 102
Materials and Methods.................................................................................. 103
Results .......................................................................................................... 107
Discussion ..................................................................................................... 107

CHAPTER 7. RESCUE OF PREGNANCY IN BEEF CATTLE AFTER A

LUTEOLYTIC DOSE OF PROSTAGLANDIN2α ..................................... 113
Introduction.................................................................................................... 113
Materials and Methods .................................................................................. 114
Results .......................................................................................................... 118
Discussion ..................................................................................................... 129

CHAPTER 8. SUMMARY AND CONCLUSIONS......................................................... 133

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REFERENCES.............................................................................................................. 135

APPENDIX A: BO-A STOCK SOLUTION ..................................................................... 168

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APPENDIX B: BO-B STOCK SOLUTION ..................................................................... 169

APPENDIX C: CR1AA STOCK SOLUTION.................................................................. 170

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VITA .............................................................................................................................. 171
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 LIST OF TABLES
3.1. Characteristics of mixed-breed beef heifers that reached the
pre-determined target weight (345 kg) and exposed to two estrous
synchronization protocols using either altrenogest (ALT) or melengestrol
acetate (MGA) progestin treatments ................................................................... 49

3.2. Effects of estrous synchronization treatments on the percentage of heifers

exhibiting estrus, interval to the onset of estrus following prostaglandin
(PGF2α) administration, duration of estrus and number of mounts per female in
mixed-breed beef heifers .................................................................................... 50

3.3. Time from prostaglandin (PGF2α) administration to the onset of estrus, mean
duration of estrus and number of mounts per female during estrus for
altrenogest (ALT)-treated and melengestrol acetate (MGA)-treated pregnant
and nonpregnant beef heifers ............................................................................. 53

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3.4. Pregnancy rates, calving rates and mean birth weight of calves produced
from heifers synchronized with either altrenogest (ALT) or melengestrol
acetate (MGA)..................................................................................................... 54

4.1.
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Treatments for experimental groups in Experiment 4.1 and Experiment 4.2 ...... 68

4.2. Pregnancy rates for females treated with progesterone (P4) or altrenogest
(ALT) on days 3, 4 and 5 of the estrous cycle..................................................... 71
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4.3. Serum progesterone (P4) levels in progestin-treated (altrenogest, ALT)

and nontreated females on days 3, 4, 5 and 6 after onset of estrus in
Experiment 4.2. ................................................................................................... 74
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5.1. Number of P4-treated and control 8-cell bovine embryos in Experiment 5.1
that reached the morula, blastocyst and hatched blastocyst developmental
stages in respective treatments........................................................................... 88

5.2. Number of P4-treated and control 8-cell bovine embryos in Experiment 5.1
cultured in respective treatments that reached morula, early blastocyst,
blastocyst or expanded blastocyst stages on day-6 post-insemination............... 89

5.3. Number of P4-treated and control 8-cell bovine embryos in Experiment 5.1
cultured in respective treatments that reached early blastocyst, blastocyst,
expanded blastocyst, or hatched blastocyst stages on day-8 post-
insemination ........................................................................................................ 90

5.4. Mean diameter of blastocyst stage bovine embryos in Experiment 5.1

cultured in respective treatments on day-7 post-insemination ............................ 91

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5.5. Number of P4-treated and control 8-cell bovine embryos in Experiment
that reached the morula, blastocyst and hatched blastocyst developmental
stages in respective treatments .......................................................................... 93

5.6. Number of grade 1 or 2 bovine blastocyst staged embryos in Experiment 5.2

on day-7 post-insemination ................................................................................. 94

5.7. Mean diameter of blastocyst staged bovine embryos in Experiment 5.2

cultured in respective treatments on day-7 post-insemination ............................ 95

5.8. Number of P4-treated and control 8-cell bovine embryos in Experiment 5.2
cultured in respective treatments that reached early blastocyst, blastocyst,
expanded blastocyst, or hatched blastocyst stage on day-8 post-
insemination ........................................................................................................ 96

5.9. The mean red score per embryo per treatment over the four observatory
periods of embryo development.......................................................................... 97

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 LIST OF FIGURES
3.1. Estrous synchronization protocols were arranged so that prostaglandin
(PGF2α) was administered simultaneously in both treatments. This allowed
for estrus detection and AI to occur concurrently in both treatment groups. ....... 46

3.2. Distribution of time to the onset of estrus following prostaglandin (PGF2α)

administration for altrenogest (ALT)-treated and melengestrol acetate
(MGA)-treated heifers. ........................................................................................ 51

3.3. Artificial insemination (AI) scores assigned to heifers that exhibited estrus
that were synchronized with either altrenogest (ALT) or melengestrol
acetate (MGA)..................................................................................................... 52

3.4. Sex ratio of calves produced from heifers synchronized with either altrenogest
(ALT) or melengestrol acetate (MGA) treatments. .............................................. 56

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4.1. Experimental protocol for classifying females as fertile and repeat breeder
(RPB) cows.. ....................................................................................................... 63

4.2. The experimental procedure for altrenogest (ALT) treatment of heifers in

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Experiment 4.1. ................................................................................................... 65

4.3. The experimental procedure for cows in replicate I of Experiment 4.2 ............... 67
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4.4. The experimental procedure for cows in replicate II of Experiment 4.2 .............. 69

4.5. Serum progesterone (P4) levels in pregnant (preg) and nonpregnant

(nonpreg) RPB cows administered 15 mg of P4 on days 3, 4 and 5 after
onset of estrus..................................................................................................... 73
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4.6. Serum progesterone (P4) levels in pregnant (preg) and nonpregnant

(nonpreg) “fertile” control (cont) cows on days 3, 4 and 5 after onset
of estrus .............................................................................................................. 76

6.1. Fluorence of sectioned bovine uterine tissue. Note the specific staining of
P4-receptors along the luminal lining of uterine tissue. ..................................... 108

6.2. Autofluorence of unstained IVF-derived bovine hatched blastocysts. This

indicates the staining procedure is not applicable in bovine embryos.. ............ 109

6.3. Autofluorence of stained IVF-derived bovine hatched blastocysts. Note the

nonspecific staining pattern that precludes the determination of the
presence of P4 receptors................................................................................... 110

7.1. The circulatory progesterone (P4) pattern constructed from mature

crossbred beef cows (n=12) 30 to 40 days of gestation following
prostaglandin (PGF2α) treatment (Experiment 7.1). .......................................... 119

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7.2. Development of follicles ≥10 mm in pregnant crossbred beef cows and
their subsequent luteinization after the administration of 2,500 IU of hCG
following the initial prostaglandin (PGF2α) treatment, as recorded via
ultrasonography. ............................................................................................... 121

7.3. First calf (bull calf weighing 35 kg) produced from a crossbred beef cow
(No. 1) that was administered a luteolytic dose of prostaglandin and
supplemented progesterone (P4) starting 2 hours following prostaglandin
administration for 24 days. The pregnancy was continued on hCG-induced
luteal tissue at ~45 days of pregnancy.............................................................. 122

7.4. The circulatory progesterone (P4) patterns constructed from mature

crossbred beef cows (n=12) at 80 to 90 days of gestation following
prostaglandin (PGF2α) treatment (Experiment 7.2). ......................................... 123

7.5. The second calf (heifer calf weighing 23 kg) and third calf (bull calf

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weighing 34 kg) produced from cows (No. 1 and 2) that were administered
altrenogest 2 hours after a luteolytic dose of prostaglandin (PGF2α) was
administered. The pregnancies were maintained on exogenous progesterone
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(P4 )until luteal tissue was induced by hCG administration. At 19 days, post-
PGF2α administration, exogenous P4 was discontinued and the pregnancies
were continued to term from hCG-induced luteal tissue. .................................. 125
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7.6. The circulatory progesterone (P4) pattern constructed from mature
crossbred beef heifers 30 to 40 days of gestation following prostaglandin
(PGF2α) treatment (Experiment 7.3). ................................................................. 126

7.7. The fourth calf (heifer calf weighing 36 kg at birth) and fifth calf (bull calf
weighing 38 kg at birth) (A) sixth calf (heifer calf weighing 33 kg at birth)
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(B) and seventh calf (bull calf weighing 35 kg at birth) (C) produced from
cows that were administered altrenogest 6 hours (1 calf) 12 hours (1 calf)
and 18 hours (2 calves) after a luteolytic dose of prostaglandin (PGF2α)
was administered. The pregnancies were maintained on exogenous
progesterone (P4) until luteal tissue was induced by hCG administration.
At 21 to 49 days post-PGF2α administration, exogenous P4 was discontinued
and the pregnancies were continued to term on induced luteal tissue. ............ 128

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 ABSTRACT
There has been great controversy involving progesterone (P4) levels during early
pregnancy in cattle. The objectives of these experiments were to determine the effect of
an early low dose administration of P4 or altrenogest (ALT) on pregnancy rates in repeat
breeder (RPB) females, if an increase in pregnancy rates could result from a direct effect
of P4 on the embryo and if ALT could support pregnancy in the absence of a functional
CL. Firstly, ALT was evaluated for use as a progestin in cattle by synchronizing estrus in
beef heifers. There were no differences in the number of females displaying behavioral
estrus or in pregnancy rates when synchronized with ALT or MGA. A second
experiment was designed to determine the effect of P4 or ALT supplementation during
days 3 to 5 on pregnancy rate in RPB cattle. It was determined that 15 mg of P4 or ALT

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during days 3 to 5 increased pregnancy rates compared with nontreated breeding
periods. A third experiment was designed to determine if P4 exerted a direct effect on
the embryo. In vitro produced (IVP) embryos were cultured in the absence of a co-
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culture system. At day 3, post-insemination, embryos were cultured in the presence of
P4 and evaluated on days 6 to 9. On day 7 post-insemination, there were significantly
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more grade 1 blastocysts from the P4 group compared with other treatment groups.
Also, embryo developmental rates were increased when cultured in the presence of P4
and more of these embryos developed to the hatched blastocyst stage compared with
other treatment groups. After a direct effect of P4 on developing IVP bovine embryos
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existed, it was determined that these embryos did not possess P4 receptors. Finally, it
was demonstrated that ALT could support pregnancy in the absence of a functional CL.
These experiments demonstrated that ALT could serve as a progestin in cattle and when
administered in low doses during early pregnancy could improve pregnancy rates in
RPB cows. These results are likely due to a direct effect of P4 on the embryo; however,
this mechanism is by means other than binding the PR.

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 CHAPTER 1
INTRODUCTION
It is well known that progesterone (P4) has been shown to be necessary for the
establishment and maintenance of pregnancy in mammals. In addition, P4 has been
proven essential for the initiation of reproductive function in the female. In cattle, P4
treatment of prepubertal females has been demonstrated to hasten the initiation of
cyclicity in heifers (Short et al., 1976; Heitzman et al., 1979). Also, P4 treatment of post-
partum cows has decreased the number of days to mating (Brown et al., 1972; Yelich et
al., 1988; Yelich et al., 1995a; Patterson et al., 1995). Wilmut et al. (1980) reported that a
normal period of P4 exposure during the luteal phase was necessary to achieve normal
pregnancy rates during the subsequent estrual period. However, the effects of elevated

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P4 during early pregnancy on the developing bovine embryo are still under investigation.
Elevated P4 levels during corpus luteum (CL) formation (metestrus) may have
effects on the developing embryo. Increased circulating P4 levels at the time the embryo
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is in the oviduct has been reported to hasten the rate of embryo transport into the uterus
in mice (Kendle and Lee, 1980) and cattle (EL-Banna and Hafez, 1970; Crisman et al.,
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1980). This treatment hastened embryo transport along with stimulation of uterine cell
proliferation and differentiation, as well as increased blood flow to the bovine uterus
(Johnson et al, 1997; Rider et al., 1998; Gray et al., 2001). An early rise in P4 has been
reported to alter the pattern of protein synthesis, secretion and overall increase uterine
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protein secretions in cattle (Garret et al., 1988). Furthermore, it has been noted that
when early circulating P4 increased there were increased embryo developmental rates
(Lawson et al., 1983; Lawson and Cahill, 1983; Garrett et al., 1987; Garrett et al., 1988).
Thus, with earlier embryo entry into the uterus, there will be a stimulated, receptive
uterus to support further embryo growth and differentiation.
An early increase in P4 levels in pregnant or nonpregnant cows, however, has not
been consistently demonstrated (Shemesh et al., 1968; Pope et al., 1969; Henricks et
al., 1970; Henricks et al., 1971; Sreenan and Diskin, 1983). Ashworth et al. (1989) and
Mann and Lamming (1996) reported that hastened CL development and function would
likely increase pregnancy rates in cattle. There is indirect evidence of faster CL
development positively impacting pregnancy rates, when comparisons were made of the
reproductive performance of pubertal and mature females (Davies and Beck, 1993; Silva

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et al., 2002). In sheep, ewe lambs had lower levels of P4 during metestrus and diestrus
and lowered reproductive performance compared with mature ewes (Davies and Beck,
1993). However, research has failed to demonstrate differences between fertile beef and
dairy cows regarding early P4 levels during metestrus or diestrus (Shemesh et al., 1968;
Pope et al., 1969; Robertson and Sarda, 1971; Bulman and Lamming, 1978; Geisert et
al., 1988). The question of whether or not there is an early increase in P4 levels during
metestrus (presumably from earlier developed luteal tissues) in pregnant females
compared with nonpregnant females remains to be answered. Lukaszewska and
Hansel (1980) reported that P4 levels were significantly higher in pregnant females
compared with nonpregnant females by day 8 (estrus = day 0). Later, Hwang et al.
(1988) demonstrated that the bovine embryo produces luteotrophic prostaglandins.

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These luteotrophins had been previously reported to cause increased P4 levels in the
ewe (Pratt et al., 1979). These reports demonstrate that viable bovine or ovine embryos
have the ability to increase P4 levels during early pregnancy.
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The next logical step was to administer P4 to mated females in an attempt to
increase pregnancy rates. Unfortunately, this approach has resulted in varied success
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rates in cattle. Even with the lack of success, there have been reports of increased
pregnancy rates over controls with P4 supplementation from day 1 to day 7 following
onset of estrus in dairy cattle (Henrick, 1953; Dawson, 1954; Wiltbank et al., 1956;
Johnson et al., 1958). However, these studies used various levels of P4 supplementation
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and are primarily veterinary case reports. The majority of these reports, however,
indicated that P4 supplementation from day 6 onward (estrus = day 0) failed to increase
pregnancy rates (Sreenan and Diskin, 1983; Munro and Bertram, 1990; Van Cleef et al.,
1991; Stevenson and Mee, 1991; Mann et al., 1998).
Attempts to increase P4 levels by administration of luteotrophins have also failed
to significantly increase pregnancy rates over that of control females. Administration of
luteotrophins (human chorionic gonadotrophin, hCG or gonadotrophin releasing
hormone, GnRH) during mid-cycle (day 7 or later) to artificially inseminated dairy and
beef cattle have significantly increased circulating P4 levels (Wiltbank et al., 1961;
Rajamahendran and Sianangama, 1992; Lulia et al., 1994). However, the majority of
these studies report no difference in pregnancy rates between treated and nontreated
females (Holness et al., 1982; Sreenan and Diskin, 1983; Walton et al., 1990; Sheldon

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and Dobson, 1993). These studies, taken collectively, indicate that increasing
endogenous P4 levels during mid to late luteal phase does not increase embryo survival
rates. These findings are noteworthy because it is at this time that the embryo is
releasing luteotrophic substances (Lewis et al., 1982; Hwang et al., 1988; Lewis, 1989).
The previous findings led to two hypotheses for the role of P4 in the successful
development during pregnancy: (1) P4 increase must occur during early metestrus for a
successful pregnancy to be established and (2) elevated P4 during the luteal phase
exerts a beneficial effect directly on embryonic growth and survival. The second
hypothesis has recently gained some support. Mann et al. (1996) demonstrated that
elevated P4 levels during the early luteal phase (day 4 and later) resulted in a reduced
luteolytic signal and thereby, allowed the embryo to have a greater chance of sending an

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optimal signal for maternal recognition of pregnancy. Furthermore, it was demonstrated
that elevated P4 levels result in conversion of uterine prostaglandin synthesis from PGF2α
(luteolytic) to PGE2 (luteotrophic), elevated interferon tau (a maternal recognition signal)
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synthesis by the conceptus and a need for lower amounts of interferon tau to prevent
luteal regression (Mann et al., 1998).
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At this time it appears that these two hypotheses are not mutually exclusive and
therefore, the further development of the first hypothesis was part of the objective of this
dissertation. This objective was based on the findings of Gustaffason and Larson (1983)
who demonstrated that when embryos were collected from cows of lowered fertility on
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day 7 (estrus = day 0) and transferred to fertile cows, this resulted in reduced embryo
survival. Conversely, when embryos were collected on day 7 (day 0 = estrus) from fertile
cows and transferred to cows with lowered fertility, embryo survival was normal
(Gustaffason and Larson, 1985). These findings suggested that early embryonic
developmental (day 3 until sustained luteal function) under the influence of elevated P4
could possibly exert an effect on increasing embryonic survival. Also, there has been a
need to determine if P4 can exert a direct effect on the embryo.
Another objective of this dissertation was to determine if, the progestin,
altrenogest could serve as a biologically active progestin in ruminant species, such as
the cow. By determining whether altrenogest could serve a bovine progestin, it could be
used to further evaluate the effect of progestins on early embryo development, as well
as, maintaining pregnancy in beef cattle.

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 CHAPTER 2
LITERATURE REVIEW
Role of Oral Progestins in Control of Reproduction
MGA in Control of Reproduction
Melengestrol Acetate (MGA) is an orally active progestin commonly used for
estrus suppression and as a growth promotant in beef cattle. For MGA to be orally
active, the progestin has been chemically modified at carbons 6 (methyl and double
bond additions), 16 (methylene addition) and 17 (acetoxy addition) (Lauderdale, 1983).
These modifications allow MGA to remain chemically active as it transverses the rumen
into the circulation.
MGA was originally developed for use as a growth promotant in feedlot heifers

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and has been shown to consistently improve weight gain and feed efficiency (Bloss et
al., 1966). Hafez (1987) subsequently reported that MGA improved weight gain and feed
efficiency by preventing behavioral estrus and blocking ovulation, while allowing follicular
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development to continue on the ovaries. This resulted in sustained estrogen (E2) levels,
which stimulated a synergistic effect on growth hormone (GH) release that resulted in
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increased nitrogen retention and increased protein synthesis.
In cattle, MGA is ~30 times more potent than progesterone (P4) in pregnancy
maintenance and 125 times more potent than P4 for suppressing estrus (Lauderdale,
1983). When fed at a rate of 0.25 mg per head per day, estrus was suppressed in all
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heifers and at a rate of 0.50 mg per head per day follicular growth and development
would still occur in the absence of a corpus luteum (CL) (Bloss et al., 1966). However,
Young et al. (1966) reported that when MGA was fed (for 154 days) at a rate of 0.20 mg
per head per day behavioral estrus was only suppressed in 12 of 20 females, while at a
rate of 0.40 mg per head per day, only 3 of 20 females exhibited behavioral estrus and
at a rate of 0.60 mg per head per day no females displayed behavioral estrus.
Furthermore, Zimbelman and Smith (1966a) indicated that 0.42 mg per female per day
of MGA was the minimum requirement to prevent ovulation and behavioral estrus in beef
cattle. An important finding indicated that MGA was equally effective when administered
orally or intramuscularly and when administered orally, MGA was absorbed rather than
degraded in the rumen and/or intestines (Zimbelman and Smith, 1966b). Prior to its use
for estrous cycle synchronization, MGA was reported to maintain pregnancy in bilaterally

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ovariectomized cows (Zimbelman and Smith, 1966c). A dose of 4 mg per female per day
of MGA was found to be the minimum dose required to maintain pregnancy in bilaterally
ovariectomized cows. This was 20 times greater than the dose required to prevent
ovulation in beef cattle (Zimbelman, 1963). More recently, MGA has been used to
support pregnancy in females where detection of endogenous P4 levels were important
(Wright et al., 1994; Bridges et al., 2000).
Use of MGA to control estrus in cycling cows utilizes long term (14 to 18 days)
daily administration of MGA at doses ranging from 0.5 to 1.0 mg of MGA per head per
day (Zimbelman and Smith 1966b; Zimbelman and Smith, 1968a; Zimbelman and Smith,
1968b; Roche and Crowley, 1973; Patterson et al., 1989). In these studies, the interval
from removal of MGA from the ration to the onset of estrus ranged from 3 to 7 days

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(Zimbelman and Smith, 1968a; Zimbelman and Smith, 1968b; Roche and Crowley,
1973; Patterson et al., 1989). It should be noted that decades before, Zimbelman and
Smith (1966b) reported low pregnancy rates among MGA-treated females following
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artificial insemination (AI). Subsequent reports verified a decrease in pregnancy rates
that occurred in long-term MGA-treated females when compared with those of
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nontreated control females (Hill et al., 1971; Lamond et al., 1971). There have been two
reports of no decrease in pregnancy rates following AI in dairy cows administered long-
term MGA treatment (Roussel et al., 1969; Boyd, 1970), although decreases in
pregnancy rates among AI dairy cows have been reported with long-term administration
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of P4 (≥14 days) and other progestins (Trimberger and Hansel, 1955; Ray et al., 1961;
Hansel, 1967; Wiltbank et al., 1967; Lamond et al., 1971; Wettemann and Hafs, 1973;
Roche, 1974a; Roche, 1974b).
With long-term MGA treatment failing to produce respectable pregnancy rates
among cattle following AI, alternative protocols were developed for the use of MGA. An
attempt to improve pregnancy rates, following long-term treatment with MGA, by
administration of hCG or LH at the time of AI failed to improve pregnancy rates in MGA-
treated females (Zimbelman and Smith, 1968a; Zimbelman and Smith, 1968b; Roche
and Crowley, 1973). Researchers aware that long-term progestin treatments decreased
pregnancy rates following AI, continued searching alternative approaches using MGA.
Soon after, a short-term treatment (≤14 days) with MGA was developed, which produced
pregnancy rates similar to control treated females although the degree of synchrony was

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greatly reduced (Wiltbank and Kasson, 1968; Roche, 1974a; Sreenan and Mulvehill,
1975). It was later demonstrated that long-term administration of MGA or other
progestins induced persistent dominant follicles in cattle (Yelich et al., 1997; Cavalieri et
al., 1998; McDowell et al., 1998). This was caused by P4 down-regulating LH receptors,
which are required for ovulation and final maturation of the oocyte (Batra and Miller,
1985; Thiery and Martin, 1991). In addition to the decreased viability of the oocyte, a
persistent dominant follicle altered the steroid hormone secretion pattern and thus,
secretory patterns of the oviduct thereby adversely affect fertilization in vivo (Binelli et
al., 1999).
To circumvent the decreased fertility associated with long-term MGA
administration and to prevent the loss of synchrony with short-term MGA administration,

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a short-term MGA protocol was combined with administration of PGF2α. Although Fike et
al. (1997) demonstrated that infertility due to persistent dominant follicles could be
overcome by preventing ovulation of the persistent follicle and inducing ovulation of a
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second follicle this procedure was subsequently not practical for on-farm use. Using the
combination of short-term MGA feeding and PGF2α females were administered MGA for
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5 days and administered PGF2α following the end of MGA treatment. However, Moody et
al. (1978) reported that when using this approach pregnancy rates remained low. When
the MGA treatment was extended to 7 days and combined with PGF2α, pregnancy rates
among beef cattle still remained low (Patterson et al., 1986; Beal et al., 1988). A protocol
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was then developed where MGA was administered for 14 to 16 days and PGF2α was
administered 16 to 17 days following the last feeding of MGA (Brown et al., 1988). This
protocol resulted in increased pregnancy rates which were attributed to a 17 day delay
from MGA removal and the administration of PGF2α. This delay would allow for the
majority of the females to be in diestrus at the time of PGF2α administration (King et al.,
1982). This amendment of the delay from last feeding of MGA to PGF2α administration
was combined with a more abbreviated period of MGA administration (~14 days) to
further improve pregnancy rates following estrous cycle synchronization and AI in cattle.
Mauck et al. (1988) compared the 14-day MGA feeding period with a 7-day
feeding period and determined that the 14-day protocol was superior in pregnancy rates
and degree of synchrony. Later, Coleman et al. (1990) evaluated the effect of a 21-day
MGA treatment period with or without subsequent PGF2α administration (14 days later)

6
and found that pregnancy rates were markedly reduced without PGF2α administration.
Today, the optimal MGA protocol for beef cows and heifers is a 14-day MGA treatment
period followed by PGF2α administration 15-days later for cows and 17-days later for
heifers (Kesler et al., 1996).
MGA has also been utilized for estrous cycle synchronization of postpartum
cows. Using the current MGA protocol (14-day MGA administration followed 17 days
later by administration of PGF2α) and AI in postpartum cows acceptable estrual
response, pregnancy rates and increased weight gain were achieved (Yelich et al.,
1988). Yelich et al. (1995a) concluded that MGA was effective in postpartum females
provided that synchronization was initiated when cows had a good body condition score.
In addition, it was reported that 48-hour calf removal increased synchronized pregnancy

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rates in postpartum cows synchronized with MGA (Yelich et al., 1995b). The pregnancy
rates achieved following synchronization with MGA and AI in postpartum cattle were in
agreement with the pregnancy rates reported for postpartum cows by Patterson et al.
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(1995).
In prepubertal and pubertal heifers, synchronization rates and pregnancy rates
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were consistently higher following MGA-estrous cycle synchronization and AI compared
with nontreated control heifers and heifers undergoing estrous cycle synchronization
with other progestins (Deutscher et al., 1989; Goehring, 1989; Jaeger et al., 1992;
Patterson and Corah, 1992).
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There have been attempts to test the effectiveness of MGA in non-ruminant

species. Loy and Swan (1966) demonstrated that 10 to 20 mg per day per head of MGA
or 100 mg injections of MAP (6α-methyl-17α-acetoxyprogesterone) were not effective in
controlling estrus or ovulation in the mare. It was concluded that P4 was catabolized to
the urinary metabolite 5α allopregnane rather than its primary urinary metabolite 5β
pregnane. This may have altered the progesterone compound rendering it inert in the
mare. Additional studies demonstrated that although MGA or norgestomet would support
pregnancy in bilaterally ovariectomized cows, however, they were ineffective in
maintaining pregnancy in bilaterally ovariectomized mares (McKinnon et al., 2000).

7
Altrenogest for Control of Reproduction in the Mare
Altrenogest (allyltrenbolone, ALT) is an orally active progestin developed for use
in the horse. Altrenogest has been chemically modified by addition of a hydroxyl group
and 3 carbon chain placed on carbon 17 of the progestin (Peters, 1992). Research has
demonstrated that ALT has low anabolic activity and is 20 times less potent than
testosterone or similar progestins when compared for effects on muscle growth in
castrated rats (Peters, 1992). It was further noted that 50% of altrenogest residue
becomes bound to binding proteins and becomes biologically inactive. Based on these
findings (Peters, 1992), the withdrawal period for ALT (known commercially as
Regumate) for equids is 15 days.
There have been no deleterious effects reported of ALT administration to healthy

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cycling mares when fed for 86 days at a rate of 0.044 mg per kg of body weight, 0.132
mg per kg of body weight or 0.220 mg per kg of body weight (Shideler et al., 1983). This
observation was based on evaluations for differences in white blood cell counts, platelet
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number, creatinine levels, cholesterol, signs of inflammation and overall organ
appearance following necropsy.
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The initial development of ALT was for controlling estrus in mares. Squires et al.
(1979) demonstrated that when ALT was orally administered to estrual mares (early in
the breeding season) at a rate of 0.044 mg per head per day for 12 days, behavioral
estrus ceased within 3 days following initiation of ALT treatment and estrus remained
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inhibited for the duration of treatment. However, ovulation was not inhibited in all ALT-
treated mares during this time. Following the cessation of ALT treatment, ALT-treated
mares had a mean interval to estrus of 4.5 days and had a significantly shorter time
interval from the end of treatment to ovulation and the onset of estrus to ovulation
compared with nontreated mares.
Squires et al. (1979) also reported that initially transitional mares treated with
ALT had a high incidence of continuous estrus compared with a high incidence of
discontinuous estrus seen in control-nontreated transitional mares. In this initial testing
of ALT, Squires et al. (1979) tested for the most efficient stage of estrous cycle to begin
administration of ALT for estrous cycle synchronization and it was reported that the
interval from ALT-treatment to estrus was shorter when ALT-treatment was initiated
during diestrus compared with that of estrus. Although ALT was effective in the control of

8
estrual behavior and in estrous synchronization, the inability of ALT to block ovulation in
all mares made this 12-day treatment period unlikely to be the most effective regime,
since it did not allow for sufficient time for CL regression to occur before the end of
treatment (Squires et al., 1979).
Further testing of ALT in transitional mares revealed that it was effective in
abbreviating the transition period in these females (Turner et al., 1981; Webel and
Squires, 1982). When transitional mares were administered ALT (0.044 mg/head/day)
for 15 days there was a significant decrease in the length of estrus and the number of
inseminations required without a decrease in pregnancy rate (Turner et al., 1981). Webel
and Squires (1982) reported similar effects of ALT-treatment in transitional mares and
demonstrated that ALT-treatment during late transition significantly decreased the

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interval from treatment to pregnancy.
In the mare, the effect of ALT-treatment on follicular development appears to be
dependant on the size of follicles present on the ovary at time of ALT administration. If
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ALT was administered when follicles ≥ 20 mm were present, follicular growth was
suppressed and the number of large follicles (>30 mm) and follicle diameter was
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reduced (Turner et al., 1981; Webel and Squires, 1982; Squires et al., 1983). In these
reports of a reduction in development of large follicles (>30 mm) and follicle diameter
usually occurred during a reported increased FSH levels towards end of treatment
period (Turner et al., 1981 and Squires et al., 1983). Subsequently, Wiepz et al. (1988)
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demonstrated that ALT and not norgestomet (another progestin) suppressed follicular
activity in late transitional mares and the rise in serum LH following ALT removal was
greater than detected in nontreated control females.
Although ALT had been shown to improve breeding efficiency in transitional
mares, the failure to block ovulation in the mare remained a problem. When ALT was
administered to estrual mares, ovulation occurred in 75% (9 of 12) of treated females
and CL were formed in all of these females (Squires et al., 1983). However, when PGF2α
was administered to ALT-treated cycling, ovulation in diestrous mares was blocked in
60% (3 of 5) of the mares and behavioral estrus was suppressed in all mares (Lofsedt
and Patel, 1989). It was also noted in the cycling mares that large follicles could develop
during ALT treatment and that ALT had only 60% of the binding affinity of P4 in the
hypothalamus and that amount may not be sufficient to block gonadotrophin release. In

9
support of this theory, it was shown that P4 in doses of 100 mg per head per day or
greater administered mid-cycle were successful in inhibiting behavioral estrus and
ovulation, but 50 mg per head per day of P4 would inhibit behavioral estrus only (Loy and
Swan, 1966). The discrepancies reported on the efficiency of blocking ovulation and
follicular dynamics are probably due to the dose administered as well as the phase of
reproductive transition at which ALT was administered.
ALT has also been utilized for asynchronous embryo transfer in the mare (Pool et
al., 1987). In females treated with ALT immediately following ovulation, that received a
day-6 or day-7 embryo on day 2 to 5 of ALT-treatment could became pregnant. In the
same experiment it was noted that when ALT-treatment was initiated at day-9 or greater
following ovulation, there was an increased pregnancy rate when recipient females

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received day-6 or day-7 embryos. These findings demonstrated that early pregnancy
could be supported with ALT administration. Furthermore, these findings demonstrate
that ALT-treatment could increase the number of females in a group available for use as
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embryo transfer recipients. Also, supplementation with ALT has been demonstrated to
prevent embryo loss in 60-day or less pregnant mares that were administered
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Salmonella typhimurium (endotoxin) (Daels et al., 1991).
Not only has ALT been demonstrated to support early pregnancy in the absence
of luteal tissue, it has been reported to support pregnancy through gestation in the
ovariectomized mare (Hinrichs et al., 1985; Hinrichs and Kenny, 1987; Hinrichs et al.,
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1987). Hinrichs et al. (1985), Hinrichs and Kenny (1987) and Hinrichs et al. (1987)
reported that pregnancies could be established in ovariectomized recipient mares with
supplementation of 300 mg of P4 daily until ~90 days of gestation. However, when 22 mg
of ALT daily was used to support pregnancy, it was ineffective at maintaining pregnancy
to term (Hinrichs et al., 1986). This finding was in contrast to that of McKinnon et al.
(1988), who demonstrated that 22 to 25 mg of ALT administered daily to ovariectomized
pregnant mares was capable of supporting pregnancy to term.
With ALT-treatment prior to and after embryo transfer there was concern
regarding postnatal effects on the developing offspring (Naden et al., 1990a; Naden et
al., 1990b). With a gestation of ~336 days, mares as with cattle must become pregnant
while nursing an offspring. Therefore, it would be of importance to determine detrimental
effects of ALT on lactation and foal growth. However, there have been no reports of

10
detrimental effects of ALT on the developing offspring. Following a series of GnRH
challenges in stud colts produced from ALT-treated dams (ALT administered from day-
20 to day-325 of gestation) there were no differences as a result of treatment of the dam
with ALT (Naden et al., 1990a). In addition, no differences were found in the time to
puberty or in hypothalamic function of fillies produced from dams treated with ALT from
day-20 to day-325 of gestation (Naden et al., 1990b). Furthermore, Sigler et al. (1989)
demonstrated that treatment with 0.044 kg ALT per head per day to lactating mares had
no effect on milk yield, milk composition or foal growth as compared with untreated
control mares.
Altrenogest in Control of Reproduction in Swine
In an early report on the use of ALT for the synchronization of estrus and

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ovulation in gilts, 33 of 38 gilts exhibited estrus within 2 to 7 days following the last ALT
administration, when 12.5 mg of ALT per head per day was administered for 19 days
(Davis et al., 1979). Although an increase in ovulation rate with no decrease in
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fertilization rates was noted an increased incidence of cystic follicles occurred in ALT-
treated gilts. It was proposed that the increase in cystic follicles may have been a
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response to the failure of these females to consume the entire dose of ALT. Following
this initial testing of ALT in gilts, it was demonstrated that the minimum dose required for
effective synchronization of estrus in gilts or sows was 10 to 20 mg of ALT per head per
day (Kraeling et al., 1981), while doses of 20 to 40 mg of ALT per head per day were the
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most effective for synchronization of estrus and ovulation, without induction of follicular
or luteal cysts. In this study, the interval from the last treatment with ALT to the onset of
estrus was significantly longer for gilts (5.4±0.3 days) compared with that of sows
(6.0±0.2 days).
In conjunction to determining optimal dosage of ALT, research continued on
determining the optimal length of ALT administration. Davis et al. (1979) reported an
increased ovulation rate in gilts treated with 12.5 mg of ALT for 19 consecutive days,
while Pursel et al. (1981) demonstrated that gilts treated with 15 mg of ALT for 18
consecutive days had a significantly greater live litter size at birth and weaning
compared with nontreated control gilts. In a later study, synchronized pregnancy rates
were not different for ALT-estrous cycle synchronization (70.7%) compared with
nontreated controls (73.5%).

11
 The consistent efficiency of estrous cycle synchronization and ovulation in gilts
resulting from ALT treatment, allowed for improved timed insemination opportunities in
swine breeding herds. Davis et al. (1985) reported that pregnancy rates, litter size and
live pigs at farrowing were not different for gilts treated with 15 mg of ALT for 18 days
and time inseminated at 5, 6 and 7 days following ALT-treatment cessation. It should be
noted that the timing of insemination after ALT withdrawal is influenced by breed of gilts
or sows (Martinat-Botté et al., 1985). It was later reported that ALT-estrous cycle
synchronization could increase the number of gilts detected in estrus compared with the
nontreated control gilts (Davis et al., 1987). An increased litter size has been reported in
ALT-treated gilts mated at their pubertal estrus (Davis et al., 1987), although there have
been other reports that ALT treatment during the prepubertal period was ineffective in

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improving breeding success (O’Reilly et al., 1979; Kraeling et al., 1982; Wood et
al.,1992)
To further increase the precision of control of estrus and ovulation in gilts, the
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ALT-treatment was combined with gonadotropin administration. These efforts resulted in
a decrease in the time from end of ALT treatment to onset of estrus without changing
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ovulation rate or litter size (Varley et al., 1989).
The effects of ALT on ovarian activity in gilts are similar to the effects reported in
the mare. ALT treatment at 15 mg per head per day effectively suppressed behavioral
estrus, however, the time from ALT withdrawal to the onset of estrus was dose
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dependant with higher doses of ALT hastening the onset of estrus (Redmer and Day,
1981). Differences in ovulation rate were not detected in this study, although Diehl et al.
(1986) demonstrated that ALT-treatment of inbred pigs with genetically reduced
ovulation rates, increased the ovulation rate without affecting fertilization. There was a
decrease in the number of ovulatory-size follicles (>8 mm) in gilts that received 15 mg of
ALT for 18 days but not in gilts that received 2.5 mg for 18 days (Redmer and Day,
1981). In contrast, gilts that received a higher dose of ALT (15 mg) had a reduction in
the number of cystic follicles compared with gilts that received a low dose (2.5 mg).
Estradiol levels were elevated in gilts treated with low daily doses of ALT, while
the circulating LH and P4 levels were unaffected by this treatment. Conversely, E2 levels
decreased in gilts treated with high daily doses of ALT and LH and P4 levels were
unaffected (Redmer and Day, 1981). These findings were validated by those of Martinat-

12
Botté et al. (1985), who reported that the decreased E2 levels during treatment with high
doses of ALT (≥15 mg), due to a decrease in large follicles (>6 mm), which are the
predominant source of E2. In support of this theory, Guthrie and Bolt (1985)
demonstrated that a high dose of ALT (20 mg daily) prevented the estrogen-triggered LH
surge and behavioral estrus in eCG-treated gilts.
It has been hypothesized that the major factor contributing to piglet survival at
birth was fetal hypoxia occurring during parturition (Randall, 1972). Therefore, the ability
to control the time of parturition in the sow could increase the number of live births by
allowing management to increase surveillance during this time. In sows, the time of
parturition can be controlled with PGF2α but the onset of parturition often ranges from 8
to 48 hours following PGF2α treatment (Guthrie, 1985). P4 has also been used to control

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the time of farrowing in sows, however, if administration is continued past day 114 of
gestation problems with lactation and uterine involution occur (Nellor et al., 1975).
Administration of 16 mg of ALT per day from day 111 to 118 in pregnant sows followed
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by E2 treatment 6-hours following last ALT administration successfully delayed farrowing,
but there was an increase in stillborn piglets (Varley and Brooking, 1981). Following this
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observation Guthrie et al. (1984) demonstrated that 20 mg of ALT (days 109 to 112)
followed by PGF2α treatment hastened the onset of parturition after PGF2α administration
compared with females treated only with ALT. There was no increase in stillbirths
observed in the ALT+PGF2α-treated females compared with control-treated females
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during this experiment. In a subsequent report the lack of increase in stillbirths noted
with ALT-PGF2α controlled parturition protocol was validated by Guthrie et al. (1987).
After determining that ALT could function as a progestin in swine researchers
began to work in earnest to determine if ALT administration could decrease the interval
from farrowing to estrus. Treatment with ALT from 7 days prior to weaning decreased
the number of sows that experienced delayed estrus without decreasing the degree of
synchrony in the subsequent estrus following group weaning (Martinat-Botté et al.,
1985). Stevenson et al. (1985) reported that treatment with ALT for 7 days prior to
weaning (4-week lactation period) actually increased the interval to estrus 14.5±0.2 days
in ALT-treated females compared with 5.6±0.2 days for the nontreated control sows but
increased the farrowing rate of sows inseminated at their first estrus following weaning.
In early weaned sows (~2 weeks of lactation), ALT treatment increased the number of

13
post-weaned sows (97%) detected in estrus following weaning (Koutsotheodoros et al.,
1998). Also, ALT-treated sows had an increased ovulation rate and there was tendency
for increased embryo survival rate in these females found at the time of sacrifice.
Altrenogest in Control of Reproduction in Exotic Species
There have been few reports on the use of ALT in species, other than the horse
or the pig. However, Schwarzenberger et al. (1999) reported an okapi, that had
experienced five abortions due to placental deficiency, was administered 10 mg of ALT
per day from day-50 post-mating until 30 days prior to expected parturition. In this
experiment, the dose of ALT was reduced by 1 mg per day over a 10-day period ending
at the end of ALT-treatment. A female calf was born from this female and no deleterious
health affects were noted in the dam during ALT-treatment. The female calf did die 30

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days following parturition, however, this was due to kidney hypoplasia that was not
linked to ALT-treatment.
Effects of P4 on the Oviducts during Early Pregnancy
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Disruption of Fertilization
In domestic farm animals (e.g., cattle and sheep), P4 levels are relatively low
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during ovulation and fertilization. At 14 hours after ovulation in the cow, circulating P4
levels remain below 0.5 ng/ml (Henricks et al., 1970; Wettemann and Hafs, 1973) and by
day 3 post-ovulation levels are still below 2 ng/ml (Henricks et al., 1971; Wettemann and
Hafs, 1973), however, by 6 days following ovulation P4 levels in both pregnant or
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nonpregnant cows are ≥3 ng/ml and peak around day 14 post-ovulation (Henricks, et al.,
1970, Wettemann and Hafs, 1973).
Low P4 levels during ovulation and fertilization have been shown to be beneficial
to fertility in many species. Chang (1967) demonstrated that in vivo fertilization could be
effectively disrupted by administration of P4, at the time of fertilization in rabbits. In
addition, it was noted that when rabbits were treated during estrus with P4, unfertilized
ova and degenerate embryos were often recovered from the uterus. Chang (1967)
suggested that the P4 treatment hastened embryo transport from the oviduct to the
uterus prior to the embryo developing to a stage capable of surviving in the uterus. In
another polytoccus species, the pig, increased P4 levels shortly prior to ovulation was
proposed to be the cause of increased polyspermy at fertilization (Day and Polge, 1968).
Hunter (1968) demonstrated that P4 administration to hamsters 3 days prior to the time

14