CHANGES IN TEMPORAL LEPTIN CONCENTRATIONS AND OTHER
METABOLIC FACTORS IN PRIMIPAROUS, POSTPARTUM,
ANESTROUS, SUCKLED, BEEF COWS
EXPOSED TO BULLS
by
Jesse Riley Olsen
A thesis submitted in partial fulfillment of
the requirements for the degree
of
Master of Science
in
Animal and Range Sciences
MONTANA STATE UNIVERSITY
Bozeman, Montana
August, 2009
©COPYRIGHT
by
Jesse Riley Olsen
2009
All Rights Reserved
ii
APPROVAL
of a thesis submitted by
Jesse Riley Olsen
This thesis has been read by each member of the dissertation committee and has
been found to be satisfactory regarding content, English usage, format, citation,
bibliographic style, and consistency, and is ready for submission to the Division of
Graduate Education.
Dr. James G. Berardinelli
Approved for the Department of Animal and Range Sciences
Dr. Bret E. Olson
Approved for the Division of Graduate Education
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
In presenting this thesis in partial fulfillment of the requirements for a doctoral
degree at Montana State University, I agree that the library shall make it available to
borrowers under rules of the library.
If I have indicated my intention to copyright this thesis by including a
copyright notice page, copying is allowable only for scholarly purposes, consistent with
“fair use” as prescribed in the U.S. Copyright Law. Requests for permission for extended
quotation from or reproduction of this thesis in whole or in parts may be granted
only by the copyright holder.
Jesse Riley Olsen
August, 2009
iv
ACKNOWLEDGEMENTS
I would like to thank my parents, Peter and Linda Olsen for their love and
encouragement throughout my academic career at Montana State University. As well as,
a special thanks to Dr. Duane Keisler for his collaboration with the leptin assay; my
fellow graduate colleagues, Jarrod R. C. Wilkinson and Shaun A. Tauck for their
insurmountable support, assistance, and input throughout this thesis; and undergraduate
interns, Luke Runnion, Tim Gibbs, Katie Philips, Riley Wedlake, and Kyla Hendry.
Also, I would like to express my sincerest thanks to my major professor, Dr. James G.
Berardinelli, for his time, guidance, encouragement of new ideas, patience, constructive
criticism, and friendship throughout the course of my graduate training. This study was
supported by Award No. 2007-35203-17743, NRI Competitive Grants Program,
CSREES, and the USDA, the Montana NSF EpsCOR program, the Montana Agricultural
Experiment Station, and is a contributing project to Multistate Research Project, W1112,
Reproductive Performance in Domestic Ruminants. I would also like to thank my
graduate committee members, Drs. Milan Shipka and Dennis Hallford for their valuable
time, assistance, and encouragement during the course of my studies and in the
preparation of this thesis.
v
TABLE OF CONTENTS
1. INTRODUCTION .........................................................................................................1
2. LITERATURE REVIEW ..............................................................................................4
Physiology of the Postpartum Anestrous Cow ..............................................................4
Hypothalmic Neurohormone ...................................................................................4
Gonadotropin-Releasing Hormone (GnRH) ................................................4
Adenohypophyseal Gonadotropins ..........................................................................5
Luteinizing Hormone (LH) ..........................................................................5
Follicle Stimulating Hormone (FSH)...........................................................6
Ovarian Steroids.......................................................................................................7
Estrogen .......................................................................................................7
Progesterone .................................................................................................7
Role of the Hypothalamic Hypophyseal-Ovarian Axis ...........................................8
Hypothalmic Feedback Effect of Estrogen ................................................10
Factors Known to Influence the Postpartum Anestrous Period ...................................11
Nutrition .................................................................................................................12
Effect of Suckling ..................................................................................................15
Other Factors ..........................................................................................................18
Effect of Bull Biostimulation on the Postpartum Anestrous Period ............................19
Metabolic Regulation and Reproduction .....................................................................22
Metabolic Regulation and the Hypothalmic-Hypophyseal Ovarian Axis .............23
Cortisol.......................................................................................................23
Tri-iodothyronine (T3) and Thyroxine (T4) ..............................................24
Role of Leptin in Regulation of Energy Homeostasis and Reproduction ..............25
Leptin in Energy Homeostasis ...................................................................25
Leptin Regulation on Puberty and Gonadotropin Secretion ......................27
Direct Ovarian Effects of Leptin................................................................30
Ovarian Steroid Regulation on Leptin Secretion .......................................30
3. STATEMENT OF PROBLEM ....................................................................................32
4. EXPERIMENT 1: CONCENTRATIONS OF GLUCOSE, NEFA, THYROXINE,
AND TRIIODOTHYRONINE IN PRIMIPAROUS, ANESTROUS, SUCKLED
BEEF COWS EXPOSED TO BULLS ........................................................................34
Introduction ..................................................................................................................34
Materials and Methods .................................................................................................34
Animals and Treatments ........................................................................................35
Facilities .................................................................................................................35
Nutrition .................................................................................................................36
vi
TABLE OF CONTENTS-CONTINUED
Intensive Blood Sampling Procedures ...................................................................36
Metabolite and Hormone Assays ...........................................................................37
Statistical Analyses ................................................................................................38
Results ..........................................................................................................................38
Discussion ....................................................................................................................40
5. EXPERIMENT 2: BIOSTIMULATORY EFFECT OF BULLS ON TEMPORAL
PATTERNS OF LEPTIN CONCENTRATIONS AND RESUMPTION OF LUTEAL
ACTIVITY IN PRIMIPAROUS, POSTPARTUM, ANESTROUS, SUCKLED BEEF
COWS ..........................................................................................................................43
Introduction ..................................................................................................................43
Materials and Methods .................................................................................................44
Animals and Treatments ........................................................................................44
Facilities .................................................................................................................45
Nutrition .................................................................................................................45
Blood Sampling for Progesterone, Leptin, and Resumption of Luteal Activity ....46
Statistical Analyses ................................................................................................48
Results ..........................................................................................................................48
Discussion ....................................................................................................................52
6. EXPERIMENT 3: EFFECT OF DURATION OF DAILY BULL EXPOSURE ON
LEPTIN CONCENTRATIONS DURING RESUMPTION OF OVULATORY
ACTIVITY IN PRIMIPAROUS, POSTPARTUM, ANESTROUS, SUCKLED BEEF
COWS ..........................................................................................................................54
Introduction ..................................................................................................................54
Materials and Methods .................................................................................................55
Animals and Treatments ........................................................................................55
Facilities and Daily Bull Exposure ........................................................................56
Nutrition .................................................................................................................57
Blood Sampling, Leptin and Progesterone Concentrations, and Resumption of
Ovarian Activity.....................................................................................................58
Statistical Analyses ................................................................................................59
Results ..........................................................................................................................60
Discussion ....................................................................................................................64
vii
TABLE OF CONTENTS-CONTINUED
GENERAL DISCUSSION ................................................................................................67
Hypothesis for a Metabolic Component or Pathway to the
Biostimulatory Effect of Bulls .....................................................................................68
LITERATURE CITED ......................................................................................................72
viii
LIST OF TABLES
Table
Page
1. Number of cows per treatment and least square means for days from calving
to start of the experiment, cow BW at the start of treatment, calf birth weight
(BW), BCS, calf sex ratio, and dystocia score for primiparous, suckled beef
cows exposed to bulls (EB) or not exposed to steers (ES)…...……..…………..39
2. Least squared means of glucose, NEFA, thyroxine (T4), and triiobothyronine
(T3) concentrations for primiparous, suckled beef cows exposed to bulls
(EB) and steers (ES)………………………………...…………………………..40
3. Number of cows per treatment and least square means for calving date, cow
BW, calf BW, BCS, calf sex ratio, and dystocia score for first calf suckled
beef cows exposed (BE) or not exposed (NE) to bulls at the start of the
experiment………………………………………………..……………...……..49
4. Number of cows per treatment and least squares means for intervals from
calving to resumption of luteal activity (RLA) and proportions of cows that
RLA during the experiment for primiparous, anestrous, suckled, beef cows
exposed to bulls for 12-h daily (BE12), 6-h daily (BE6), or not exposed to
bulls (NE) for 45 d starting 51.5 ± 2.3 d (± SE) after calving…...……………..61
ix
LIST OF FIGURES
Figure
Page
1. Pattern of progesterone concentrations used to determine occurrence of
resumption of ovarian cycling activity. Panel A represents a cow that
resumed cycling activity. Panel B represents a cow that did not resume
ovarian cycling activity...........................................................……..…………..47
2. Percentages of primiparous, suckled cows exposed (BE) or not exposed (NE)
to bulls that resumed luteal activity (%RLA) at the start of the experiment
and after the 30-d exposure period. Bars that lack common letters within
each cycling classification differ, P < 0.05; cycling start of experiment,
2 = 1.02, d.f. = 1; cycling at start of breeding season,2 = 9.1, d.f. = 1.........49
3. Least squares means of leptin concentrations for 6-d intervals in cows exposed
to bulls (BE) and cows not exposed to bulls (NE) that resumed ovulatory
activity after start of experiment (D 0). Bars that lack common letters within
and between each interval differ, P < 0.05. Interval by treatment interaction;
P < 0.05............………………………………………………..………………..50
4. Percentages of primiparous, suckled cows exposed (BE) or not exposed (NE)
to bulls that had not started to cycle by start of experiment (D0) and resumed
luteal activity (%RLA) during intervals D 0 to 6, D 9 to 15, and D 18 to 24 of
the experiment. Bars that lack common letters within and between each
interval differ, P < 0.05; interval D 0 to 6, 2 = 1.99, d.f. = 1; interval D 9 to
15,2 = 8.53, d.f. = 1; interval D 18 to 24,2 = 8.53, d.f. = 1; and between
intervals D 0 to 6 and D 9 to 15 for BE cows,2 = 6.2, d.f. = 1..................…..51
5. Facilities where cows were housed during experiment and schedule of daily
bull exposure..........................................................................................………..57
6. Least squares means of leptin concentrations for 10-d intervals in cows exposed
to bulls for 0 (NE), 6 (BE6), and12 (BE12) h daily. Bars that lack common
letters within and between each interval differ, P < 0.05. Interval by treatment
interaction, P = 0.06; treatment interaction, P < 0.0001................……………..62
7. Least squares means of leptin concentrations for 10-d intervals in cows
exposed to bulls for 0 (NE), 6 (BE6), and12 (BE12) h daily. Non-cycling
NE, BE6 and BE12 data are samples collected before the resumption of
ovulatory activity. Bars that lack common letters within and between each
interval differ, P < 0.05. Interval by treatment interaction, P = 0.08;
treatment interaction, P < 0.0001…….................................................................63
x
LIST OF FIGURES - CONTINUED
Figure
Page
8. Percentages of primiparous, suckled cows exposed to bulls for 0 (NE), 6
(BE6), and12 (BE12) h daily that resumed luteal activity (%RLA) during
intervals D 0 to 10, D 11 to 20, D 21 to 30, and D 31 to 40 of the experiment.
Bars that lack common letters within each interval differ, P < 0.05; interval
D 0 to 10, 2 = 3.37, d.f. = 2; interval D 11 to 20,2 = 2.84, d.f. = 2;
interval D 21 to 30,2 = 6.38, d.f. = 2; ; interval D 31 to 40,2 = 8.12,
d.f. = 2 and the overall interaction, P < 0.05;2 = 382.7, d.f. = 11....................64
xi
ABSTRACT
Exposing cows to bulls or excretory products of bulls stimulates resumption of
ovarian cycling activity in postpartum, suckled, anestrous cows. This biostimulatory
effect may be mediated by pheromones produced by bulls that stimulate physiological
changes in metabolic regulation of the hypothalamic-pituitary-ovarian (HPO) axis of
cows. In Experiment 1, the hypotheses tested were that concentrations of glucose,
NEFA, thyroxine (T4), tri-iodothyronine (T3), and T3:T4 ratios do not differ between
cows exposed to bulls or steers. The biostimulatory effect of bulls was associated with
lower mean concentrations of NEFA in postpartum cows. Experiment 2 was designed to
determine if continuous (24-h daily) bull exposure alters temporal patterns of leptin
concentrations in postpartum, anestrous cows. Cows exposed to bulls that resumed
cycling activity after the start of the experiment tended to have higher leptin
concentrations by the end of the 30-d exposure period than cows not exposed to bulls.
However, it was not known if these changes were related to resumption of ovarian
cycling activity in postpartum, anestrous cows. Experiment 3 tested the hypothesis that
temporal leptin concentrations may depend upon duration of daily bull exposure. Cows
had higher daily leptin concentrations and resumed ovarian cycling activity sooner as
duration of daily bull exposure increased. In conclusion, as duration of daily bull
exposure increases, the biostimulatory effect of bulls alters temporal leptin concentrations
and this change may facilitate or support the function of the HPO axis and accelerate
resumption of ovarian cycling activity in primiparous, postpartum, suckled, anestrous
cows.
1
CHAPTER 1
INTRODUCTION
The goal of cow-calf operations is to wean one calf per cow per year (Yavas and
Walton, 2000). This can be referred to as reproductive efficiency. Postpartum anestrus,
or the time after calving during which cows do not exhibit estrus and cannot become
pregnant, decreases reproductive efficiency in beef cattle. Generally, after a gestation of
approximately 280 d, cows are required to establish a pregnancy within 80 to 85 d after
calving. This problem is more pronounced in first-calf suckled beef cows that require 15
to 25 d longer to resume ovarian cycling activity than multiparous cows (Short et al.,
1994). Many management strategies have been developed to help reduce the interval of
postpartum anestrus. Unfortunately, these strategies can be costly, labor intensive,
unsustainable, and socially unacceptable. The use of the biostimulatory effect of bulls
may be an effective management strategy to reduce postpartum anestrus that is cost
effective, labor saving, sustainable, and socially acceptable to both producers and
consumers. Although, implementation of the use of the biostimulatory effect of bulls in
practical situations and the mechanism by which this effect is mediated is not well
understood.
The biostimulatory effect of bulls involves interactions between bulls and cows in
which the physical presence of bulls stimulates the resumption of ovarian cyclic activity
in multiparous and primiparous suckled beef cows (Zalesky et al., 1990; Custer et al.,
1990). Berardinelli and Joshi (2005) reported that more cows exposed to bulls 55 d
2
postpartum resumed ovarian cycling activity within 20 d of exposure than cows exposed
to bulls starting on d 15 or d 35. This result indicates that the ability of cows to respond
to the biostimulatory effect of bulls increases as time after parturition increases. Bull
exposure is known to alter cortisol concentrations and the characteristics of their pulsatile
patterns (Tauck et al., 2007; Tauck, 2008).
Furthermore, there is the possibility that the biostimulatory effect of bulls to
accelerate resumption of ovulatory activity involves metabolic changes in cows that are
induced by changes in cortisol concentrations, specifically changes involved with adipose
metabolism (Landaeta-Hernandes et al.; 2004, Wilkinson et al., 2007). Experiment 1 of
this thesis focuses on the influence of the biostimulatory effect of bulls on concentrations
of thyroid hormones, thyroxine (T4) and tri-iodothyronine (T3), glucose, and nonesterified fatty acids (NEFA). Research conducted within the last ten years has shed light
on the physiological mechanisms by which energy balance modulates reproductive
functions. Leptin, an adipocyte-derived hormone or adipokine, is secreted by white
adipose tissue and is an important factor in the control of energy balance, satiety, and
food intake, and has been shown to have many levels of modulation within the
hypothalamic-hypophyseal-gonadal axis (HPO; for review see, Tene-Sempere, 2007). In
Experiment 2 and 3, we investigated the capacity of the biostimulatory effect of bulls to
alter temporal leptin concentration patterns during postpartum anestrus and resumption of
ovarian cycling activity.
This review of literature will encompass: 1) an overview of the endocrinology of
postpartum cows and factors that influence the postpartum interval to resumption of
3
ovarian cycling activity; 2) a review of the biostimulatory effect of bulls on the
resumption of ovarian cycling activity of postpartum cows; and 3) an in depth review of
literature related to metabolic regulation, specifically by leptin, of reproduction in
mammals.
4
CHAPTER 2
LITERATURE REVIEW
Physiology of the Postpartum Anestrous Cow
Endocrine control of the reproductive system includes the function of the
hypothalamus, anterior pituitary gland and the ovaries. Gonadotropins are hormones
secreted by the anterior pituitary gland that modulate processes of the ovary. Ovarian
function includes: the development, maturation and release of the female gamete; and, the
synthesis and secretion of protein and steroid hormones. Reproductive events in the
female are regulated by a complex system of interactions between hormones secreted
within the hypothalamic-pituitary-ovarian (HPO) axis. This section will review
hormones of the hypothalamus, anterior pituitary, and ovary which play a major role in
regulating reproductive function in the postpartum anestrous female bovine.
Hypothalmic Neurohormone
Gonadotropin-Releasing Hormone (GnRH). Gonadotropin-releasing hormone is
a 10 amino acid neurohormone released from a subset of the hypothalamic
neurosecretory neurons into the hypothalamo-hypophyseal portal circulation to the
adenohypophysis. The primary function of GnRH is to stimulate secretion of
gonadotropic hormones from the anterior pituitary gland. The release of the
gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) from
the anterior pituitary (listed below), particularly when measured in the portal blood, are
5
characterized as pulsatile or otherwise reflecting a rapid increase followed by a decrease
in circulating concentrations (Caldani et al., 1993; Padmanabhan et al., 2002a).
However, pulsatile release is not spontaneous but induced by GnRH. Furthermore,
gonadotropin secretion is regulated by the interaction of hypothalamic GnRH with
gonadotropes and the feedback of gonadal steroids and peptides (Caldani et al., 1993).
Additionally, other intra-adenohypophyseal factors modulate the secretion of LH and
FSH (Padmanabhan et al., 2002a).
Adenohypophyseal Gonadotropins
Luteinizing Hormone (LH). The most consistent factor reported to precede
resumption of ovarian cycling activity in anestrous female bovids is the onset and
increase in LH pulsatility (Walters et al., 1982). The gonadotropin LH is a dimeric
glycoprotein secreted by luteotropes of the anterior pituitary (for review see, Hafez and
Hafez, 2000). During the luteal phase of the estrous cycle of cows, LH is secreted into
the general circulation in a temporal pattern characterized as having low frequency and
high amplitude pulses. During proestrus, the period proceeding estrus, the pulsatile
patterns in LH changes to that of low amplitude and high frequency LH release. There
have been many studies on LH pulsatility prior to ovulation and it is well established that
this is the appropriate pattern of LH release that leads to the preovulatory surge of LH
and causes ovulation (Hafez and Hafez, 2000).
An increase in LH pulse frequency is observed before anestrous cows resume
ovarian cycling activity (Walters et al., 1982; Humphery et al., 1983; Peters and
6
Lamming, 1990). Furthermore, the pulse frequency, amplitude (Rawlings et al., 1980;
Garcia-Winder et al., 1984; Garcia-Winder et al., 1986; Savio et al., 1990; Wright et al.,
1990), and average plasma concentration (Walters et al., 1982; Humphrey et al., 1983;
Garcia-Winder et al., 1984; Garcia-Winder et al., 1986; Nett et al., 1988) of LH have
been shown to increase with time after calving. Once LH pulse frequency, amplitude,
and concentration change to patterns observed in proestrus, a surge of LH is released
from the anterior pituitary which in turn causes ovulation and resumption of normal
ovarian cycling activity in postpartum anestrous cows.
Follicle Stimulating Hormone (FSH). Follicle stimulating hormone is also a
dimeric gonadotropin glycoprotein like LH; however, it is synthesized and secreted by
folliculotropes of the anterior pituitary and is responsible for the recruitment and
development of secondary and tertiary follicles of the ovary (for review see, Hafez and
Hafez, 2000). Follicle stimulating hormone and LH differ molecularly within the βsubunit, but α-subunit composition is the same for both FSH and LH.
Follicular selection and dominance appear to be dependent on FSH (for review
see, Hafez and Hafez, 2000). Concentrations of FSH have been shown to increase
shortly after parturition (Moss et al., 1985; Crowe et al., 1998); however, these
fluctuations do not result in ovulation. In a cow with normal ovarian cycling activity,
FSH secretion is controlled by the negative feedback effects of follicular inhibin. As one
follicle begins to become dominant, the granulosa cells within this dominant follicle
release inhibin, inhibiting the release of FSH from pituitary folliculotropes causing
smaller follicles within that cohort begin atresia. The dominant follicle then becomes
7
FSH independent-LH dependent and the appropriate temporal LH release pattern
stimulates the final maturation and development of the largest or “dominant” follicle. In
anestrous cows, maturation, development, and ovulation of follicles does not occur
because of the inappropriate release of LH. Dominant follicles in this type of hormone
milieu fail to synthesize and secrete estradiol-17β and do not grow beyond 7 to 9 mm in
diameter (Gong et al., 1995; Gong et al., 1996). As a result, these follicles do not ovulate
Ovarian Steroids
Estrogen. Estradiol-17β, the dominant ovarian estrogen, is synthesized primarily
by the granulosal cells of the ovarian antral follicles. Shortly after parturition, circulating
concentrations of estradiol-17β are very low due to the ovaries being devoid of large
follicles (Arije et al., 1974; Humphrey et al., 1983; Crowe et al., 1998). However,
follicular waves that develop a dominant follicle (DF) begin to develop 5 to 11 d after
parturition (Crowe et al., 1998). Thereafter, estradiol-17β concentrations remain
relatively constant and low throughout postpartum anestrus (Carruthers et al., 1980;
Chang et al., 1981), regardless of any effect suckling and milk production may have upon
secretion (Carruthers and Hafs, 1980). Again, only the appropriate LH signal can
stimulate a change in estradiol-17β concentrations, inducing a DF to become larger and
secrete higher levels of estradiol-17β. This change can induce behavioral estrus and
signal the preovulatory release of LH from the anterior pituitary.
Progesterone. Progesterone is a steroid hormone produced by the theca interna
cells of antral follicles and corpora lutea (CL; Hafez and Hafez, 2000). Following
8
parturition, progesterone concentrations decrease almost immediately and remain low
throughout postpartum anestrus. Usually, the postpartum cow exhibits a small rise in
progesterone concentration 3 to 7 days prior to the resumption of ovulatory activity (Arije
et al., 1974; Stevenson and Britt, 1979; Rawlings et al., 1980; Humphrey et al., 1983;
Werth et al., 1996). This increase in progesterone concentrations is followed by a decline
to baseline concentrations before the first postpartum estrus and is often referred to as a
“short cycle”. Short cycles are due to ovulation and formation of a CL (Castenson et al.,
1976; Stevenson and Britt, 1979); however, the CL formed after this ovulation is short
lived and produces small amounts of progesterone (Corah et al., 1974). Behavioral estrus
does not generally accompany this ovulation probably because there is no preovulatory
increase in estrogen. A priming effect by progesterone is believed to be responsible for
the full expression of estrus in response to estrogen in the bovine (Kieborz-Loos et al.,
2003). Mann and Lamming (2000) showed that the shortened lifespan of the CL is most
likely caused by low concentrations of estradiol-17β before ovulation which, in turn,
causes the retention of oxytocin receptors in the endometrium of the uterus; this allows
for a premature increase in prostaglandin-F2α (PGF2α) and lysis of the CL.
Role of the Hypothalamo-Hypophyseal-Ovarian Axis
Considerable effort has gone into the study of the relationship between the
postpartum anestrous interval to resumption of ovulatory activity and the physiological
mechanisms responsible for its occurrence and subsidence (for reviews see, Peters and
Lamming, 1986; Short et al., 1990; Williams, 1990; Yavas and Walton, 2000).
Physiological controls of the postpartum period are located in the complex interactions of
9
neuroendocrine-endocrine relationships within the hypothalamo-hypophyseal-ovarian
axis. Simply, the hypothalamus stimulates the pituitary which, in turn, stimulates the
ovary, and again in turn, stimulates a feedback and exerts control over the hypothalamus
and adenohypophysis. The following is an overview of the interaction between the
hypothalamus, pituitary, and ovary during the postpartum, anestrous periods of the
bovine.
Normal ovarian function is dependent upon stimulation by gonadotropins secreted
by the adenohypophysis, also called the anterior pituitary gland. The hypothalamus
contains neurons that are known as neurosecretory neurons or peptidergic neurons
because they synthesize peptides (neurohormones) that are secreted into the blood,
instead of a synaptical space. Gonadotropin secretion is modulated by the secretion of
GnRH. Once GnRH binds to its receptors on gonadotropes, it stimulates the synthesis
and secretion of gonadotropins. More specifically, GnRH stimulates pituitary luteotropes
and folliculotropes to secrete LH and FSH, respectively. Generally, GnRH neurons
intrinsically secrete GnRH in small bursts or pulses; the intrinsic pattern of release is
known as the GnRH “pulse generator”. A pulse of GnRH will stimulate a concomitant
pulse in LH but not FSH (for review see, Hafez and Hafez, 2000). Therefore, factors that
modulate the GnRH pulse generator will influence the secretion pattern of LH release as
well.
Luteinizing hormone pulsatility is an important factor in explaining the failure of
DF to ovulate soon after parturition. Luteinizing hormone has direct stimulatory effect
on the DF and in a cow exhibiting normal ovarian cycling activity, temporal release of
10
LH is characterized by low frequency and high amplitude pulses (Walters et al., 1982;
Peters and Lamming, 1990). This temporal release pattern extends the life of the DF and
causes it to grow beyond 7 to 9 mm into a pre-ovulation size of greater than 9 mm
(Fortune et al., 1991; Savio et al., 1993; Gong et al., 1995; Rhodes et al., 1995; Gong et
al., 1996). During postpartum anestrus, LH patterns are characterized by low frequency
and high amplitude pulses (Rawlings et al., 1980; Humphery et al., 1983; Garcia-Winder
et al., 1984; Garcia-Winder et al., 1986; Savio et al., 1990; Wright et al., 1990). The
temporal release pattern of LH is the primary endocrine cause of postpartum anestrus.
Therefore, to understand the physiological mechanisms involved with regulating
resumption of ovarian cycling activity in cows, one must understand those mechanisms
that limit pulsatile LH release.
Hypothalmic Feedback Effect of Estrogen. Secretion of gonadotropin releasing
hormone (GnRH), a critical neurohormone produced by the hypothalamus that stimulates
the release of both LH and FSH, is greatly influenced by production of gonadal steroids
(Fernandes et al., 1978; Jagger et al., 1987). Progesterone is high during the luteal phase
of the estrous cycle, however as progesterone begins to decrease during the early
proestrous phase of the estrous cycle, low estradiol concentrations cause neurosecretory
neurons to release GnRH with an increased frequency, causing release of LH from the
anterior pituitary in low amplitude, high frequency manner (Rawlings et al., 1980;
Humphery et al., 1983; Garcia-Winder et al., 1984; Garcia-Winder et al., 1986; Savio et
al., 1990; Wright et al., 1990, Evans et al., 1994), described as the positive feedback
mechanism of estradiol. The release of LH in this temporal manner stimulates the
11
continued growth and development of the DF in the ovary, causing the synthesis and
secretion of estadiol-17β. Once estrogen concentrations reach a threshold, luteotropes are
stimulated for the episodic or preovulatory release of LH. This episodic release of LH
assures development and ovulation of the dominant follicle. Concomitantly, the sexual
behavior center of the brain detects the rise in estrogen concentration and stimulates
behavioral estrus.
During postpartum anestrus, circulating estrogen concentrations do not change
dramatically (Carruthers et al., 1980; Chang et al., 1981). Neurosecretory neurons
responsible for the tonic release of GnRH are quite sensitive to low constant levels of
estradiol-17β (Acosta et al., 1983) causing a decrease in the pulsatile release of LH from
the anterior pituitary. This is known as the negative feedback effect of estrogen. As time
after calving increases, sensitivity of neurosecretory neurons to estrogen declines,
allowing GnRH pulses to occur more frequently. This increased GnRH pulse frequency
stimulates the appropriate low amplitude, high frequency LH secretion required for
ovulation and resumption of ovarian cycling activity following parturition.
Factors Known to Influence the Period of Postpartum Anestrus
The primary factors influencing the length of time from calving to the resumption
of ovarian cycling activity are nutrition, suckling stimuli, and parity. Other factors that
affect this interval are breed, environmental stress, dystocia, and social factors (for
review see, Short et al., 1990; Yavas and Walton, 2000).
12
Nutrition
Basal metabolism, activity, growth and basic energy stores have priority over
reproduction (Grimard et al., 1997; Guedon et al., 1999), and a negative energy balance
would reduce availability of glucose for use in processes such as resumption of ovarian
cycling activity and maintenance of a pregnancy (Yavas and Walton, 2000). Such a
negative metabolic state would increase the mobilization of energy reserves stored as
adipose tissue. Additionally, energy balance greatly influences the length of postpartum
anestrus in the bovine, whereby low energy intake before and after calving increases the
time interval from calving to resumption of ovarian cycling activity (Dunn et al., 1969;
Wiltbank, 1970; Falk et al., 1975; Dunn et al., 1985). Furthermore, if cows remain on a
low plane of nutrition after calving, they may fail to resume ovarian cycling activity
throughout the breeding season (Wiltbank, 1970). On the other hand, the interval to
resumption of ovarian cycling activity can be shortened if cows are fed a higher plane of
nutrition after calving (Bellows and Short, 1978; Ducker et al., 1985; Henricks and Rone,
1986). As a consequence, cows fed a high plane of nutrition have a greater chance of
becoming pregnant before the end of the breeding season than cows fed a low plane of
nutrition (Bellows and Short, 1978; Henricks and Rone, 1986; DeRouen et al., 1993).
A moderate to high plane of nutrition is important for the resumption of ovarian
cycling activity after calving (Dunn et al., 1969; Wiltbank, 1970; Falk et al., 1975; Dunn
et al., 1985). However, there are conflicting reports regarding the prepartum and
postpartum nutrition regimens affecting postpartum ovarian cycling activity. Houghton
13
et al. (1990) reported that cows fed a low energy diet before calving and switched to a
high energy diet after calving had shorter postpartum intervals to estrus and more cows
cycling than cows fed the low energy diet prepartum and low energy diet postpartum.
Contrary to these results, Dunn and Kaltenbach (1980) concluded that prepartum
nutrition was more important than postpartum nutrition in reducing the postpartum
interval.
These results beg to question as to how nutrition might affect follicular
development. Henricks and Rone (1986) reported that cows fed a high energy diet had
more medium- and small-sized follicles and higher estradiol-17β concentrations than
cows fed a low energy diet. Also, it has been observed that increasing the energy intake
beginning either two wk before parturition (Lammoglia et al., 1996), at parturition (Beam
and Butler, 1997; DeFries et al., 1998), or at four wk after calving (Khireddine et al.,
1998) increased the number of antral follicles in the ovaries. Further evidence has shown
that feeding a higher energy ration at parturition prolongs the life of the corpus luteum
(Williams, 1989).
Progesterone, the hormone of pregnancy, prepares the uterus for embryonic
implantation and sustains pregnancy after implantation. Gombe and Hansel (1973) and
Beal et al. (1978) found that progesterone concentrations were lower for first-calf cows
fed a restricted-energy diet. The low concentrations of progesterone in these cows could
have been due in part to the low rate of follicular development caused by poor nutrition
documented by Henricks and Rone (1986) and Perry et al. (1991). Also, energyrestricted cows had smaller corpora lutea with lower progesterone contents on d 10 of the
14
third postpartum cycle (Gombe and Hansel, 1973). This indicates that energy intake can
be directly related to the ability of the cow to conceive and maintain pregnancy (Dunn et
al., 1969).
Percent body fat at the time of and after parturition can also influence the length
of postpartum anestrus in cows (Bartle et al., 1984; Richards et al., 1986). Cows of good
body condition at time of parturition resume ovarian cycling activity and conceive sooner
compared to thin cows that have longer intervals from calving to resumption of ovarian
cycling activity and therefore a greater difficulty conceiving before the end of the
breeding season (DeRouen et al., 1994; Spitzer et al., 1995; Vizcarra et al., 1998). Body
condition score positively correlates with follicular development early postpartum (Ryan
et al., 1994), pituitary LH concentrations at 30 d after calving (Connor et al., 1990), and
influences circulating concentrations of insulin-like growth factor-1 (IGF-1), LH pulse
frequency, and postpartum interval to resumption of ovulatory activity after weaning
(Bishop et al., 1994). However, Wright et al. (1990) reported that average LH
concentrations in systemic circulation were not affected by body condition.
Severe nutrient restriction has been shown to have a negative effect on LH
secretion in sexually-mature and prepubertal mammals, including cattle (Day et al.,
1986a; McCann and Hansel, 1986) and sheep (Foster et al., 1989; Keisler and Lucy,
1996). Restricted and control-fed heifers respond to administration of exogenous GnRH
with an LH surge-like release; however, the release of LH was lower in restricted than in
control heifers (Day et al., 1986a). These results indicate that under-nutrition suppresses
pulsatile LH secretion by impairing hypothalamic GnRH release. Thus, the diminished
15
pulsatile release of LH observed during low energy balance has been generally accepted
as the result of a reduced frequency of GnRH pulses (Williams, 1999). However, under
nutrition may have a modulatory effect directly at the level of the luteotropes in the
anterior pituitary. Simultaneous blood sampling for LH from the adenohypophyseal
portal system and the jugular vein has demonstrated that there is a greater reduction in
frequency of LH pulses in feed-restricted female lambs than the reduction in frequency of
GnRH pulses (I’Anson et al., 2000). This suggests low amplitude pulses of GnRH did
not always result in concomitant LH pulses and that gonadotropes tend to have a
decreased sensitivity to GnRH in long-term, feed-restricted lambs. Although, short-term
fasting had effects on indicators of metabolic status, such as insulin, IGF-I, and nonesterified fatty acids (NEFA) (McCann and Hansel, 1986; Kadokawa and Yamada,
1999), there was no affect on circulating LH concentrations in mature cows. However, in
prepubertal heifers, 48 to 72 h of total feed restriction reduces LH pulsatility (Amstalden
et al., 2000; Maciel et al., 2003a). Taken together, energy balance, maturity of the central
reproductive axis, and interactions with the negative feedback of estradiol may be major
factors in influencing the postpartum anestrous period in cows. Good body condition and
medium to high energy rations fed pre- or postpartum are necessary for cows to resume
ovarian cycling activity, conceive, and maintain pregnancy after calving.
Effect of Suckling
If nutrition is not a limiting factor, suckling stimuli are the primary factor
contributing to prolonged postpartum anestrus in beef and dairy cows (Short et al., 1990;
Williams, 1990; Stagg et al., 1998). Cows suckled once daily and non-suckled cows have
16
shorter postpartum anestrous intervals to resumption of ovarian cycling activity than
cows suckled twice daily, cows suckled normally, and cows suckled intensively (Smith
and Vincent, 1972; Laster et al., 1973; Carter et al., 1980; Odde et al., 1980; La Voie et
al., 1981; Randel, 1981; Reeves and Gaskins, 1981; Ramirez-Godinez et al., 1982;
Garcia-Winder et al., 1984; Houghton et al., 1990). Removal of the calf at birth and
weaning calves early causes a significant reduction in the length of postpartum anestrus
compared to cows suckling calves (Bellows et al., 1974; Walter et al., 1982; Williams,
1990; Short et al., 1990); whereas, intensive suckling can prolong postpartum anestrus in
cows (Wettemann et al., 1986; McNeilly, 1988).
Suckling stimuli have the potential to modulate the neuroendocrine-endocrine
physiological events that control the resumption of ovarian cycling activity. Cows who
have been weaned from their calves have higher LH concentrations than suckled cows
(Walters et al., 1982; Carter et al., 1980). Non-suckled cows and restricted-suckled cows
exhibit a sustained increase in LH concentrations within 20 d after calving, while cows
subjected to intensive suckling exhibit no sustained increase until 48 d postpartum
(Garcia-Winder et al., 1984). Also, LH mean concentration, peak frequency (Carruthers
and Hafs, 1980; Whisant et al., 1986), and amplitude (Carruthers and Hafs, 1980; Chang
et al., 1981; Whisnant et al., 1986) increase significantly within 48 h post-weaning
compared to non-weaned, suckled cows. Additionally, suckled cows have lower
frequency and amplitude of LH release than milked cows (Carruthers and Hafs, 1980).
Therefore, suckling may affect the hypothalamus and GnRH neurosecretory system
17
reducing the tonic release of LH causing the postpartum anestrous state of ovarian
quiescence.
Inhibition of LH release by suckling may involve more than mammarysomatosensory pathways. The physical presence of the calf may also be needed to elicit
this effect. Williams et al. (1993) found that LH pulse frequency increased within 9 to 13
days after weaning when compared to that of suckled cows. Also, these investigators
found that denervation of the mammary gland failed to increase the LH pulse frequency.
Therefore, the physical presence of a calf appeared to be a component of the inhibitory
effect of the suckling stimulus and suggested that cows recognize calves through
olfactory and visual cues. This is known as the cow-calf bond (Williams and Griffith,
1995; Lamb et al., 1997).
In summary, it is possible to model the mechanism by which suckling stimuli fits
into the endocrinology of the postpartum anestrous cow. Following parturition, suckling
stimuli is intense due to the close relationship to and the physical presence of the calf.
Suckling stimuli causes the GnRH pulse generator of the hypothalamus to be more
sensitive to the negative feedback effects of estrogen; resulting in high amplitude, low
frequency patterns of GnRH and causes high amplitude, low frequency pulses of LH.
This type of LH pattern signals the ovary to remain anovular. As time increases
postpartum and the calf grows older, it becomes more nutritionally and socially
independent, reducing suckling stimuli. There is then a decrease in the sensitivity of the
GnRH pulse generator to the negative feedback effect of estrogen, causing a change in
18
the release of LH in a high frequency, low amplitude manner. This type of pulsatile
pattern in LH signals to the ovary to resume ovarian cycling activity.
Other Factors
The following is a summary of other factors contributing to postpartum anestrus
reviewed by Short et al. (1990). Multiparous cows have shorter anestrous intervals than
do primiparous cows. The reason for this is believed to be a result of a combination of
increased incidence of dystocia and greater nutrient demands on primiparous cows due to
energy requirements for growth, lactation, and production of a calf. Dystocia in both
primiparous and multiparous cows increases the length of postpartum anestrus. The
greater degree of difficulty during parturition causes more damage to the reproductive
tract and increases amount of physical stress. Stress increases energy demand which, in
turn, causes prolonged periods of anestrus after parturition. Stress can come in many
forms; studies have shown that stressing the cow with extreme heat or cold will prolong
postpartum anestrus.
Breed or breed-type also plays a role in the length of postpartum anestrus.
Continental cattle breeds have higher growth and milking genetic potentials, and longer
postpartum anestrous intervals than do British breeds. Increased milking and growth
potentials cause an increase in nutrient demands, which may result in longer intervals
from calving to resumption of ovarian cycling activity.
19
Effect of Bull Biostimulation on the Postpartum Anestrous Period
Exposing cows to the physical presence of bulls with intent to hasten the
resumption of ovarian cycling activity was first documented in early breeding
experiments (Nersesjan, 1962; Sipilov, 1964; Ebert et al., 1972; Macmillan et. al., 1979;
Zalesky et al., 1984; Berardinelli et. al., 1987; Scott and Montgomery, 1987; Custer et.
al., 1990; Naasz and Miller, 1990; Stumpf et. al., 1992; Cupp et. al., 1993; Hornbuckle et.
al., 1995; Fernandez et al., 1996; Peres-Hernandez et al., 2002; Anderson et al., 2002).
This biostimulatory effect of bulls is described as the influence of bulls on the
reproductive function of cows that is mutually beneficial for successful reproduction.
Bull biostimulation interacts with other factors known to influence the length of
postpartum anestrus (Stumpf et al., 1992; Hornbuckle et al., 1995). Stumpf et al. (1992)
showed cows exposed to bulls and fed a low plane of nutrition had postpartum anestrous
intervals that were 14 d shorter than cows fed a low plane of nutrition and not exposed to
bulls. In the same study, cows fed a high plane of nutrition that were exposed to bulls
had postpartum anestrous intervals to resumption of ovarian cycling activity 6 d shorter
than non-exposed cows also fed on a high plane of nutrition. These data indicate that the
biostimulatory effect of bulls shortens postpartum anestrus to a greater extent under
nutrient-restricted conditions in cows. This suggests the possibility that an increase in
sensitivity to the biostimulatory effect of bulls is associated with a decrease in energy
balance.
The mechanism by which the bull stimulates the hypothalamo-hypophsealovarian control axis is not well understood. Data from previous research showed that an
20
increase in LH pulse frequency occurred directly before postpartum anestrous cows
resumed ovarian cycling activity (Walters et al., 1982; Peters and Lamming, 1990), and
this change in LH secretion patterns is believed to be required for the resumption of
ovarian cycling acitivity after calving. Cows exposed continuously to bulls and cows
exposed to bulls intermittently had higher mean LH concentrations and greater frequency
of LH pulses than cows that were not exposed to bulls (Fernandez et al., 1996).
Additionally, Baruah and Kanchev (1993) administered bull urine oronasally to cows and
found that mean LH and FSH concentrations increased in the blood within 80 min after
urine application. However, Fernandez et al., (1996) and Baruah and Kanchev (1993)
reported that exposing cows to bulls intermittently or administering bull urine oronasally
to cows did not decrease the length of postpartum anestrus. These data indicate that the
quantity of stimulation was not sufficient to cause a biostimulatory effect. Taken
together, these studies support the hypothesis that the mechanism whereby the
biostimulatory effect of bulls acts involves the hypothalamo-hypophseal-ovarian axis and
allows one to speculate that a pheromone(s) may be involved with this effect.
Berardinelli et al. (2004) explored the hypothesis that a social bond may be
formed between the bull and cow, causing the biostimulatory effect of bulls. This notion
was tested by comparing the length of postpartum anestrus of cows exposed to familiar
bulls, then switched to exposure to unfamiliar bulls, and cows exposed to familiar
ovariectomized (OVX) cows and switched to exposure of unfamiliar OVX cows. Cows
exposed to familiar and unfamiliar bulls had shorter intervals from calving to the
resumption of ovarian cycling activity than cows exposed to OVX cows. However,
21
familiarity had no effect on the length of postpartum anestrus. Therefore, social
interaction and bonding between bulls and cows may not be an important factor that
mediates the biostimulatory effect of bulls. Instead, it appears that the physical presence
of bulls acts independent of social interaction and is probably related to a pheromonal
factor. Berardinelli and Joshi, (2005) tested this idea by comparing intervals to
resumption of ovarian cycling activity in cows that were: 1) exposed to continuous
presence of bulls, 2) exposed to excretory products of bulls for 12 h daily, 3) exposed to
their own excretory products for 12 h daily, or 4) not exposed to bulls, excretory products
of bulls or cows beginning 35 d after calving. Cows exposed to bulls or excretory
products of bulls or their own excretory products had shorter intervals to resumption of
cycling activity than cows not exposed to bulls, excretory products of bulls or excretory
products of cows. These data indicate that the biostimulatory effect of bulls is mediated
through exposure to excretory products of bulls. Previously, Burns and Spitzer (1992)
showed that length of postpartum anestrus for cows exposed to testosterone-treated cows
and cows exposed to bulls did not differ but were shorter than that for control cows. The
data from these experiments indicate that the biostimulatory effect of bulls could be
mediated through a pheromone(s) present in bull excretory products, and that production
and excretion of this pheromone could be androgen-dependent.
There is evidence that the biostimulatory effect of bulls involves activation of the
hypothalamo-hypophyseal-adrenal axis as well. Tauck et al. (2007) found that cows
exposed to bulls for 30 d had greater cortisol concentrations and shorter postpartum
intervals to resumption of ovulatory activity than cows not exposed to bulls. Recently,
22
research in the same laboratory evaluated the effect of acute bull exposure on
characteristics of temporal cortisol and LH concentration patterns. They found that
cortisol pulse frequency was lower, cortisol pulse duration was longer, and LH pulse
frequency was higher in postpartum anestrous cows exposed continuously to bulls within
10 d of the start of the exposure period and after a 2 d intensive sampling acclimatization
period (Tauck, 2008). Furthermore, Landaeta-Hernandez et al. (2004) found that nonesterified fatty acids (NEFA) were 33% higher in suckled cows exposed to bulls than in
non-exposed suckled cows. It is possible that these results could be related to an increase
in cortisol concentrations associated with peri-ovulatory events in resumption of ovarian
cycling activity in postpartum cows (Humphreys et al., 1983). A well known catabolic
effect of cortisol is the liberation of NEFA and amino acids from skeletal muscle and
adipose tissue to be used as substrates for hepatic gluconeogenensis (for review see,
Hadley and Levine, 2007). These findings indicate that the biostimulatory effect of bulls
activates or alters the hypothalamic-hypophyseal-adrenal axis and the hypothalamichypophyseal-ovarian axis prior to the resumption of ovulatory activity in postpartum,
anestrous, beef cows. Furthermore these results indicate the potential for adrenal
involvement in the mechanism of bull exposure to accelerate the resumption of ovulatory
activity in postpartum, anovular cows via alterations in metabolic processes.
Metabolic Regulation and Reproduction
Energy balance or metabolic status of an organism is defined by the availability of
energy and nutrients to the tissues. An organism’s intrinsic perception of this status is a
23
critical component in the modulation of various biological functions. In mammals, it is
very well known that reproduction, the ability to produce and rear a viable offspring, is
dependent upon availability of nutrients to support growth, onset of puberty and sexual
maturity, copulation, pregnancy, lactation, and rearing. Energy balance is of even greater
importance in the female, wherein pregnancy and lactation are a considerable energetic
drain. However, the physiological basis for the coupling of regulation of energy balance
and reproductive function has been only briefly explained in relatively recent research.
Reproduction depends upon the development and function of proper
neuroendocrine control. The HPO axis has three levels of regulation: the secretion of
GnRH from neurosecretory neurons in the hypothalamus, LH and FSH from the anterior
pituitary, and hormonal products or sex steroids, from the gonads. These factors all
participate in the aforementioned positive and negative feed-forward and feed-back loops
in the regulation of proper reproductive function. This section will review some of the
many central and peripheral metabolic modulatory factors, both stimulatory and
inhibitory, that participate in the regulation of reproductive function at all levels of the
HPO axis.
Metabolic Regulation and the Hypothalmic-Hypophyseal-Ovarian Axis
Cortisol. Cortisol , as well as other adrenal glucocorticoids, is associated with
stress and can affect carbohydrate, lipid, and protein metabolism. Actions of
glucocorticoids on the liver are anabolic and increase the synthesis of a variety of
gluconeogenic enzymes within hepatocytes. On the other hand, glucocortisoids enhance
24
catabolic actions in skeletal muscle and adipose tissue. Inhibition of glucose uptake in
peripheral tissues by glucocorticoids causes a void of metabolic substrate for ATP
production, resulting in the proteolysis of muscle proteins and lipolysis of fat. Once
mobilized, free fatty acids and amino acids become substrates available for use in hepatic
gluconeogenesis (for review see, Hadley and Levine, 2007).
Hormone milieu in total at the time of stress may influence the effect of cortisol
on function of the HPO axis. Additionally, the effects of cortisol on the HPO axis seem
to be attenuated in the presence of gonads and their products (Tilbrook et al., 1999; Daley
et al., 2000; Stackpole et al., 2006). Cortisol has been shown to be involved with the
physiological mechanism by which postpartum anestrous interval is shortened due to the
biostimulatory effect of bulls. Tauck et al. (2007) found that cows exposed to bulls had
greater cortisol concentrations and shorter postpartum intervals than cows not exposed to
bulls. Recently, research in the same laboratory further evaluated the effect of acute bull
exposure on characteristics of temporal cortisol and LH concentration patterns. They
found cortisol pulse frequency was lower and pulse duration was longer while coinciding
with a higher LH pulse frequency in postpartum anestrous cows continuously exposed to
bulls (Tauck, 2008). These findings indicate that the biostimulatory effect of bulls
activates or alters the HPA axis and the HPO axis prior to the resumption of ovulatory
activity in postpartum, anestrous, beef cows. This suggests the potential for adrenal
involvement in the mechanism of bull exposure to accelerate the resumption of ovulatory
activity in postpartum, anovular cows.
Tri-iodothyronine (T3) and Thyroxine (T4). Thyroid hormones, T3 and T4, are
25
required for normal growth and development, stimulate thermogenesis and oxygen
consumption, are altered by energy balance, and are believed to play a permissive role in
the function of many other hormones (for review see, Hadley and Levine, 2007).
Thyroid hormone synthesis and secretion is regulated by thyrotropin (thyroid stimulating
hormone; TSH) secretion from the anterior pituitary (Hafez and Hafez, 2000).
Thyrotropin shares the same α-subuinit as the adenohypophyseal gonadotropes;
luteinizing hormone (LH) and follicle stimulating hormone (FSH) (Hadley and Levine,
2007). Additionally, TSH secretion is controlled by hypothalamic thyrotropin-releasing
hormone (TRH). Therefore, the thyroid hormones T3 and T4 are modulators of
metabolic processes and are closely related to modulators of reproductive function within
the hypothalamic-pituitary axis.
Role of Leptin in Regulation of Energy Homeostasis and Reproduction
Leptin in Energy Homeostasis. Energy balance is defined by the equilibrium
between food intake and energy expenditure, with energy homeostasis being the most
tightly regulated functions of the organism (Casanueva and Dieguez, 1999). It wasn’t
until the mid-1990s that our knowledge of the endocrine control mechanism for the
control of energy stores was elucidated, exclusively due to the discovery of the adipocyte
derived hormone leptin (Zhang et al., 1994; Friedman and Halaas, 1998; Rosenbaum and
Leibel, 1998; Casanueva and Dieguez, 1999; Ahima et al., 2000;). The 167-amino acid
hormone synthesized and secreted by adipocytes has been implicated in communicating
nutritional status to the brain and is involved in regulation of food intake, metabolism,
26
and reproduction (Houseknecht et al., 1998). It has been widely stated that serum leptin
concentrations inform the brain about the level of adipocity or the availability of
metabolic fuels (Schneider, 2004). The adipokine is expressed in placenta (Hoggard et
al., 1997a), stomach (Bado et al., 1998), skeletal muscle (Wang et al., 1998), brain
(Morash et al.,1999; Wiesner et al., 1999), adenohypophysis (Morash et al., 1999) and
leptin mRNA is detected in the cerebral cortex, cerebellum, hypothalamus, and pineal
gland (Morash et al., 1999).
There is a great deal of evidence supporting the idea that leptin increases the
intracellular availability and oxidation of free-fatty acids (FFA). Treatment with leptin
decreases de novo FFA synthesis, increases intracellular FFA esterification to
triglycerides, increases triglyceride breakdown and FFA oxidation, and increases FFA
exportation to nonadipocytes in rats (William et al., 2002). Ultimately, this results in a
greater efflux of FFAs from adipocytes thereby reducing FFA oxidation within
adipocytes and increasing availability to nonadipocytes (William et al., 2002). If leptin
positively influences reproductive function by increasing fuel oxidation and availability,
then it is possible that leptin would no longer improve reproductive function as metabolic
fuels became scarce. This is the case in rats restricted to 70% of their ad libitum food
intake wherein leptin failed to improve reproductive function, however leptin fully
reversed the effects of fasting in rats restricted to only 80% of ad libitum intake (Cheung
et al., 1997). Furthermore, treatment with leptin can shorten the duration of lactational
diestrus in rats exposed to acute food deprivation but not in rats exposed to prolonged or
chronic food restriction (Woodside et al., 1998 and 2000). This suggests that leptin can
27
facilitate reproductive function but only under adequate metabolic fuel availability.
Effects of leptin in control of food intake appear to be within the hypothalamus,
where leptin in known to modulate orexigenic neurons as well as anorexigenic neurons
(for review see, Sahu, 2004). In addition, these neuronal pathways are subject to other
modulators of food intake that are either synergistic or antagonistic towards the effects of
leptin on energy homeostasis (Zigman and Elmquist, 2003). Thus, knowing leptin’s
potential as a pleiotropic modulator of a vast array of endocrine functions involved with
the hypothalamus (Wauters et al., 2000), it is considered a “genuine” neuroendocrine
integrator, communicating control of biological functions to the organism’s state of
energy balance (Tene-Sempere, 2007).
Leptin Regulation on Puberty and Gonadotropin Secretion. In agreement that
metabolic control of reproduction is much more restrictive in the female due to the
energetic drain of pregnancy and lactation, it has been suggested that leptin plays a
prominent role in control of female reproductive function (Tene-Sempere, 2007).
Homozygous mutation for the gene that encodes leptin, ob gene, results in obesity,
infertility, and atrophy of reproductive organs in mice (Barash et al., 1996). However,
these manifestations can be reversed via treatment with exogenous leptin (for review see,
Schneider, 2004). Leptin treatment induced weight loss and development of reproductive
organs, and restored reproduction in male (Mounzih et al., 1997) and female (Barash et
al., 1996) ob/ob mice. In normal prepubertal mice, leptin treatment induced earlier onset
of estrous cycles (Ahima et al., 1997; Chehab et al., 1997).
Circulating concentrations of leptin are correlated positively with adiposity and
28
increases in body weight in normal mice (Frederich et al., 1995), humans (Ahren et al.,
1997), sheep (Delavaud et al., 2000), cattle (Ehrhardt et al., 2000), and developing heifers
(Garcia et al., 2002a). Exogenous treatment with leptin reduces feed intake and the
effects of negative energy balance on reproduction, including the onset of puberty, length
of the estrous cycle, length of lactational diestrus, gonadotropin and gonadal steroid
levels, and pulsatile LH secretion in mice, rats, hamsters, and non-human primates (for
review see, Schneider, 2004). Additionally, treatments of leptin to within physiological
ranges reverse the effects of food deprivation on the estradiol–induced LH and prolactin
surges in rats (Watanobe et al., 1999).
Initially, direct actions by leptin on GnRH neurons were suggested after the
discovery of leptin receptor (LR) expression within immortalized GnRH-secreting cell
line, GT1-7 (Magni et al., 1999). However, under physiological conditions it has been
accepted that GnRH receptors do not express the LR in rats (Watanobe et al., 2002).
Thus, it has been suggested that the influence of leptin on the GnRH and LH surges occur
through intermediate neurons projecting from LR-containing neurons to ultimately
synapse upon GnRH neurons (for review see, Schneider, 2004). These pathways have
been postulated to include neuropeptide-Y (NPY), pro-opiomelanocortin (POMC), and
cocaine- and amphetamine-regulated transcript (CART) secreting neurons, all of which
are well known to influence GnRH neuron function (Schneider, 2004). Messenger RNA
for the LR has been detected in NPY neurons (Mercer et al., 1996; Finn et al., 1998;
Williams et al., 1999). Expression of NPY in the hypothalamus is increased in ob/ob
mice and treatment with leptin diminishes hypothalamic NPY in this mutant mouse
29
(Stephens et al., 1995). Interestingly, feed restriction increases NPY mRNA in sheep
(McShane et al., 1993), and intra-cerebral-ventricular (ICV) infusion of NPY decreases
circulating concentrations of LH in sheep (McShane et al., 1992) and cows (Gazal et al.,
1998). In addition, ICV infusion of leptin prevented NPY-induced feed intake in rats
(Sahu, 1998). However, in the ovariectomized, estradiol implanted cow, pretreatment
with leptin did not prevent the NPY-mediated decrease in plasma concentrations of LH
(Garcia et al., 2002b).
Short-term fasting reduces circulating concentrations of leptin in rodents and
ruminants (Amstalden et al., 2000; Nagatani et al., 2000). In rats, prepuberal heifers
(Amstalden et al., 2000), castrated lambs (Nagatani et al., 2000), and ovariectomized gilts
(Whisnant and Harrel, 2001) the fasting-mediated reduction in mean concentrations of
leptin is associated with decreased pulsatility of LH. Moreover, administration of leptin
to castrated, estradiol-implanted lambs (Nagatani et al., 2000), and peripubertal heifers
(Maciel et al., 2003a) during fasting prevents the reduction in frequency of LH pulses. In
ovariectomized, estradiol-implanted cows under acute nutrient restriction, leptin increases
mean concentrations of LH (Zieba et al., 2002). However, when higher doses (10 times
greater) are used, leptin loses its ability to stimulate LH. Similarly, long-term
undernourished, ovariectomized ewes, but not normal-fed ewes, exhibited increased
secretion of LH after ICV infusions of recombinant human leptin (Henry et al., 1999;
Henry et al., 2001).
More recent research has elucidated much of the neuroendocrine networks for the
signaling of leptin to GnRH neurons. One example of this is the discovery that leptin
30
directly targets kisspeptin neurons within the hypothalamus in mice (Smith et al., 2006).
Kisspeptin neurons are one of the most potent stimulators of the GnRH/LH axis known
thus far (Tena-Sempere, 2006). Furthermore, gene expression of kisspeptin is decreased
during undernutrition while administration of kisspeptin attenuates negative effects of
undernutrition on the onset of puberty. These results indicate the potential that changes
in endogenous leptin concentrations participate in metabolic regulation in reproduction.
However, it is commonly assumed that the majority of modulation by leptin occurs
directly upon the HPO axis. It is important to note that leptin greatly upregulates energy
expenditure, thermogenesis, and fuel oxidation (Schneider, 2004), therefore, has the
potential to influence reproduction simply by means of regulating metabolic fuel
availability.
Direct Ovarian Effects of Leptin. Many studies of different species have
documented the ability of leptin to modulate ovarian steroidogenesis, most of which
suggest an inhibitory action by leptin (Spicer and Francisco, 1991; Zachow and
Magoffin, 1997; Agarwal et al., 1999; Ghizzoni et al., 2001; Kendall et al., 2004). This
contradicts apparent stimulatory and permissive effects upon the hypothalamus and
pituitary. In addition to leptin receptor (Ob-R) mRNA expression within granulosal and
thecal cells of the human ovary (Karlsson et al., 1997) and long (Ob-Rb) and short (ObRa) forms of leptin receptor in the rat ovary, the adipokine, leptin, itself appears to be
expressed in the ovaries and in concentrations that fluctuate with ovarian cyclic activity
(Loffler et al., 2001; Ryan et al., 2002; Archanco et al., 2003; Ryan et al., 2003). Though
many studies have found contradictory results as to positive or negative effects of ovarian
31
leptin on follicular development (Duggal et al., 2000; Craig et al., 2004; Olatinwo et al.,
2005), Burkan et al. (2005) interestingly reported that leptin induced LH-independent
ovulation in GnRH deficient mice.
Ovarian Steroid Regulation on Leptin Secretion. Expression and secretion of
leptin, leptin receptor, and biological actions of leptin may be influenced by sex steroids.
Leptin mRNA decreases in adipose tissue after ovariectomy in rats and estradiol
stimulates mRNA expression and secretion of leptin in adipocytes in culture (Machinal et
al., 1999). Central infusion of leptin increases mean concentrations of LH and FSH in
ovariectomized rats treated with estradiol (Walczewska et al., 1999). However,
ovariectomized rats without estradiol treatment did not respond to central infusions of
leptin and high doses of estradiol attenuated the leptin-induced increase in the release of
LH (Walczewska et al., 1999). Furthermore, estradiol may influence expression of leptin
receptor. Expression of the long form of leptin receptor (Ob-Rb) in the hypothalamus of
rats decreases following estradiol treatment (Bennett et al., 1998).
32
CHAPTER 3
STATEMENT OF THE PROBLEM
As pointed out in the review of literature, the biostimulatory effect of bulls
appears to be mediated by pheromones that influence reproductive behavior and function
of cows. Stumpf et al. (1992) found that cows of lower body condition had a greater
reduction in postpartum anestrous interval when exposed to bulls than cows of higher
body condition exposed to bulls. Furthermore, Tauck (2008) described the activation and
involvement of the hypothalamic-pituitary-adrenal axis with the biostimulatory effect of
bulls on primiparous, postpartum, anestrous, suckled beef cows. One interpretation of
these observations may be that bull exposure alters metabolic regulation and energy
homeostasis, ultimately influencing the HPO axis and accelerating resumption of ovarian
cycling activity in primiparous, postpartum, anestrous, suckled cows.
As stated in the review of literature, energy balance is of particular importance to
the postpartum, lactating, and still growing primiparous cow. Many metabolic hormones
and metabolites serve as indicators of energy balance and have the ability to modulate
functions of reproduction by influencing the HPO axis. One of which, leptin, is a peptide
hormone secreted by adipose tissue and is accepted as an indicator of adiposity and
satiety. Leptin is known to influence feed intake, onset of puberty, as well as influencing
the gonadotropic hormone secretion patterns. The pulsatile pattern of luteinizing
hormone, a gonadotropin secreted from the anterior pituitary, is what must be altered in
order for the postpartum anestrous cow to resume ovarian cycling activity.
33
The goals of this research were: 1) to determine if thyroid hormones (T3 and T4),
glucose, or NEFA are involved with the physiological response of cows to the
biostimulatory effect of bulls, 2) determine the role of leptin in the biostimulatory effect
of bulls on the function of the HPO axis and resumption of ovarian cycling activity, and
3) to determine if duration of daily bull exposure is a factor involved with the appropriate
manner whereby bull-pheromonal stimuli alters temporal leptin concentrations and
accelerates resumption of ovarian cycling activity in primiparous, postpartum, anestrous,
suckled, beef cows.
34
CHAPTER 4
EXPERIMENT 1: CONCENTRATIONS OF GLUCOSE, NEFA, THYROXINE, AND
TRI-IODOTHYRONINE IN PRIMIPAROUS, ANESTROUS, SUCKLED BEEF COWS
EXPOSED TO BULLS
Introduction
Resumption of luteal function in primiparous, anovular, suckled beef cows after
calving is accelerated if cows are exposed to bulls (Custer et al., 1990) or excretory
products of bulls (Berardinelli and Joshi, 2005). Olsen et al. (2006) found that cortisol
concentrations increase in cows exposed to bulls. Cortisol is intimately involved with
metabolic regulation of energy utilization (Hadley and Levine, 2007). There is the
possibility that the biostimulatory effect of bulls to accelerate resumption of ovulatory
activity involves metabolic changes in cows that are induced by changes in cortisol
concentrations. The objective of this study was to determine if exposing cows to bulls
alters systemic glucose, NEFA, and thyroid hormones concentrations in primiparous,
postpartum, anestrous, suckled beef cows. The specific hypothesis tested in this
experiment were that systemic concentrations of glucose, NEFA, thyroxine (T4),
triiodothyronine (T3), and T3:T4 ratios do not differ in postpartum, anestrous, suckled
beef cows exposed daily for 5 h to bulls or steers for 9 d.
Materials and Methods
This experiment was conducted at the Montana State University Bozeman
Agricultural Research and Teaching Facility. Animal care, handling, and protocols used
35
in this experiment were approved by the Montana State University Agriculural Animal
Care and Use Committee.
Animals and Treatments
Sixteen spring-calving 2-yr-old Angus X Hereford primiparous, suckled beef
cows, 2 mature Angus X Hereford bulls, and 2 yearling Angus X Hereford steers were
used in this experiment. Cows and calves were maintained in a single pasture and had no
contact with bulls or their excretory products during pregnancy and from calving until the
start of the experiment.
Average calving date for these cows was Feb. 4, 2006. At the start of the
experiment (D 0) cows averaged 67 ± 3.5 d postpartum. One week before the start of the
experiment cows were stratified by body weight, BCS, calf birth weight, calving date, sex
of calf and dystocia score and assigned randomly to two treatments: exposed to bulls
(EB, n = 8) or steers (ES, n = 8) 5 h daily for 9 d (D 0 to 8).
Facilities
Cows were housed within pens in separate lot areas at the Bozeman Livestock
Teaching and Research Center. Pens within the north lot were used for maintaining EB
cows while pens within the south lot were used for maintaining ES cows. During the
daily sample collection and exposure period cows were moved into individual stalls
within open-air sheds adjacent to pens that housed cows in each treatment. Sheds were
similar in structure, area and light density. Light density within sheds was tested using a
Minolta Autometer Pro Photometer and tarps were used to manipulate the light density so
36
that cows in each sampling area were exposed to the same amount of light. During daily
sample collections and exposure periods cows were halter-restrained within aligned (sideby-side) stalls, and bulls or steers were contained in front of cows. Bulls and steers were
unrestrained and allowed to eat, roam, come into contact with the frontal aspect of cows
and interact with cows individually within each stanchion. Excretory products of bulls
and steers were not removed from the exposure area throughout the experimental period.
Nutrition
Cows had free access to good quality, chopped mixed-grass alfalfa hay, and any
pasture grasses that were available before the start of the experiment. Once cows and
calves were moved into pens they were given free access to the same hay, 0.5 kghd-1d-1
cracked barley, water, and a trace mineral-salt supplement. The TDN of the diet
exceeded the NRC requirement for lactating beef cow with a mature weight of 545 kg by
approximately 18% (NRC, 1996). Bulls were fed 0.5 kg of cracked barely and good
quality, chopped mixed-grass alfalfa hay. Steers were fed a finishing ration that consisted
of 70% concentrate and 30% roughage throughout the experiment.
Intensive Blood Sampling Procedures
Two days before the start of the experiment, each cow was fitted with an
indwelling jugular catheter. Blood samples (~7 mL) were collected daily from each cow
at 15-min intervals (starting at Time 0 and ending at 360 min) for 6 h (1000 to 1600 h)
beginning 1 h before the 5-h exposure period each day during the 9 d period (D 0 to 8).
Serum was harvested for each sample and stored at −20°C. Blood samples were
37
collected by the same technician for cows in each treatment throughout the experiment.
A total of 143 (3.9%) samples were not collected during this experiment due to noncooperative animals, coagulation within catheters, or catheter replacement.
Metabolite and Hormone Assays
It was determined from a preliminary analysis that randomly selected samples
throughout the intensive bleeding period were representative of the daily mean
concentrations. Glucose concentrations in serum for Times 15 and 225 min on D 2 and 8
were determined using a commercially available, hand-held glucose/ketone monitor
(Precision Xtra™, MediSense/Abbott Laboratories, Bedford, MA). In order to
compensate for assay drift, high and low serum pools were repeated 5 times every 20
samples. Mean high and low pool concentrations were 135.8 and 56.5 mg/dL with an
inter-assay CV of 4.5 and 6.3%. Concentrations of NEFA at Times 15 and 300 min on
all days were quantified with a commercially available enzymatic-colorimetric assay (HR
Series NEFA – HR [2], Wako Diagnostics, Richmond, VA). Inter- and intra-assay CV
were 5.8 and 4.6% for a serum pool that contained 0.415 mmol/L; and, 14 and 5.6%,
respectively, for a serum pool that contained 0.100 mmol/L. Thyroxine (T4) and
triiodothyronine (T3) concentrations at Time 225 min on each day were assayed using
solid-phase RIA kits (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA).
The intra-assay CV for serum pools that contained 155 and 105 ng/mL of T4 were 5.8
and 4.9%, respectively. The intra-assay CV for serum pools that contained 10.3 and 4.5
ng/mL of T3 were 11.2 and 24.5%, respectively.
38
Statistical Analyses
Glucose and NEFA concentrations(reasoning for times, presentation) were
analyzed by separate ANOVA for a completely random design using PROC GLM of
SAS (SAS Inst. Inc., Cary, NC). The model included treatment, time, day, animal within
treatment, and all possible interactions. Animal within treatment variance component
was used to test the effect of treatment. Thyroxine, T3, and T3:T4 ratio concentrations
were analyzed by separate ANOVA for a completely random design using PROC GLM
of SAS (SAS Inst. Inc., Cary, NC). The model included treatment, day, animal within
treatment, and all possible interactions. Animal within treatment variance component
was used to test the effect of treatment. Means were separated by Bonferroni multiple
comparison tests.
Results
Stratification factors, days form calving to start of the experiment, cow BW at
start of the experiment, calf BW, BCS of cows, calf sex ratio, and dystocia score did not
differ (P > 0.30) between EB and ES cows (Table 1).
39
Table 1. Number of cows per treatment and least square means for days from calving to start of the
experiment, cow BW at the start of treatment, calf birth weight (BW), BCS, calf sex ratio, and
dystocia score for primiparous, suckled beef cows exposed to bulls (EB) or exposed to steers (ES)
Treatment
SEMa
P value
67
3.5
0.94
526
531
36
0.76
Calf BW (kg)
31
32
3.5
0.61
BCSc
5.3
5.3
0.23
0.92
e
0.62
0.34
Variable
EB
ES
8
8
Day postpartum
67
Cow BW (kg)
n
b
Calf sex ratio
d
Dystocia Score
f
0.50
0.63
0.25
1.1
1.0
0.16
a
SEM = Standard error of the mean.
Days from calving to start of the experiment.
c
BCS; 1 = emaciated, 9 = obese.
b
Calf sex ratio = ratio of male to female calves, 1 = male and 0 = female. Tested with ϰ2 analysis.
d
ϰ2 value.
e
f
Dystocia Score: 0 = No assistance to 5 = Caesarean section.
There was no interaction among or between treatments, days, and times for
glucose concentrations. Glucose concentrations did not differ between Times 15 and 225
min, or D 2 and 8 or between EB and ES cows (Table 2). There was no interaction
among or between treatments, days, and times for NEFA concentrations. Concentrations
of NEFA did not differ between Times 15 and 300 min or among days (0 to 8).
However, NEFA concentrations were greater (P < 0.05) in ES cows (0.262 ± 0.024
mmol/L) than in EB cows (0.197 ± 0.020 mmol/L) over the course of the experiment
(Table 2). There was no interaction between treatments and days for concentrations of
40
T4 and T3, or T3:T4 ratios. Concentrations of T4 and T3, and T3:T4 ratios did not differ
among days or between EB and ES cows (Table 2).
Table 2. Least squared means of glucose, NEFA, thyroxine (T4), and triiobothyronine (T3)
concentrations for primiparous, suckled beef cows exposed to bulls (EB) and steers (ES)
Treatment
SEMa
P value
82.4
6.2
0.79
0.197
0.262
0.099
0.06
T4, ng/mLc
17.7
14.0
7.9
0.15
T3, ng/mLc
9.5
8.8
3.5
0.41
T3:T4 ratio
0.68
0.79
0.36
0.25
Variable
EB
ES
8
8
Glucose, mg/dLb
81.5
NEFA, mmol/L
n
a
SEM = Pooled standard error of the mean.
Glucose concentrations for samples obtained at Time 15 and 225 min on Days 2 and 8.
c
T4 and T3 concentration for samples obtained at Time 225 min on each day of the experiment.
b
Discussion
In the present experiment we evaluated concentrations of glucose on D 2 and 8 at
Times 15 and 225 min, NEFA concentrations every day at Times 15 and 300 min, and
T4, T3 concentrations and T3:T4 ratios every day at Time 225 min. Blood samples were
collected every 15 min for 6 h daily for 9 d. Concentrations of glucose, NEFA, T4, and
T3 for a full collection period were evaluated in 1 or 2 cows and on 1 or 2 days chosen
randomly. We found that the patterns of glucose, NEFA, T4, and T3 concentrations did
exhibit pulsatile rhythms, in fact they exhibited a random pattern of change throughout
the sampling period. Thus, we randomly chose D 2 and 8 and Times 15 and 225 min to
41
evaluate changes in glucose concentrations: Times 15 and 300 min for all days for
NEFA concentrations and Time 225 on all days for T4 and T3.
Exposing anestrous, suckled beef cows to bulls for 5 h daily over a period of 9 d
did not influence glucose, T4, T3 concentrations and T3:T4 ratios. However, exposing
anestrous cows to bulls decreased concentrations of NEFA compared to concentrations of
NEFA in cows exposed to steers. This result of the present study for NEFA
concentrations in suckled cows exposed to bulls is quite different from the result reported
by Landaeta-Hernandez et al. (2004). They found that concentrations of NEFA were
33% higher in suckled cows exposed to bulls than in non-exposed suckled cows. The
reason for this difference is not clear; however, the data reported by Landaeta-Hernandez
et al. (2004) may have been confounded by the fact that more bull exposed cows had
resumed ovulatory activity by the end of the experiment than non-exposed cows. This
may have been due to an increase in cortisol concentrations that are associated with periovulatory events of resumption of ovulatory activity in postpartum cows (Humphreys et
al., 1983). A well-known catabolic affect of cortisol is the liberation of NEFA and amino
acids from skeletal muscle and adipose tissue to be used as substrates for
gluconeogenesis in the liver (for review see, Hadley and Levine, 2007). Whereas, in the
present study all samples were collected in anestrous cows that had not resumed
ovulatory activity and this difference may be related to NEFA concentrations in anestrous
cows compared to those of cows during the peri-ovulatory period.
The primary objective of this experiment was to determine if exposing anestrous,
suckled beef cows to bulls alters glucose, NEFA, and thyroid hormones that may directly
42
or indirectly affect carbohydrate and lipid metabolism. This idea was based on the
following observations that alterations in carbohydrate and lipid metabolism can either
delay or accelerate the resumption of ovulatory activity in postpartum, anestrous, suckled
beef cows (for reviews see, Randel, 1990; Short et al., 1990). Exposing postpartum cows
to bulls increased systemic cortisol concentrations relative to cows not exposed to bulls
(Tauck et al., 2007). Cortisol is a hormone known to influence carbohydrate and lipid
metabolism (for review see, Hadley and Levine, 2007). Recently, Landaeta-Hernandez et
al. (2004) reported that systemic concentrations of NEFA were greater in postpartum
cows exposed to bulls than in cows not exposed to bulls. One could hypothesize that the
biostimulatory effect of bulls to reduce the interval of postpartum may be mediated by
inducing changes in cortisol concentrations which in turn may directly affect metabolic
processes of postpartum, anestrous, suckled beef cows. This in turn could alter the
hypothalamic release patterns of GnRH stimulating follicular development and ovulation.
Thus, the affect of bull exposure on anestrous cows appears to be related to a decrease in
catabolism of adipose tissue and may be involved in the mechanism whereby bulls
accelerate the resumption of ovulatory activity.
43
CHAPTER 5
EXPERIMENT 2: BIOSTIMULATORY EFFECT OF BULLS ON TEMPORAL
PATTERNS OF LEPTIN CONCENTRATIONS AND RESUMPTION OF LUTEAL
ACTIVITY IN PRIMIPAROUS, POSTPARTUM, ANESTROUS, BEEF COWS.
Introduction
Resumption of luteal function in primiparous, anovular, suckled beef cows after
calving is accelerated if cows are exposed to bulls (Custer et al., 1990) or excretory
products of bulls (Berardinelli and Joshi, 2005). Tauck et al. (2007) found that cortisol
concentrations increase in cows exposed to bulls. Cortisol is intimately involved with
metabolic regulation of energy utilization (Hadley and Levine, 2007). Furthermore, there
is the possibility that the biostimulatory effect of bulls to accelerate resumption of
ovulatory activity involves metabolic changes in cows that are induced by changes in
cortisol concentrations, specifically changes involved with adipose metabolism
(Landaeta-Hernandes et al., 2004, Wilkinson et al., 2007; Tauck, 2008). Stumpf et al.
(1992) reported that cows of low body condition have a greater reduction in postpartum
interval to resumption of luteal activity when exposed to bulls than cows of high body
condition. The physiological mechanisms by which energy balance modulates
reproductive functions have recently been elucidated within the last decade. Leptin, an
adipocyte-derived hormone or adipokine, is secreted by white adipose tissue and is an
important factor on the control of energy balance, satiety, food intake and has been
shown to have many levels of modulation upon the hypothalamic-hypophyseal-gonadal
axis (for review see Tene-Sempere, 2007). The objectives of this study were to determine
44
if 1) short-term bull exposure (30 d) increased the number of cows cycling before the
start of the breeding season; and 2) bull exposure alters temporal leptin concentration
patterns during postpartum anestrus and resumption of luteal activity. The specific
hypothesis tested in this experiment was that the proportion of cows that resume luteal
activity and systemic concentrations of leptin do not differ in postpartum, anestrous,
suckled beef cows continuously exposed or not exposed to bulls.
Materials and Methods
Animals and Treatments
Fifty-three spring-calving two-yr-old, primiparous, suckled, Angus Hereford
crossbred beef cows and four mature, epididymectomized Angus x Hereford bulls were
used in this experiment conducted at the Montana State University Bozeman Agricultural
Research and Teaching Facility. Animal care, handling, and protocols used in this
experiment were approved by the Montana State University Agricultural Animal Care
and Use Committee.
Cows and calves were maintained in a single pasture and had no contact with
bulls or their excretory products during pregnancy and from calving until the start of the
experiment. Average calving date for these cows was Feb. 16, 2003. At the start of the
experiment (D 0) cows averaged 63 d (± 14 d; ± SE) postpartum; D 30 of the experiment
was day of start of estrous synchronization (ES). One week before the start of treatment
cows were stratified by calving date, cow BW, calf birth weight, calf sex, dystocia score,
and BCS. Once cows were stratified they were assigned randomly to one of two
45
treatments; exposure to mature bulls (BE; n = 26) or no bull exposure (NE; n = 27) from
the start of the experiment (D 0) until the start of ES (D 30).
Facilities
Two lots were used for this experiment, designated north and south by their
geographic location. Each lot contained four pens (41 m x 18 m) that were identical in
east-west configuration, bunk space, aspect, slope, and connection to open-shed shelters.
Lots were approximately 0.35 km apart, and the arrangement was such that the prevailing
wind blew from south to north. Animals housed in one lot were not able to see or smell
animals housed in the other lot; however, there was a possibility that sounds made by
animals in one lot could be heard by animals in the other lot. These lots and arrangement
have proven to be effective in previous experiments involving bull-cow interactions
(Fernandez et al., 1993; 1996). Pens in the south lot had not held bulls for more than six
years. Pens in the north lot had not held bulls for ten months.
Nutrition
Cows had free access to good quality, chopped mixed-grass alfalfa hay, and any
pasture grasses that were available before the start of the experiment. Once cows and
calves were moved into pens they were given free access to the same hay, 0.5 kg/hd/d
cracked barley, water, and a trace mineral-salt supplement. The TDN of the diet
exceeded the NRC requirement for lactating beef cows with a mature weight of 545 kg
by approximately 18% (NRC, 1996). Bulls were fed the same ration as cows.
46
Blood Sampling for Leptin,
Progesterone, and Resumption of Luteal Acitivity
Blood samples were collected from each cow by jugular venepuncture at 3-d
intervals from the start of the experiment to the start of the breeding season. Serum
concentrations of leptin were determined in triplicate and were quantified by using a
competitive liquid-liquid phase, double-antibody leptin RIA procedure described
previously (Delavaud et al., 2000). The intra and inter assay coefficients of variation were
2.01% and 3.22%, respectively. Serum was assayed for progesterone concentration using
solid-phase RIA kit (Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA).
Intra- and interassay CV’s for a serum pool that contained 0.8 ng/mL were 4.8 and 11%,
respectively; and for a pool that contained 7.0 ng/mL were 2.0 and 5.4%, respectively.
Progesterone concentrations patterns were used to assess the resumption of ovarian
cycling activity. A rise in progesterone concentration of > 0.5 ng/ml in three consecutive
samples that exceeded 1 ng/mL provided evidence that cows resumed ovarian cycling
activity during the experimental period.
Progesterone (ng/mL)
47
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0
3
6
9
12
15
18 21
24 27
30
24
30
Progesterone (ng/mL)
Day of treatment
7
6.5
6
5.5
5
4.5
4
3.5
3
2.5
2
1.5
1
0.5
0
0
3
6
9
12
15
18
21
27
Day of treatment
Figure 1. Pattern of progesterone concentrations used to determine occurrence of
resumption of ovarian cycling activity. Panel A represents a cow that resumed cycling
activity. Panel B represents a cow that did not resume ovarian cycling activity.
48
Graphic representation of the pattern of progesterone concentrations used to
determine the occurrence of resumption of ovarian cycling activity is illustrated in Figure
1.
Statistical Analyses
Comparisons of calving date, calf birth weight, calf sex ratio, dystocia score, and
body condition score were analyzed by analysis of variance for a completely random
design using PROC GLM of SAS (SAS, Cary, NC). The model included treatment.
Means were separated by PDIFF procedure of SAS.
Leptin concentrations were analyzed by PROC MIXED repeated measures
analysis of SAS. The model included TRT, Animal as experimental unit, Day as the
repeated measure, and the TRT by Day interaction and means were separated using
Bonferroni Multiple Comparison tests.
Results
Average calving date, cow BW, calf birth weight, calf sex ratio, dystocia score,
and BCS did not differ between BE and NE cows (Table 3). Proportions of BE (50%)
and NE (44%) cows cycling at the start of the experiment did not differ (P = 0.68; Figure
2). However, a greater proportion (P < 0.05) of cows exposed to bulls (BE; 100%) were
cycling by the end of the 30-d exposure period than cows not exposed to bulls (NE;
70.4%; Figure 2).
49
Table 3. Number of cows per treatment and least square means for calving date, cow
BW, calf BW, BCS, calf sex ratio, and dystocia score for first calf suckled beef cows
exposed (BE) or not exposed (NE) to bulls at the start of the experiment
Treatment
Variable
BE
NE
SEMa
P value
n
26
27
Calving dateb
47
46
14.37
0.93
Cow BW (kg)
518
509
41.72
0.40
Calf BW (kg)
37
37
3.89
0.66
BCS
4.7
4.8
1.11
0.98
Calf sex ratioc
0.53
0.59
0.49
0.96
Dystocia scored
1.08
1.15
0.39
a
SEM = Standard error for means.
b
Day of year.
c
Calf sex ratio = ratio of male to female calves.
d
Dystocia Score: 0 = No assistance to 5 = Caesarean section.
0.91
BE
NE
y
100%
90%
% RLA
80%
x
70%
60%
50%
x
x
40%
30%
20%
Start of experiment
End of experiment
Figure 2. Percentages of primiparous, suckled cows exposed (BE) or not exposed (NE)
to bulls that resumed luteal activity (%RLA) at the start of the experiment and after the
30-d exposure period. Bars that lack common letters within each cycling classification
differ, P < 0.05; cycling start of experiment, 2 = 1.02, d.f. = 1; cycling at start of
breeding season,2 = 9.1, d.f. = 1.
50
Temporal patterns of leptin concentrations did not differ between BE and NE
cows. However, there was a treatment by day interaction (P < 0.05; Figure 3) for leptin
concentrations in BE and NE cows that began to cycle after D 0. Leptin concentrations
increased on D 15 and remained stable until D 21, then increased again by D 27.
Whereas, concentrations of leptin in NE cows increased on D 18, decreased on D 21, then
increased by D 27. In order to better understand this treatment by day interaction, the
experiment period was grouped into three intervals; D 0 to 6, D 9 to 15, and D 18 to 24.
Here, leptin concentrations during the 18 to 24 d period were greater (P < 0.05; Figure 3)
in BE cows than in NE cows that resumed luteal activity after the start of the experiment.
Leptin concentrations during 0 to 6 d and 9 to 15 d did not differ (P > 0.10; Figure 3).
NE
10
Leptin, ng/mL
9.5
9
BE
SEM; P < 0.05
y
x, y
x, y
8.5
x
x
x
9 to 15 d
18 to 24 d
8
7.5
7
0 to 6 d
Interval
Figure 3. Least squares means of leptin concentrations for 6-d intervals in cows exposed
to bulls (BE) and cows not exposed to bulls (NE) that resumed ovulatory activity after
start of experiment (D 0). Bars that lack common letters within and between each
interval differ, P < 0.05. Interval by treatment interaction; P < 0.05.
51
In cows that had not resumed luteal activity before the start of the experiment
(D0), proportions of BE (62%) and NE (33%) cows that had resumed luteal activity
during the D 0 to 6 interval did not differ (P > 0.10; Figure 4). However, a greater
proportion (P < 0.05) of cows exposed to bulls (BE; 100%) resumed luteal activity by the
intervals D 9 to 15 and D 18 to 24 than cows not exposed to bulls (NE; 46.7%; Figure 4).
BE
100%
NE
y
y
90%
% RLA
80%
70%
x
60%
x
50%
40%
x
x
30%
20%
0 to 6
9 to 15
18 to 24
Interval
Figure 4. Percentages of primiparous, suckled cows exposed (BE) or not exposed (NE)
to bulls that had not started to cycle by start of experiment (D0) and resumed luteal
activity (%RLA) during intervals D 0 to 6, D 9 to 15, and D 18 to 24 of the experiment.
Bars that lack common letters within and between each interval differ, P < 0.05; interval
D 0 to 6, 2 = 1.99, d.f. = 1; interval D 9 to 15,2 = 8.53, d.f. = 1; interval D 18 to
24,2 = 8.53, d.f. = 1; and between intervals D 0 to 6 and D 9 to 15 for BE cows,2 =
6.2, d.f. = 1.
52
Discussion
Long-term bull exposure (> 60 d) reduces the postpartum interval to resumption
of ovarian cycling activity in first-calf suckled beef cows (Custer et al., 1990, Fernandez
et al., 1993). In this experiment cows exposed to mature bulls for 30 d increased the
proportion of cows that started to cycle by 24% relative to cows that were not exposed to
bulls. Macmillan et al. (1979) reported a greater proportion of cows exposed to bulls
than cows not exposed to bulls for 21 d before the breeding season showed estrus during
the 60 d breeding season. Similarly, Scott and Montgomery (1987) reported that more
cows exposed to bulls for 20 d before the breeding season began cycling than cows not
exposed to bulls. These results indicate that short-term bull exposure has the same effect
as long-term exposure. Short-term bull exposure may be an effective strategy to increase
the proportion of first-calf suckled cows that are cycling at the beginning of the breeding
season.
Additionally, we asked, “Does bull exposure for 30 d influence temporal leptin
concentration patterns during postpartum anestrus and the resumption of ovarian cycling
activity?” In the previous experiment of this thesis we found that there is a decrease in
NEFA concentrations associated with the biostimulatory effect of bulls. These two
experiments are complimentary wherein we find that there is a difference between
treatments of bull-exposed and non-exposed in leptin concentrations by day, wherein
leptin concentrations are higher in cows exposed to bulls for 18to 24 d than cows not
exposed. Furthermore, Tauck et al. (2007) and Tauck (2008) described adrenal activation
and involvement associated with exposure to bulls whereby cows exposed to bulls had
53
higher cortisol concentrations; and increased duration, decreased frequency of cortisol
pulses, respectively. Cortisol is well known to be involved with lipid metabolism as well.
This suggests that there may be a metabolic component to the biostimulatory effect of
bulls in hastening the resumption of ovarian cycling activity, particularly in the area of
lipid mobilization and metabolism.
We conclude that short-term bull exposure of primiparous suckled beef cows
altered leptin concentrations such that temporal leptin concentrations were higher in cows
after 18 d of bull exposure.
54
CHAPTER 6
EXPERIMENT 3: EFFECT OF DURATION OF DAILY BULL EXPOSURE ON
LEPTIN CONCENTRATIONS DURING RESUMPTION OF OVULATORY
ACTIVITY IN PRIMIPAROUS, POSTPARTUM, ANESTROUS, BEEF COWS
Introduction
The biostimulatory effect of bulls involves interactions between bulls and cows in
which the physical presence of bulls stimulates the resumption of luteal activity in
multiparous and primiparous suckled beef cows (Custer et al., 1990). Bull exposure is
known to alter cortisol concentrations and the characteristics of their pulsatile patterns
(Tauck et al. 2007). Furthermore, there is the possibility that the biostimulatory effect of
bulls to accelerate resumption of luteal activity involves metabolic changes in cows that
are induced by changes in cortisol concentrations, specifically changes involved with
adipose metabolism (Landaeta-Hernandes et al., 2004, Wilkinson et al., 2007). Stumpf et
al., 1992 reported that cows of low body condition have a greater reduction in postpartum
interval when exposed to bulls than cows of high body condition. In the previous
experiments of this thesis, the biostimulatory effect of bulls was shown to decrease
NEFA and increase leptin concentrations in cows after short-term (5h/d for 9d and 24h/d
for 30d; respectively) bull exposure. Taken together these results indicate a metabolic
component to the biostimulatory effect of bulls, specifically at the level of the adipocytes
in cows. However, little is known as to whether this component is influenced more by
the biostimulatory effect of bulls or simply by the resumption of luteal activity in the
cow. This begs the questions: “Is the alteration or increase associated with bull exposure
55
in temporal leptin concentrations an all-or-nothing response or is it dose-dependent and is
this change in temporal leptin concentrations influenced by cycling status”?. Leptin, an
adipocyte-derived hormone or adipokine, is secreted by white adipose tissue and is an
important factor on the control of energy balance, satiety, food intake and has been
shown to have many levels of modulation upon the hypothalamic-hypophyseal-gonadal
axis (for review see Tene-Sempere, 2007). In this study, our objective was to investigate
the effect of daily duration of bull exposure to alter the temporal leptin concentration
patterns during postpartum anestrus and the resumption of luteal activity. The specific
hypothesis tested in this experiment were that systemic concentrations of leptin do not
differ in postpartum, anestrous, suckled beef cows exposed to bulls for 12, 6, or 0 h daily.
Materials and Methods
This experiment was conducted at the Montana State University Bozeman Area
Research and Teaching Facility. Animal care, handling, and protocols used in this
experiment were approved by the Montana State University Institutional Agricultural
Animal Care and Use Committee.
Animals and Treatment
Thirty-nine, spring-calving, two-yr-old Angus X Hereford primiparous, suckled
beef cows and four mature epididymectomized Angus X Hereford bulls were used in this
experiment. Cows and calves were maintained in a single pasture and had no contact
with bulls or their excretory products from the previous breeding season until the start of
the experiment (D 0). Average calving date for these cows was Feb. 18, 2008. Before
56
the start of the experiment, cycling status of each cow was rated by two ultrasound
examinations of each ovary for the presence or absence of a corpus luteum. The first and
second ultrasonic examinations were conducted 10 and 2 d, respectively, before the start
of the experiment. Cows that did not exhibit the presence of a corpus luteum on either
ovary in both ultrasound examinations were used in this experiment.
The interval from calving to D 0 averaged 51.5 ± 2.3 d. Two d before the start of
the experiment cows were stratified by calving date, calf birth weight, dystocia score,
cow body weight, cow BCS, and sex of calf and assigned randomly to be exposed to bulls
for 12 h daily (BE12; n = 15), 6 h daily (BE6; n =14) or not exposed to bulls (NE; n = 10)
for 45 d.
Facilities and Daily Bull Exposure.
Cows were housed within pens in separate lot areas. Pens within the south lot
were used to maintain BE12 and BE6 cows while pens within the north lot were used to
maintain NE cows. A common holding pen, approximately 0.35 km from the lot that
housed NE cows and 30 m from the lot that housed BE12 and BE6 cows, was used to
house bulls before and after daily exposure periods. During daily exposure periods two
bulls were moved from the common holding pen into the pen that housed BE12 cows and
two bulls were moved into the pen that housed BE6 cows. Cows in each treatment were
exposed to bulls at 0700 h each day for 45 d (D 0 to 44). At 1900 and1300 h bulls were
removed from pens that housed BE12 and BE6 cows, respectively, and housed in the
common holding pen until the following day. Cows in each treatment could not see or
57
smell bulls before or after daily exposure periods. Figure 5 below illustrates pen
arrangements and method of daily bull exposure.
Figure 5. Facilities where cows were housed during experiment and schedule of daily
bull exposure.
Nutrition.
Cows had free access to good quality, chopped mixed-grass alfalfa hay, and any
pasture grasses that were available before the start of the experiment. Once cows and
calves were moved into pens they were given free access to the same hay, 0.5 kghd-1d1 cracked corn, water, and a trace mineral-salt supplement. Average body weight of
cows was 467.5 ± 37.7 kg. The TDN of the diet was 110% of the recommended energy
requirement for lactating beef cows with a mature weight of 533 kg (NRC, 1996). Bulls
were fed the same diet as cows.
58
Blood Sampling, Leptin and Progesterone
Concentrations, and Resumption of Luteal Activity.
Blood samples were collected from each cow by jugular venepuncture every other
day from D 0 to the end of the experiment (D 44). Serum concentrations of leptin were
determined in triplicate and were quantified by using a competitive liquid-liquid phase,
double-antibody leptin RIA procedure described previously (Delavaud et al., 2000). The
intra and inter assay coefficients of variation were 2.01% and 3.22%, respectively. Serum
was assayed for progesterone concentration in duplicate using solid-phase RIA kits
(Siemens Medical Solutions Diagnostics, Los Angeles, CA, USA) validated for bovine
serum in our laboratory (Custer et al., 1990). Intra- and interassay CV for a serum pool
that contained 2.2 ng/mL of progesterone were 10.2 and 15.4%, respectively, and 8.9 and
11.8%, respectively, for a pool that contained 5.75 ng/mL. Progesterone concentrations
in these samples were used to determine the interval from calving to resumption of luteal
activity (RLA), interval from the start of the experiment to RLA, and the proportion of
cows that RLA during the experiment. An increase of progesterone concentration, above
the average progesterone baseline of individual cows in three consecutive samples that
exceeded 1 ng/mL was used to determine the occurrence of RLA. Intervals from calving
and D 0 to RLA were determined by the number of days from the treatment to the lowest
inflection point before a rise in three consecutive samples that exceeded 1 ng/mL.
Progesterone concentrations and RLA were confirmed by transrectal ultrasonographic
examination of ovaries of each cow using a Titan ultrasound with a 7.5 to 10 MHz rectal
transducer every other day throughout the 45-d exposure period used in this experiment
59
(SonoSite Inc., Bothell, WA, USA). The presence of a corpus luteum in the same
anatomical position of an ovary in 4 successive scans was used as evidence to confirm
RLA. Cows that failed to exhibit a rise in progesterone over three consecutive samples
and did not have a corpus luteum in their ovaries were assigned an interval from calving
or the start of treatment to the end of the experiment.
Statistical Analyses.
Intervals from D 0 to RLA were evaluated by ANOVA for a completely
randomized design using PROC GLM of SAS (SAS, Cary, NC). The model included
treatment (TRT) and means were separated using Bonferroni Multiple Comparison tests.
Linear regression analyses were used to determine the relationship between intensity of
bull exposure (hours/d) and intervals (days) from calving and the start of the experiment
to RLA using the PROC REG procedure of SAS. Data for intervals from D 0 to RLA
showed heterogeneous variances among treatments using Bartlett’s Box F-test.
Therefore, data from the start of the experiment to RLA that were used in ANOVA were
transformed using the Empirical Method by intervals to the power of 10.3. Least squares
means and standard errors of means for intervals and D 0 to RLA, reported herein, were
transformed to original values after analysis. Differences in proportions of cows that
resumed OA during the experiment were analyzed by chi-square using the PROC FREQ
procedure of SAS.
Leptin concentrations were analyzed by PROC MIXED repeated measures
analysis of SAS. The model included TRT, Animal as experimental unit, Day as the
repeated measure, and the TRT by Interval interaction and means were separated using
60
Bonferroni Multiple Comparison tests. Linear regression analyses between leptin
concentrations and hours of daily bull exposure were conducted using PROC REG
procedure of SAS.
Results
Interval from D 0 to RLA was shorter (P < 0.05) for BE12 cows than for NE
cows, 30.6 d and 43.0 d, respectively. However, interval from D 0 to RLA did not differ
(P > 0.10) between BE6 cows, 35.0 d, and either BE12 or NE cows. The proportion of
BE12 and BE6 cows that RLA during the experiment did not differ (P > 0.10), 60 and
64.3%, respectively. The proportion of cows that RLA for either BE6 cows or BE12
cows were greater than for NE cows, 10% (Table 2).
61
Table 4. Number of cows per treatment and least squares means for intervals from
calving to resumption of luteal activity (RLA) and proportions of cows that RLA
during the experiment for primiparous, anestrous, suckled, beef cows exposed to bulls
for 12-h daily (BE12), 6-h daily (BE6), or not exposed to bulls (NE) for 45 d starting
51.5 ± 2.3 d (± SE) after calving.
Variable
n
Interval from D0
to RLA, db
NE
10
43.0y
Treatment a
BE6
14
35.0x,y
BE12
15
30.6x
SEM
P -Value
10.6
<0.05
Proportion that
10.0y
64.3x
60.0x
8.0c
<0.05
RLA, %
a
Means and proportions that lack common superscript differ (P < 0.05).
b
Cows that failed to RLA were assigned an interval from calving to the end of the
experiment.
c 2
X value.
There was a treatment by day interaction (P < 0.05) for leptin concentrations.
Temporal patterns of leptin concentrations differed among NE, BE6, and BE12 cows.
However, leptin concentrations tended to be greater (P = 0.09) in cows that RLA than
cows that did not RLA, indicating this interaction may be due to cycling status. In order
to better understand this treatment by day interaction, the experiment period was grouped
into four intervals; D 0 to 10, D 11 to 20, D 21 to 30, and D 31 to 40. Figure 6 illustrates
patterns of leptin concentrations by treatment, wherein leptin exhibits a similar pattern in
all levels of daily bull exposure. However, it appears that as the duration of bull exposure
increases, treatments exhibit leptin patterns at slightly higher levels, respectively.
62
NE
9
BE6
BE12
z
SEM
z
z
Leptin, ng/mL
8
y
7
6
y
y
y
x, y
y
y
x, y
x
5
0 to 10 d
11 to 20 d
21 to 30 d
31 to 40 d
Interval
Figure 6. Least squares means of leptin concentrations for 10-d intervals in cows
exposed to bulls for 0 (NE), 6 (BE6), and12 (BE12) h daily. Bars that lack common
letters within and between each interval differ, P < 0.05. Interval by treatment interaction,
P = 0.06; treatment interaction, P < 0.0001.
Bull exposed groups, both BE6 and BE12, had higher (P < 0.05) leptin
concentrations over the D 0 to 10 interval than NE cows. Leptin concentrations seemed
to increase through the first 20 d of the experiment and then level out. Furthermore,
leptin concentrations from D 11 to 20, D 21 to 30, and D 31 to 40 were greater (P < 0.05)
for BE12 cows than BE6 and NE cows. Again, there is the possibility that these effects
could be attributed to the proportion cycling within each treatment. However, when
samples taken after animals had resumed luteal activity were removed from the analysis,
there was a similar significant treatment by day interaction (P < 0.05) as well (Figure 7).
Again to better explain this interaction the experiment period was grouped into four
intervals; D 0 to 10, D 11 to 20, D 21 to 30, and D 31 to 40.
63
non-cycling NE
9
non-cycling BE6
z
non-cycling BE12
z
SEM
Leptin, ng/mL
8
y, z
y
y
7
6
y
x, y
y
y, z
x, y
y, z
x
5
0 to 10 d
11 to 20 d
21 to 30 d
31 to 40 d
Interval
Figure 7. Least squares means of leptin concentrations for 10-d intervals in cows
exposed to bulls for 0 (NE), 6 (BE6), and12 (BE12) h daily. Non-cycling NE, BE6 and
BE12 data are samples collected before the resumption of ovulatory activity. Bars that
lack common letters within and between each interval differ, P < 0.05. Interval by
treatment interaction, P = 0.08; treatment interaction, P < 0.0001.
Similarly, concentrations of leptin increased faster within the first 20 d than
through the rest of the experiment and were greater (P < 0.05) for bull exposed groups,
both BE6 and BE12, than for NE cows during the D 0 to 10 interval. Additionally, leptin
concentrations during intervals D 11 to 20 and D 21 to 30 were greater for BE12 cows
than both BE6 and NE cows. However, in this analysis of samples from cows before
they resumed luteal activity, leptin concentrations from D 21to 30 were greater (P < 0.05)
in BE12 than both BE6 and NE cows. However, it appears that during the D 31 to 40
interval treatments did not differ (P > 0.10). There was a tendency for leptin
concentrations to be greater (P = 0.09) in cows that had resumed luteal activity during the
experiment than cows that did not resume OA, 8.0 and 6.9 ng/mL, respectively.
64
Furthermore, there was a tendency for a linear relationship (b1 = 0.123 ng/h; P = 0.09)
between mean leptin concentrations and hours of daily bull exposure.
NE
BE6
BE12
70%
y
y
60%
y
%RLA
50%
40%
y
30%
x
20%
x
10%
0%
x
x
x
x
0 to 10 d
x
x
11 to 20 d
21 to 30 d
31 to 40 d
Interval
Figure 8. Percentages of primiparous, suckled cows exposed to bulls for 0 (NE), 6
(BE6), and12 (BE12) h daily that resumed luteal activity (%RLA) during intervals D 0 to
10, D 11 to 20, D 21 to 30, and D 31 to 40 of the experiment. Bars that lack common
letters within each interval differ, P < 0.05; interval D 0 to 10, 2 = 3.37, d.f. = 2;
interval D 11 to 20,2 = 2.84, d.f. = 2; interval D 21 to 30,2 = 6.38, d.f. = 2; ; interval
D 31 to 40,2 = 8.12, d.f. = 2 and the overall interaction, P < 0.05;2 = 382.7, d.f. =
11.
Discussion
The purpose of this study was to evaluate changes in leptin concentrations in
primiparous, postpartum, anovular, suckled, beef cows that were exposed to bulls for
durations of 0, 6, and 12 hours daily. Even though, mean leptin concentrations did not
65
differ among treatments, we found that there was a treatment by day interaction for leptin
concentrations. This data indicates that the temporal patterns of leptin differed among
cows exposed to bulls for 0, 6, and 12 hours daily.
Interestingly, across all treatments, leptin concentrations tended to be greater in
cows that had resumed luteal activity during the experimental period (D 0 to D 44) than
in cows that had not resumed luteal activity by the end of the experiment. Specifically,
cows that had resumed luteal activity during the experimental period had a mean leptin
concentration of 8.0 ng/mL, whereas cows that did not resume luteal activity by the end
of the experiment (D 44) had a mean leptin concentration of 6.9 ng/mL. This suggests
that the treatment by day interaction seen with leptin concentrations is perhaps more
directly related to the cycling status of the animal and indirectly influenced by the
biostimulatory effect of bulls to hasten the resumption of luteal activity in the postpartum
anestrous cow.
If cycling status is more responsible for mean leptin concentrations, one could
expect that the proportion of cows cycling would correlate to mean leptin concentrations.
However, we found a tendency for a positive linear relationship between the level of bull
exposure (0, 6, and 12 hours daily) and mean leptin concentrations, wherein the
proportion of cows exposed to bulls for 6 and 12 hours daily did not differ, 64.3 and
60.0% respectively; but were significantly greater than the 10% cycling of cows not
exposed to bulls. Though it is only a tendency, this relationship suggests the possibility
that the biostimulatory effect of bulls may have a direct influence on leptin
concentrations. In order to better understand the treatment by day interaction, the
66
experiment period was grouped into four periods; D 0 to 10, D 11 to 20, D 21 to 30, and
D 31 to 40. In Fig. 6, mean leptin concentrations of each period are represented for cows
exposed to bulls for 0, 6, and 12 hours daily. This illustrates a graded curve by duration
of bull exposure on leptin concentrations, wherein leptin exhibits a similar pattern in all
treatments. However, it seems that as the duration cows are exposed to a bull increases,
treatments indicate leptin patterns at slightly higher levels, respectively. Again, there is
the possibility that this could be attributed to the proportion cycling within each
treatment. However, when samples taken after animals had resumed ovulatory activity
were removed from the analysis, there was a similar tendency for a treatment by interval
interaction as well (Fig. 7). These interactions are further supported by the tendency for a
linear relationship between mean leptin concentrations and duration of daily bull
exposure.
In conclusion, daily bull exposure reduced the interval to resumption of luteal
activity and altered the temporal pattern of leptin concentrations in the postpartum,
anovular, suckled, beef cows. This suggests that the biostimulatory effect of bulls may
have a metabolic component. The results of this study suggest that leptin concentrations
are increased by both the biostimulatory effect of bulls and the resumption of luteal
activity in postpartum, suckled cows. Further investigations are necessary to explain how
the presences of bulls influence leptin; and to understand the role of changes in leptin
concentration patterns during the resumption of luteal activity in the postpartum,
primiparous, anestrous, suckled, beef cow.
67
CHAPTER 9
GENERAL DISCUSSION
In Experiment 1 of this thesis, we reported non-esterified fatty acids (NEFA) were
significantly lower in cows that were exposed to bulls than cows not exposed. If leptin
concentrations are high, this would be a signal of satiety to the brain and would be
concurrent with a decrease in the mobilization of NEFA (Tena-Sempere, 2007). T hese
results are complementary of the findings of Experiments 2 and 3 wherein we find that
bull exposure not only results in an increase temporal leptin concentrations, but it does so
in a dose-dependent manner. However, Landaeta-Hernandez et al., (2004) found that
NEFA concentrations were 33% higher in suckled cows exposed to bulls than in nonexposed cows. Perhaps this difference is due to the greater proportion in resumption of
luteal activity of cows that were exposed to bulls than cows not exposed to bulls as
reported by Landaeta-Hernandez et al., (2004).
The conclusions of Experiments 1, 2, and 3 of this thesis all suggest that there is a
metabolic component involved with the biostimulatory effect of bulls. Specifically, the
physical presence of bulls seems to decrease the mobilization of free-fatty acids (or
NEFA) and over time increase temporal leptin concentrations. One interpretation of
these results could be that within this metabolic component of the biostimulatory effect
there is a physiological change representative of an increasing energy state for the cow.
If so, it is possible that cows are “tricked” into perceiving a higher energy state than if
they were not exposed to bulls. The physiological advantage to this perception is the
68
potential to change LH pulsatility and hasten the resumption of luteal activity in the
postpartum anestrous cow through action of leptin on neural inputs and within the
hypothalamus; as described by Schneider, (2004).
In both Experiments 2 and 3, temporal leptin concentrations were increased
shortly after the start of bull exposure. Continuous (24h/d) bull exposure in Experiment 2
tended to increase temporal leptin concentrations by 18 to 20 d after the start of bull
exposure wherein 100% of cows exposed to bulls and 46% of non-exposed cows had
resumed luteal activity by 9 to 15 d from the start of bull exposure. Furthermore, in
Experiment 3 cows exposed to bulls for 12h/d (BE12) and 6h/d (BE6) had higher
temporal leptin concentrations than non-exposed (NE) cows within the first 10 d of bull
exposure and had mean intervals from the start of the experiment to the resumption of
luteal activity of 31, 35, and 43d; respectively. This indicates a definite relationship
between the biostimulatory effect of bulls to hasten the resumption of luteal activity
between the anestrous cow and changes in temporal leptin concentrations. There is the
possibility that the observed changes in leptin concentrations could be related simply to
cycling status, specifically the presence of progesterone in the blood. However, from the
analysis of non-cycling cows in Experiment 3 it appears that there is a dose-dependent
response to bull exposure in leptin concentrations in the absence of luteal activity.
Hypothesis for a Metabolic Component
or Pathway to the Bull Biostimulatory Effect of Bulls
Reproductive efficiency increases as the interval of postpartum anestrous
decreases. Exposing postpartum, anestrous cows to bulls accelerates the resumption of
69
ovulatory activity and may be used as a strategy to increase reproductive efficiency. The
mechanism by which bulls decrease the postpartum, anestrous interval may be related to
changes in metabolic regulation of adipose tissue. This is the first report wherein the
presence of bull alters a metabolic processes related to adipose metabolism in
postpartum, anestrous cows. The results suggest that leptin concentrations are increased
by both the biostimulatory effect of bulls and the resumption of ovulatory activity.
Further investigations are necessary to explain how the daily presence of bulls influences
the secretion of leptin, and to further understand the patterns of leptin concentrations
during the resumption of ovulatory activity in the postpartum, primiparous, anestrous,
suckled, beef cow.
The following diagram (adapted from Schneider, 2004; Tena-Sempere, 2007; and
Tauck, 2008) reviews the hypothetical relationship between bull biostimulation,
metabolic hormones, and the hypothalamic-pituitary-ovarian (HPO) axis.
70
The above diagram depicts the many metabolic endocrine pathways that can
influence the HPO, LH pulsatility, and ultimately cause the resumption of ovarian cycling
activity in postpartum, anestrous cows. Again from the literature review, the positive or
negative feedback by each of these hormones can change and is dependent upon the
energy state and the specific hormone milieu. However, it is clear that a change in
metabolic fuels, energy balance, and metabolic hormones can greatly influence the
function of the HPO and/or metabolic hormone sensitivity in effectors of GnRH neurons.
As aforementioned, energy homeostasis is a very tightly regulated system with many
redundant pathways and levels of modulation upon the HPO. However, it would be
advantageous for reproduction if it was possible that bull exposure stimulates the HPO
axis by “tricking” the cow’s perception of this system into that of a higher energy state.
Is it then possible that noted changes in adrenal secretion of cortisol (Tauck, 2008) in
cows under the biostimulatory effect sets into motion a permissive or positive influence
upon the effect by other metabolic hormones (Schneider, 2004 and Tena-Sempere, 2007)
on the HPO to ultimately alter LH pulsatility?
Further investigations are necessary to understand the potential role of
changes in leptin concentration patterns in hastening the resumption of luteal activity in
the postpartum, primiparous, anestrous, suckled, beef cow. In future research specific to
the influence of bull exposure on leptin concentrations, questions that must be asked are
1) “Is the change in temporal leptin concentrations due to an increase in secretion and
synthesis or to a decrease in its degredation”?; 2), “Does an increase in leptin during bull
exposure directly alter LH secretion patterns for the resumption of ovarian cycling
71
activity”?; and 3), “Does bull exposure change the expression or sensitivity of the Ob-R
(leptin receptor) within the HPO”? Further research could also investigate the effect of
bull exposure on other metabolic hormones such as insulin, ghrelin, IGF-1, etc.
72
LITERATURE CITED
Acosta, B., G.K. Tarnavsky, T.E. Platt, D.L. Hamernik, J.L. Brown, H.M. Schoenemann,
and J.J. Reeves. 1983. Nursing enhances the negative effect of estrogen on LH
release in the cow. J. Anim. Sci. 57:1530-1536.
Agarwal SK, Vogel K, Weitsman SR, Magofin DA. 1999. Leptin antagonizes the insulinlike growth factor augmentation of steroidogenesis in granulosa and thecal cells
of the human ovary. J. Clin. Endocrinol Metab. 84: 1072-1076
Ahima RS, Dushay J, Flier SN, Prabakaran D and Flier JS. 1997. Leptin accelerates the
onset of puberty in normal female mice Journal of Clinical Investigation 99 391395
Ahima RS, Saper CB, Flier JS, Elmquist JK. 2000. Leptin regulation of neuroendocrine
systems. Front Neuroendocronol. 21: 263-307.
Ahren B, Larsson H, Wilhelmsson C, Nasman B and Olsson T. 1997. Regulation of
Amstalden M, Garcia MR, Williams SW, Stanko RL, Nizielski SE, Morrison CD, Keisler
DH and Williams GL. 2000. Leptin gene expression, circulating leptin, and
luteinizing hormone pulsatility are acutely responsive to short-term fasting in
prepubertal heifers: relationships to circulating insulin and insulin-like growth
factor I. Biology of Reproduction 63 127-133
Anderson, K. A., J. G. Berardinelli, P.S. Joshi, and B. Robinson. 2002. Effects of
exposure to bull or excretory products of bulls on the breeding performance of
first-calf restricted suckled beef cows using a modified CO-synch protocol. Proc.
West. Sec. Amer. Soc. Anim. Sci. 53:457-460.
Archanco M, Muruzabal FJ, Llopiz D, Garayoa M, Gomez-Ambrosi J, Fruhbeck G,
Burrell MA. 2003. Leptin expression in the rat ovary depends on estrous cycle. J.
Histochem. Cytochem. 51: 1269-1277.
Arije, G. R., J. N. Wiltbank, and M. L. Hopwood. 1974. Hormone levels in pre- and
post-parturient beef cows. J. Anim. Sci. 39:338-347.
Bado A, Levasseur S, Attoub S, Kermorgant S, Laigneau JP, Bortoluzzi MN, Moizo L,
Lehy T, Guerre-Mille M, Marchand-Brustel Y and Lewin MJM. 1998. The
stomach is a source of leptin Nature 394 790-793
Barash IA, Cheung CC, Weigle DS, Ren H, Kabigting EB, Kuijper JL, Clifton DK and
Steiner RA. 1996. Leptin is a metabolic signal to the reproductive system
Endocrinology 137 3144-3147
73
Barkan D, Hurgin V, Dekel N, Amsterdam A, Rubinstein M. 2005. Leptin induces
ovulation in GnRH-deficient mice. FASEBJ. 19:133-135.
Bartle, S. J., J. R. Males, and R. L. Preston. 1984. Effect of energy intake on the
postpartum interval in beef cows and the adequacy of the cow’s milk production
for calf growth. J. Anim. Sci. 58:1068.
Baruah, K. K. and L. N. Kanchev. 1993. Hormonal response to olfactory stimulation
with bull urine in postpartum dairy cows. Proc. VII World Conf. Anim. Prod.
3:356-361.
Beal, W.E., R.E. Short, R.B. Staigmiller, R.A. Bellows, C.C. Kaltenbach, and T. G.
Dunn. 1978. Influence of dietary energy intake on bovine pituitary and luteal
function. J. Anim. Sci. 46:181-188.
Beam, S.W., and W.R. Butler.1997. Energy balance and ovarian follicle development
prior to the first ovulation postpartum in dairy cows receiving three levels of
dietary fat. Biol. Reprod. 56:133-142.
Bellows, R.A. and R.E. Short. 1978. Effects of precalving feed level on birth weight,
calving difficulty and subsequent fertility. J. Anim. Sci. 46:1522-1528.
Bennett PA, Lindell K, Karlsson C, Robinson ICAF, Carlsson LMS and Carlsson B.
1998. Differential expression and regulation of leptin receptor isoforms in the rat
brain: effects of fasting and oestrogen Neuroendocrinology 67 29-36
Berardinelli, J. G., M S. Roberson, and R. Adair. 1987. Shortening the anestrous period.
Montana Ag Research 4:25-28.
Berardinelli, J. G., P. S. Joshi, and S. A. Tauck. 2004. Breeding performance of firstcalf suckled beef cows exposed to familiar or unfamiliar bulls using a modified
CO-Synch protocol. Postpartum interval in first-calf suckled beef cows exposed
to familiar or unfamiliar bulls. Proc. West. Sec. Amer. Soc. Anim. Sci. 55:291293.
Berardinelli, J. G., P. S. Joshi., and S. A Tauck. 2005. Postpartum resumption of
ovarian cycling activity in first-calf suckled beef cows exposed to familiar or
unfamiliar bulls. Anim. Reprod. Sci. 90:201-209.
Bishop D.K., R.P. Wettemann, and L.J. Spicer. 1994. Body energy reserves influence the
onset of luteal activity after early weaning of beef cows. J. Anim. Sci. 72:27032708.
Burns, P.D. and J.C. Spitzer. 1992. Influence of biostimulation on reproduction in
postpartum beef cows. J. Anim. Sci. 70:358-362.
74
Caldani M, Caraty A, Pelletier J, Thiery JC and Tillet Y. 1993. LH pulsatile release and
its control. In Reproduction in Mammals and Man pp 80-96 Eds C Thibault, MC
Levasseur and RHF Hunter. Ellipses, Paris
Carruthers, T. D. and H. D. Hafs. 1980a. Suckling and four-times daily milking:
influence on ovulation, estrus and serum luteinizing hormone, glucocorticoids and
prolactin in postpartum holsteins. J. Anim. Sci. 50:919-925.
Carruthers, T.D., E.M. Convey, J.S. Kesner, H.D. Hafs, and K.W. Cheng. 1980b. The
hypothalamic-pituitary gonadotrophic axis of suckled and nonsuckled dairy cows
postpartum. J. Anim. Sci. 51:949-957.
Carter, M. L., D. J. Dierschke, J. J. Rytledge, and E. R. Hauser. 1980. Effect of
gonadotropin-releasing hormone and calf removal on pituitary-ovarian function
and reproductive performance in postpartum beef cows. J. Anim. Sci. 51:903.
Casanueva FF, Dieguez C. 1999. Neuroendocrine regulation and actions of leptin. 20:
317-363
Castenson, P. E., A.M. Sorensen Jr., C. R. Cobos, and J. L. Fleeger. 1976. Source of
-OHP preceding estrus in heifers. J. Anim. Sci.43:277.
Chang, C. H., T. Gimenez, and D. M. Henricks. 1981. Modulation of reproductive by
suckling and exogenous gonadal hormones in young beef cows post-partum. J.
Reprod. Fertil. 63: 31-38.
Chehab FF, Mounzih K, Lu R and Lim ME. 1997. Early onset of reproductive function in
normal female mice treated with leptin Science 275 88-90
Cheung CC, Clifton DK and Steiner RA. 1997. Proopiomelanocortin neurons are direct
targets for leptin in the hypothalamus Endocrinology 138 4489-4492
Cheung CC, Thornton JE, Kuijper JL, Weigle DS, Clifton DK, Steiner RA. 1997. Leptin
is a metabolic gate for the onset of puberty in the female rat. Endocrinology 138
855-858
Connor, H.C., P.L. Houghton, R.P. Lemenager, P.V. Malven, J.R. Parfet, and G.E. Moss.
1990. Effect of dietary energy, body condition and calf removal on pituitary
gonadotropins, gonadotropin-releasing hormone (GnRH) and hypothalamic
opioids in beef cows. Domest. Anim. Endocrinol. 7:403-411.
Corah, L. R., A. P. Quealy, T. G. Dunn, and C. C. Kaltenbach. 1974. Prepartum and
postpartum levels of progesterone and estradiol in beef heifers fed two levels of
energy. J. Anim. Sci.39:380-385.
75
Craig J, Zhu H, Dyce PW, Petrik J, Li J. 2004. Leptin enhances oocyte nuclear and
cytoplasmatic maturation via the mitogen-activated protein kinase pathway.
Endocrinology. 145: 5355-5363
Crowe, M. A., V. Padmanabhan, M. Mihm, I. Z. Beitins, and J. F. Roche. 1998.
Resumption of follicular waves in beef cows is not associated with periparturient
changes in follicle-stimulating hormone heterogeneity despite major changes in
steroid and luteinizing hormone concentrations. Biol. Reprod. 58:1445-1450.
Cupp, A.S., M.S. Roberson, T.T. Stumpf, M.W. Wolfe, L.A. Werth, N. Kojima, R.J.
Kittok, J.E. Kinder.1993. Yearling bulls shorten the duration of postpartum
anestrus in beef cows to the same extent as do mature bulls. J. Anim. Sci. 71:306309.
Custer, E. E., J. G. Berardinelli, R. E. Short, M. Wehrman, and R. Adair. 1990.
Postpartum interval to estrus and patterns of LH and progesterone in first-calf
suckled beef cows exposed to mature males. J. Anim. Sci. 668:1370-1377.
Daley, C. A., H. Sakurai, B. M. Adams, and T. E. Adams. 2000. Effect of stress-like
concentrations of cortisol on feedback potency of oestradiol in orchidectomized
sheep. Anim. Reprod. Sci. 59:167-178.
Day ML, Imakawa K, Zalesky DD, Kittok RJ and Kinder JE. 1986. Effects of restriction
of dietary energy intake during the prepubertal period on secretion of luteinizing
hormone and responsiveness of the pituitary to luteinizing hormonereleasing
hormone in heifers Journal of Animal Science 62 1641-1649
DeFries, C.A., D.A. Neuendorff, and R.D. Randel. 1998. Fat supplementation influences
postpartum reproductive performance in Brahman cows. J. Anim. Sci. 76:864870.
Delavaud C, Bocquier F, Chilliard Y, Keisler DH, Gertler A and Kann G. 2000. Plasma
leptin in ruminants: effect of nutritional status and body fatness on plasma leptin
concentration assessed by a specific RIA in sheep Journal of Endocrinology 165
519-526
DeRouen, S.M. , D.E. Franke, D.G. Morrison, W.E. Wyatt, D.F. Coombs, T.W. White,
P.E. Humes, and B.B. Greene. 1993. Prepartum body condition and dietary
energy on pre- and postpartum performance of beef heifers. J. Anim. Sci. 71
(Suppl. 1): 236.
DeRouen, S.M., D.E. Franke, D.G. Morrison, W.E. Wyatt, D.F. Coombs, T.W. White,
P.E. Humes and B.B. Greene. 1994. Prepartum body condition and weight
influences on reproductive performance of first-calf beef cows. J. Anim. Sci. 72:
1119-1125.
76
Ducker, M.J., R.A. Haggett, W.J. Fisher, S.V. Morant, and G.A. Bloomfield. 1985.
Nutrition and reproductive performance of dairy cattle. I. The effect of level of
feeding in late pregnancy and around the time of insemination on the reproductive
performance of first lactation dairy heifers. Anim. Prod. 41:1-12.
Duggal PS, van der Hoek, KH, Milner CR, Ryan NK, Armstrong DT, Magoffin DA,
Norman RJ. (2000). The in vivo and in vitro effects of exogenous leptin on
ovulation in the rat. Endocrinology. 141: 1971-1976.
Dunn, R.T., Jr., M.F. Smith, H.A. Garverick, and C.W. Foley. 1985. Effect of 72 hr calf
removal and / or gonadotropin releasing hormone on luteinizing hormone release
and ovarian activity in postpartum beef cows. Theriogenology 23:767.
Dunn, T.G. and C.C. Kaltenbach. 1980. Nutrition and the postpartum interval of the ewe,
sow, and cows. J. Anim. Sci. 51 (Suppl. 2): 29.
Dunn, T.G., J.E. Ingalls, D.R. Zimmerman, and J.N. Wiltbank.1969. Reproductive
performance of 2-year-old Hereford and Angus heifers as influenced by pre- and
post-calving energy intake. J. Anim. Sci. 29:719-726.
Ebert, J. J., P. Conteras, and P. Saelzer. 1972. Influence of teaser bull on puerperium
and fertility in dairy cows. VII Int. Cong. Anim. Reprod. Artif. Insem. Munich.
3:1743-1748.
Ehrhardt RA, Slepetis RM, Siegal-Willott J, van Amburgh ME, Bell AW and Boisclair
YR. 2000. Development of a specific radioimmunoassay to measure physiological
changes of circulating leptin in cattle and sheep Journal of Endocrinology 166
519-528
Falk, D.G., R.E. Christian, R.C. Bull, and R.G. Sasser. 1975. Prepartum energy effects on
cattle reproduction. J. Anim. Sci. 41:267.
Fernandes, L. C., W. W. Thatcher, C. J. Wilcox, and E. P. Call. 1978. LH release in
response to GnRH during the postpartum period of dairy cows. J. Anim. Sci.
46:443.
Fernandez, D. L., J. G. Berardinelli, R. E. Short, and R. Adair. 1996. Acute and chronic
changes in luteinizing hormone secretion and postpartum interval to estrus in
first-calf suckled beef cows exposed continuously or intermittently to mature
bulls. J. Anim. Sci. 74:1098-1103.
Fernandez, D.L, J.G. Berardinelli, R.E. Short and R .Adair.1993. The time required for
the presence of bulls to alter the interval from parturition to resumption of ovarian
activity and reproductive performance in first-calf suckled beef cows.
Theriogenology 39:411-419.
77
Finn PD, Cunningham MJ, Pau KF, Spies HG, Clifton DK and Steiner RA. 1998. The
stimulatory effect of leptin on the neuroendocrine reproductive axis of the
monkey Endocrinology 139 4652-4662
Fortune, J. E., J. Sirois, A. M. Turzillo, and M. Lavoir. 1991. Follicle selection in
domestic ruminants. J. Reprod. Fertil. Suppl. 43:187-198.
Frederich RC, Haman A, Anderson S, Lollmann B, Lowell BB and Flier JS. 1995. Leptin
levels reflect body lipid content in mice: evidence for diet-induced resistance to
leptin action Nature Medicine 1 1311-1314
Friedman JM and Halaas JL. 1998. Leptin and the regulation of body weight in mammals
Nature 395 763-770
Garcia MR, Amstalden M, Keisler DH, Raver N, Gertler A and Williams GL. 2002b.
Leptin attenuates the central effects of neuropeptide-Y on somatotropin but not
gonadotropin secretion in cows Journal of Animal Science 80 (Supplement 1) 352
Abstract 1411
Garcia MR, Amstalden M, Williams SW, Stanko RL, Morrison CD, Keisler DH,
Nizielski SE and Williams GL. 2002a. Serum leptin and its adipose gene
expression during pubertal development, the estrous cycle, and different seasons
in cattle Journal of Animal Science 80 2158-2167
Garcia-Winder, M., K. Imakawa, M. L. Day, D. D. Zalesky, R.J. Kittok, and J. E. Kinder.
1984. Effect of suckling and ovariectomy on the control of luteinizing hormone
secretion during the postpartum period of cows. Biol. Reprod. 31:771-778.
Garcia-Winder, M., K. Imakawa, M. L. Day, D. D. Zalesky, R.J. Kittok, and J. E. Kinder.
1986. Effects of suckling and low doses of estradiol on luteinizing hormone
secretion during the postpartum period in beef cows. Domest. Anim. Endocrinol.
3:79-85.
Gazal OS, Leshin LS, Stanko RL, Thomas MG, Keisler DH, Anderson LL and Williams
GL. 1998. Gonadotropin-releasing hormone secretion into third-ventricle
cerebrospinal fluid of cattle: correspondence with the tonic and surge release of
luteinizing hormone and its tonic inhibition by suckling and neuropeptide Y
Biology of Reproduction 59 676-683
Ghizzoni L, Barreca A, Mastorakos G, Furlini M, Vottero A, Ferrari B, Chrousos GP,
Bernasconi S. 2001. Leptin inhibits steroid biosynthesis by human granulosalutein cells. Horm. Metab. Res. 33: 323-328.
Gombe, S., and W. Hansel. 1973. Plasma luteinizing hormone (LH) and progesterone
levels in heifers on restricted energy intakes. J. Anim. Sci. 37:728-733.
78
Gong, J. G., B. K. Campbell, T. A. Bramley, C. G. Gutierrez, A.R. Peters, and R. Webb.
1996. Suppression in the secretion of follicle-stimulating hormone and
luteinizing hormone, and ovarian follicular development in heifers continuously
infused with a gonadotrophin-releasing hormone agonist. Biol. Reprod. 55:68-74.
Gong, J. G., T. A. Bramley, C. G. Gutierrez, A.R. Peters, and R. Webb. 1995. Effects of
a chronic treatment with a gonadotrophin-releasing hormone agonist on peripheral
concentrations of FSH and LH, and ovarian function in heifers. J. Reprod. Fertil.
105:263-270.
Grimard B., P. Humblot, J.P. Mialot, N. Jeanguyot, D. Sauvant, and M. Thibier. 1997.
Absence of response to estrus induction and synchronization treatment is related
to lipid mobilization in suckled beef cows. Reprod. Nutr. Dev. 37:129-140.
Guedon L., J. Saumande, and B. Desbals. 1999. Relationships between calf birth weight,
prepartum concentrations of plasma energy metabolites and resumption of
ovulation postpartum in Limousine suckled beef cows. Theriogenology 52:779789.
Hadley, M.E., and Levine, J.E. 2006. Adrenal steroid hormones. In Endocrinology..
Sixth ed. Pages 337-365, Pearson Prentice Hall, New Jersey, USA.
Hafez, E. S. and E., B. Hafez. 2000. Reproduction in farm animals. Seventh ed. Page 55
in chapter 4. Lippincott Williams and Wilkins, Baltimore, MD.
Henricks, D.M., and J.D. Rone. 1986. A note on the effect of nutrition on ovulation and
ovarian follicular populations in the individually fed post-partum beef heifer.
Anim. Prod. 557-560.
Henry BA, Goding JW, Alexander WS, Tilbrook AJ, Canny BJ, Dunshea F, Rao A,
Mansell A and Clarke IJ. 1999. Central administration of leptin to ovariectomized
ewes inhibits food intake without affecting the secretion of hormones from the
pituitary gland: evidence for a dissociation of effects on appetite and
neuroendocrine function Endocrinology 140 1175-182
Henry BA, Goding JW, Tilbrook AJ, Dunshea FR and Clarke IJ. 2001.
Intracerebroventricular infusion of leptin elevates the secretion of luteinizing
hormone without affecting food intake in long-term food-restricted sheep, but
increases growth hormone irrespective of bodyweight Journal of Endocrinology
168 67-77
Hoggard N, Hunter L, Duncan JS, Williams LM, Thayhurn P and Mercer JG. 1997.
Leptin and leptin receptor mRNA and protein expression in the murine fetus and
placenta Proceedings of the National Academy of Sciences USA 94 11073-11078
79
Hornbuckle, II, T, R.S. Ott, M.W. Ohl, G.M. Zinn, P.G. Weston, and J.E. Hixon. 1995.
Effects of bull exposure on the cyclic activity of beef cows. Theriogenology
43:411-418.
Houghton, P.L., R.P. Lemenager, L.A. Horstman, K.S. Hendrix, and G.E. Moss. 1990.
Effects of body composition, pre- and postpartum energy level and early weaning
on reproductive performance of beef cows and preweaning calf gain. J. Anim. Sci.
68:1438-1446.
Houseknecht KL and Portocarrero CP. 1998. Leptin and its receptors: regulators of
whole-body energy homeostasis Domestic Animal Endocrinology 15 457-475.
Humphrey, W.D., C. C. Kaltenbach, T. G. Dunn, D. R. Koritnik, and G. D. Niswender.
1983. Characterization of hormonal patterns in the beef cow during postpartum
anestrus. J. Anim. Sci. 56:445-453.
I’Anson H, Manning JM, Herbosa CG, Pelt J, Friedman CR, Wood RI, Bucholtz DC and
Foster DL 2000 Central inhibition of gonadotropin-releasing hormone secretion in
the growth-restricted hypogonadotropic female sheep Endocrinology 141 520-527
Jagger, J. P., A. R. Peters, and G. E. Lamming. 1987. Hormone responses to low-dose
GnRH treatment in post-partum beef cows. J. Reprod. Fert. 80:263.
Kadokawa H and Yamada Y (1999) Enhancing effect of acute fasting on ethanol
suppression of pulsatile luteinizing hormone release via an estrogen-dependent
mechanism in holstein heifers Theriogenology 51 673-680.
Karlsson C, Lindell K, Svenson E, Bergh C, Lind P, Billig H, Carlsson LM, Carlsson B.
1997. Expression of functional leptin receptors in the human ovary. J Clin
Endocrinol Metab. 82: 4144-4148.
Kendall NR, Gutierrez CG, Scaramuzzi RJ, Baird DT, Webb R, Campbell. 2004. Direct
in vivo effects of leptin on ovarian steroidogenesis in sheep. Reproduction. 128:
757-765.
Khireddine, B., B. Grimard, A.A. Ponter, C. Ponsart, H. Boudjenah, J.P. Mialot, D.
Sauvant, and P. Humblot. 1998. Influence of flushing on LH secretion, follicular
growth and the response to estrus synchronization treatment in suckled beef cows.
Theriogenology 49:1409-1423.
Kieborz-Loos, K. R., H. A. Garverick, D. H. Keisler, S. A. Hamilton, B. E. Salfen, R. S.
Youngquist, and M. F. Smith. 2003. Oxytocin-induced secretion of
prostaglandin F2α in postpartum beef cows: Effects of progesterone and estradiol17-β treatment. J. Anim. Sci. 81:1830-1836.
80
Kikuchi N, Andoh K, Abe Y, Yamada K, Mizunuma H, Ibuki Y. 2001. Inhibitory action
of leptin on early follicular growth differs in immature and adult female mice.
Biol Reprod. 65:66-71.
La Voie, V., D. K. Han, D. B. Foster, and E. L. Moody. 1981. Suckling effect on estrus
and blood plasma progesterone in postpartum beef cows. J. anim. Sci. 52:802.
Lamb, G. C., J. M. Lynch, D. M. Grieger, J. E. Minton, and J. S. Stevenson. 1997. Adlibitum suckling by unrelated calf in the presence or absence of a cow’s own calf
prolongs postpartum anovulation. J. Anim. Sci. 75:2762-2769.
Lammoglia, M.A., S.T. Willard, J.R. Oldham, and R.D. Randel. 1996. Effects of dietary
fat and season on steroid hormone profiles before parturition and on hormonal,
cholesterol, triglycerides, follicular patterns, and postpartum reproduction in
Brahman cows. J. Anim. Sci. 74:2253-2262.
Landaeta-Hernandez, A. J., M. Giangreco, P. Melendez, J. Bartolome, F. Bennet, D. O.
Rae, and L. F. Archbald. 2004. Effect of biostimulation on uterine involution,
early ovarian activity and first postpartum estrous cycle in beef cows.
Therigenology 61:1521-1532.
Landaeta-Hernandez, A. J., P. Melendez, J. Bartolome, D. O. Rae, and L. F. Archbald.
2006. Effect of biostimulation on the expression of estrus in postpartum Angus
cows. Theriogenology 66:710-716.
Laster, D. B., H. A. Glimp, and K. E. Gregory. 1973. Effects of early weaning on
postpartum reproduction of cows. J. Anim. Sci. 36:734.
Loffler S, Aust G, Kohler U, Spanel-Borrowski K. 2001. Evidence of leptin expression in
normal and polycystic human ovaries. Mol Hum Reprod. 7: 1143-1149.
Machinal F, Dieudonne MN, Leveveu MC, Pecquery R and Giudicelli Y. 1999. In vivo
and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for
a regional specific regulation by sex steroid hormones Endocrinology 140 15671574
Maciel MN, Zieba DA, Amstalden M, Keisler DH, Neves J and Williams GL. 2003.
Recombinant oleptin prevents fasting-mediated reductions in pulsatile LH release
and stimulates GH secretion in peripubertal heifers Proceedings of the MidWest
Section of the American Society of Animal Science 66 Abstract 263
Macmillan, K. L., A. J. Allison, and G.A. Struthers. 1979. Some effects of running bulls
with suckling cows or heifers during the premating period. New Zealand J. Exp.
Agr. 7:121.
81
Magni P, Vettor R, Pagano C, Calcagno A, Beretta E, Messi E, Zanisi M, Martini L and
Motta M. 1999. Expression of a leptin receptor in immortalized
gonadotropinreleasing hormone-secreting neurons Endocrinology 140 1581-1585
Mann,G. E. and G. E. Lamming. 2000. The role of sub-optimal preovulatory oestradiol
secretion in the aetiology of premature luteolysis during the short oestrous cycle
in the cow. Anim. Reprod. Sci. 64:171-180.
Marchlewska-Koj, A., and M. Zacharczuk-Kakietek. 1990. Acute increase in plasma
corticosterone level in female mice evoked by pheromones. Physiol. Behav.
48:577-580.
McCann JP and Hansel W. 1986. Relationships between insulin and glucose metabolism
and pituitary-ovarian functions in fasted heifers Biology of Reproduction 34 630641
McNeilly, A.S. 1988. Suckling and the control of gonadotropin secretion. Page 2323 in
The Physiology of Reproduction. E. Knobil and J Neill, ed., Raven Press, New
York.
McShane TM, May T, Miner JL and Keisler DH. 1992. Central actions of neuropeptideY may provide a neuromodulatory link between nutrition and reproduction
Biology of Reproduction 46 1151-1157
McShane TM, Petersen SL, McCrone S and Keisler DH. 1993. Influence of food
restriction on neuropeptide-Y, proopiomelanocortin, and luteinizing
hormonereleasing hormone gene expression in sheep hypothalami Biology of
Reproduction 49 831-839
Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Morgan PJ and
Trayhurn P. 1996. Coexpression of leptin receptor and preproneuropeptide Y
mRNA in arcuate nucleus of mouse hypothalamus Journal of Neuroendocrinology
8 733-735
Mora, O. A., and J. E. Sanchez-Criado. 2004. Involvement of the corticoadrenal
hormones in the pheromonal restoration activity in aging rats. Life Sciences
74:3285-3290.
Morash B, Li A, Murphy PR, Wilkinson M and Ur E. 1999. Leptin gene expression in the
brain and pituitary gland Endocrinology 140 5995-5998
Moss, G. E., J. R. Parfet, C. A. Marvin, R. D. Allrich, and M. A. Diekman. 1985.
Pituitary concentrations of gonadotropins and receptors for GnRH in suckled beef
cows at various intervals after calving. J. Anim. Sci. 60:285-293.
82
Mounzih K, Lu R and Chehab FF. 1997. Leptin treatment rescues the sterility of
genetically obese ob/ob males Endocrinology 138 1190-1193
Naasz, C.D., and H.L. Miller. 1990. Effects of bull exposure on postpartum interval and
reproductive performance in beef cows. Can. J. Anim. Sci.70:537-542.
Nagatani S, Zeng Y, Keisler DH, Foster DL and Jaffe CA. 2000. Leptin regulates
pulsatile luteinizing hormone and growth hormone secretion in the sheep
Endocrinology 141 3965-3975
Nersesjan, S. S. 1962. The use of vasectomized bulls as biological stimulators in
controlling infertility in cows. Anim. Breed. Abstr. 30:48.
Nett, T. M., D. Cermak, T. Braden, J. Manns, and G. Niswender. 1988. Pituitary
receptors for GnRH and estradiol, and pituitary content of gonadotropins in beef
cows. II. Changes during the postpartum period. Domest. Anim. Endocrinol.
5:81-89.
NRC. 1996. 7th revised (ed.) Nutrient Requirements of Beef Cattle. Natl. Acad. Press,
Washington, DC. Page 227.
Odde, K. G., H. S. Ward, G. H. Kirakofe, R. M. Mckee, and R. J. Kittok. 1980. Short
estrous cycles and associated serum progesterone levels in beef cows.
Theriogenology 14:105.
Olatinwo MO, Bhat GK, Stah, CD, Mann DR. 2005. Impact of gonadotropin
administration on folliculogenesis in prepubertal ob/ob mice. Mol Cell
Endocrinol. 245:121-127
Padmanabhan V, Karsch FJ and Lee JS. 2002. Hypothalamic, pituitary and gonadal
regulation of FSH Reproduction 59 (Supplement) 67-82
Perez-Hernandez, P., M. Garcia-Winder, and J. Gallegos-Sanchez. 2002. Bull exposure
and an increased within-day suckling interval reduced postpartum anoestrous in
dual purpose cows. Anim. Repro. Sci. 74:111-119.
Perry, R.C., L.R. Corah, R.C. Cochran, W.E. Beal, J.S. Stevenson, J.E. Minton, D.D.
Simms, and J.R. Brethour. 1991. Influence of dietary energy on follicular
development, serum gonadotropins and first postpartum ovulation in suckled beef
cows. J. Anim. Sci. 69:3762-3773.
Peters, A. R. and G.E. Lamming. 1990. Lactational anestrus in farm animals. In
Milligan S.R. (ed.), Oxford Reviews of Reproductive Biology, Vol. 12, Pages
245-288. New York: Oxford University Press.
83
Ramirez-Godinez, J. A., G. H. Kirakofe, R. R. Schalles, and G. D. Niswender. 1982.
Endocrine patterns in the postpartum beef cows associated with weaning: a
comparison of the short and subsequent normal cycles. J. Anim. Sci. 55:153-158.
Randel, R. D. 1981. Effects of once-daily suckling on postpartum interval and cow-calf
performance of first-calf Brahman X Hereford heifers. J. Anim. Sci. 53:755 -757.
Rawling, N. C., L. Weir, B. Todd, J. Manns, and J.H. Hyland. 1980. Some endocrine
changes associated with the postpartum period of the suckling beef cow. J.
Reprod. Fertil. 60:301-308.
Reeves, J. J. and C. T. Gaskins. 1981. Effect of once-a-day nursing on rebreeding
efficiency of beef cows. J. Anim. Sci. 53:889.
Rhodes, F. M., L. A. Fitzpatrick, K. W. Entwistle, and J. E. Kinder. 1995. Hormone
concentrations in caudal vena cava during the first ovarian follicular wave of the
oestrous cycle in heifers. J. Reprod. Fertil. 104:33-39.
Richards, M. W., J. C. Spitzer, and M.B. Warner. 1986. Effect of varying levels of
postpartum nutrition and body condition at calving on subsequent reproductive
performance in beef cattle. J. Anim. Sci. 62:300.
Rosenbaum M, Leibel RL. (1998). Leptin; a molecule integrating somatic energy stores,
energy expenditure and fertility. Trends Endocrinol Metab. 9: 117-124
Ryan NK, Woodhouse CM, van der Hoek KH, Gilchrist RB, Armstrong DT, Norman RJ.
(2002). Expression of leptin and its receptor in the murine ovary: possible role in
the regulation of oocyte maturation. Biol Reprod. 66:1548-1554.
Ryan, D.P., R.A. Spoon, M.K. Griffith, and G.L. Williams. 1994. Ovarian follicular
recruitment, granulose cell steroidogenic potential and growth hormone/insulinlike growth factor-I relationships in suckled beef cows consuming high lipid diets:
effects of graded differences in body condition maintained during the puerperium.
Domest. Anim. Endocrinol. 11:161-174.
Ryan, van der Hoek KH, Robertson SA, Norman RJ. 2003. Leptin and leptin receptor
expression in the rat ovary. Endocrinology. 144:5006-5013.
Sahu A. 1998. Leptin decreases food intake induced by melanin-concentrating hormone
(MCH), galanin (GAL) and neuropeptide Y (NPY) in the rat Endocrinology 139
4739-4742
Sahu A. 2004. A hypothalamic role in energy balance with special emphasis on leptin.
Endocrinology. 145: 2613-2620.
SAS Institute Inc. 2004. SAS Version 9.0. SAS Institute Inc., Cary, NC, USA.
84
Savio, J. D., M. P. Boland, N. Hynes, and J. F. Roche. 1990. Resumption of follicular
activity in the early postpartum period of dairy cows. J. Reprod. Fertil. 88:569579.
Savio, J. D., W. W. Thatcher, L. Badinga, R. L. Sota, and D. Wolfenson. 1993.
Regulation of dominant follicle turnover during the oestrous cycle in cows. J.
Reprod. Fertil. 97:197-203.
Scott, I. C. and G. W. Montgomery. 1987. Introduction of bulls induces return of cyclic
ovarian functioning post-partum beef cows. New Zealand J. Agr. Res. 30:189194.
Short, R. E., R. A. Bellows, R. B. Staigmiller, D. C. Adams, and J. G. Berardinelli. 1994.
Effects of suckling on postpartum reproduction. In: M. J. Fields, And R.S. Sand
RS (ed.), Factors Affecting Calf Crop, Pages 179-187. CRC Press, Boca Rotan,
FL.
Short, R. E., R. D. Randel, R. B. Staigmiller, and R. A. Bellows. 1990. Physiological
mechanisms controlling anestrus and infertility in postpartum beef cattle. J.
Anim. Sci. 68:799-816.
Sipilov, V. S. 1966. The use of males teasers in cattle breeding. Anim. Breed. Abstr.
35:244.
Smith JT, Acohido BV, Clifton DK, Seiner RA. 2006. KiSS-1 neurons are direct targets
fro leptin in the ob/ob mouse. J. Neuroendocrinol. 18: 298-303.
Smith, L. E. and C. K. Vincent. 1972. Effects of early weaning and exogenous hormone
treatment on bovine postpartum reproduction. J. Anim. Sci. 35:1228.
Spicer LJ, Fransisco CC. 1991. The adipose obese gene producte, leptin: evidence of a
direct inhibitory role in ovarian function. Endocrinology. 138: 3374-3379.
Spitzer, J.C., D.G. Morrison, R.P. Wettemann, and L.C. Faulkner. 1995. Reproductive
responses and calf birth and weaning weights as affected by body condition at
parturition and postpartum weight gain in primiparous beef cows. J. Anim. Sci.
73:1251-1257.
Stackpole, C. A., I. J. Clarke, K. M. Breen, A. I. Turner, F. J. Karsch, and A. J. Tilbrook.
2006. Sex difference in the suppressive effect of cortisol on pulsatile secretion of
luteinizing hormone in sheep. Endocrinology 147: 5921-5931.
Stagg, K., L. J. Spicer, J. M. Sreenan, J. F. Roche, and M. G. Diskin. 1998. Effect of
calf isolation on follicular wave dynamics, gonadotropin and metabolic hormone
changes, and interval to first ovulation in beef cows fed either of two energy
levels postpartum. Biol. Reprod. 59:777-783.
85
Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft L, Hale J,
Hoffman J, Hsiung HM, Kriauciunas A, MacKellar W, Rosteck PR Jr., Schoner
B, Smith D, Tinsley FC, Zhang XY and Heiman M. 1995. The role of
neuropeptide Y in the antiobesity action of the obese gene product Nature 377
530-532
Stevenson, J. S. and J. H. Britt. 1979. Relationship among luteinizing hormone,
estradiol, progesterone, glucocorticoids,milk yield,body weight and postpartum
ovarian activity in Holstein cows. J. Anim. Sci. 48:570-577.
Stumpf, T. T., M. W. Wolfe, P. L. Wolfe, M. L. Day, R. J. Kittok, and J. E. Kinder.
1992. Weight changes prepartum and presence of bulls postpartum interact to
affect duration of postpartum anestrus in cows. J. Anim. Sci. 70:3133-3137.
Tauck, S. A. 2008. The biostimulatory effect of bulls on the hypothalamic-pituitaryadrenal and –ovarian axis and on temporal aspects of resumption of ovarian
cycling activity in primiparous, postpartum, anestrous, suckled, beef cows. PhD
Dissertation, Montana State University, Bozeman, MT
Tauck, S. A., J. R. Olsen, J. G. Berardinelli. 2007. Adrenal involvement in the
biostimulatory effect of bulls. Reprod. Biol. Endo. 5: 33
Tilbrook, A.J., B.J. Canny, M.D. Serapiglia, T.J. Ambrose, and I.J. Clarke. 1999.
Suppression of the secretion of luteinizing hormone due to isolation/restraint
stress in gonadectomised rams and ewes is influenced by sex steroids. J.
Endocrinol. 160:469-481.
Vizcarra, J.A., R.P. Wettemann, J.C. Spitzer, and D.G. Morrison. 1998. Body condition
at parturition and postpartum weight gain influence luteal activity and
concentrations of glucose, insulin, and nonesterified fatty acids in plasma of
primiparous beef cows. J. Anim. Sci. 76:927-936.
Walczewska A, Yu WH, Karanth S and McCann SM (1999) Estrogen and leptin have
differential effects on FSH and LH release in female rats Proceedings of the
Society for Experimental Biology and Medicine 222 170-177
Walters, D. L., C. C. Kaltenbach, T. G. Dunn, and R.E. Short. 1982. Pituitary and
ovarian function in postpartum beef cows. I. Effect of suckling on serum and
follicular fluid hormones and follicular gonadotropin receptors. Biol. Reprod.
26:640-646.
Wang J, Liu R, Hawkins M, Barzilai N and Rossetti L (1998) A nutrient-sensing pathway
regulates leptin gene expression in muscle and fat. Nature 393 684-688
86
Watanobe H (2002) Leptin directly acts within the hypothalamus to stimulate
gonadotropin-releasing hormone secretion in vivo in rats. Journal of Physiology
545 255-268
Wauters M, Considine RV, van Gaal LF. 2000. Human leptin: From an adipocyte
hormone to an endocrine mediator. Eur J. Endocrinol. 143: 292-311
Werth, L. A., J. C. Whittier, S. M. Azzam, G. H. Deutscher, and J. E. Kinder. 1996.
Relationship between circulating progesterone and conception at the first
postpartum estrus in young primiparous beef cows. J. Anim. Sci. 74:616-619.
Wettemann, R.P., G.M. Hill, M.E. Boyd, J.C. Spitzer, D.W. Forrest, and W.E. Beal.
1986. Reproductive performance of postpartum beef cows after short-term calf
separation and dietary energy and protein supplementation. Theriogenology 26:411.
Whisnant CS and Harrell RJ. 2002. Effect of short-term feed restriction and refeeding on
serum concentrations of leptin, luteinizing hormone and insulin in ovariectomized
gilts. Domestic Animal Endocrinology 22 73-80
Whisnant, C.S., T.E. Kiser, F.N. Thompson, and C.R. Barb. 1986. Opioid inhibition of
luteinizing hormone secretion during the postpartum period in suckled beef cows.
J. Anim. Sci. 63:1445-1448.
Wiesner G, Vaz M, Collier G, Seals D, Kaye D, Jennings G, Lambert G, Wilkinson D
and Esler M (1999) Leptin is released from the human brain: influence of
adiposity and gender Journal of Clinical Endocrinology and Metabolism 84 22702274
William Jr WN, Ceddia RB, Curi R. 2002. Leptin controls the fate of fatty acids in
isolated rat white adipocytes. J. Endocrinol. 175: 735-744
Williams LM, Adam CL, Mercer JG, Moar KM, Slater D, Hunter L, Findlay PA and
Hoggard N. 1999. Leptin receptor and neuropeptide Y gene expression in the
sheep brain Journal of Endocrinology 11 165-169
Williams, G. L. 1990. Suckling as a regulator of postpartum rebreeding in cattle: A
review. J. Anim. Sci. 68:831-852.
Williams, G. L. and M. K. Griffith. 1995. Sensory and behavioral control of
gonadotrophin secretion during suckling-mediated anovulation in cows. J.
Reprod. Fertil. Suppl. 49:463-475.
Williams, G.L. 1989. Modulation of luteal activity in postpartum beef cows through
changes in dietary lipid. J. Anim. Sci. 67:785-793.
87
Williams, G.L., W.R. McVey Jr., and J.F. Hunter. 1993. Mammary somatosensory
pathways are not required for suckling-mediated inhibition of luteinizing hormone
secretion and delay of ovulation in cows. Biol. Reprod. 49:1329-1337.
Wiltbank, J.N. 1970. Research needs in beef cattle reproduction. J. Anim. Sci. 31:755762.
Woodside B, Abizaid A, Jafferali S. 1998. Acute food deprivation lengthens lactational
infertilitiy in rats and this effect is reduced by systemic leptin administration. Am.
J. Physiol. 274: R16553-8
Wright, I. A., S. M. Rhind, T. K. Whyte, A. J. Smith, S. R. McMillen, and R. Prado.
1990. Circulating concentrations of LH and FSH and pituitary responsiveness to
GnRH in intact and ovariectomized suckled beef cows in two levels of body
condition. Anim. Prod. 51:93-101.
Wright, I.A., S.M. Rhind, T.K. Whyte, and A.J. Smith. 1992. A note on the effects of
pattern intake and body condition on the duration of the post-partum anoestrous
period and LH profiles in beef cows. Anim. Prod.54:143-146.
Yavas, Y., and J.S. Walton. 2000. Postpartum acyclicity in suckled beef cows: A Review.
Theriogenology 54:25-55.
Zachow RJ, Magoffin DA. 1997. Direct intraovarian effects of leptin: impairment of the
synergistic action of insulin-like growth factor-I on follicle-stimulating hormonedependent estradiol-17β production by rat ovarian granulosa cells. Endocrinology.
138: 847-850.
Zalesky, D. D., M. L. Day, M. Garcia-Winder, K. Imakawa, R. J.Kittok, M. J. D’Occhio,
and J. E. Kinder. 1984. Influence of exposure to bulls on resumption of estrous
cycles following parturition in beef cows. J. Anim. Sci. 59:1135-1139.
Zhang Y, Proenca R, Maffei M, Barone M, Leopold L and Friedman JM (1994)
Positional cloning of the mouse obese gene and its human homologue Nature 372;
425-432
Zieba DA, Amstalden M, Maciel MN, Keisler DH, Raver N, Gertler A and Williams GL
(2003a) Divergent effects of leptin on luteinizing hormone and insulin secretion
are dose dependent Experimental Biology and Medicine 228 325-330
Zigman JM, Elmquist JK. 2003. From anorexia to obesity – the Yin and Yang of body
weight control. Endocrinology. 144: 3749-3756