AN ABSTRACT OF THE THESIS OF
Ursula S. Bechert for the degree of Doctor of Philosophy in Animal Science presented on
January 9, 1998. Title: Factors Affecting Prolactin Secretion in the African Elephant.
Abstract approved:
Redacted for Privacy
Fredrick Stormshak
Prolactin (PRL) is a peptide hormone that is involved in a number of diverse
physiologic roles, particularly with respect to reproduction, including: influencing sexual
and parental behaviors, onset of puberty, regulation of seasonal reproduction, follicular
maturation, ovulation, luteinization and corpus luteum (CL) function, steroidogenesis,
mammary gland development and lactation, testicular and spermatozoal function, and
immunomodulation of ovarian processes. Little is known about PRL's role in elephant
reproduction. The present research was conducted to determine seasonal changes in PRL
secretion in non-pregnant female African elephants. A corollary objective was to examine
the potential functional interrelationships between secretions of PRL, cortisol and
progesterone.
Weekly blood samples for 18 months were taken from four female African
elephants and the sera were analyzed by radioimmunoassay for progesterone, cortisol, and
PRL concentrations. Estrous cycles averaged 14 weeks in length, and estrous cycle
synchronicity was evident between pairs of elephants. The luteal phase was defined by
serum concentrations of progesterone consistently above 200 pg/ml, and averaged 9
weeks in length (range: 5-12 weeks) with a mean (± SE) concentration of 750.3 ± 171.9
pg/ml. The follicular phase was defined by serum concentrations of progesterone
consistently below 200 pg/ml, and averaged 5 weeks in length (range: 4-8 weeks) with a
mean concentration of 103.1 ± 17.5 pg/ml. Mean (± SE) serum concentration of cortisol
was 5.7 ± 1.3 ng/ml (range: 1.4-19.3 ng/ml), and concentrations of this adrenal steroid
were negatively correlated with progesterone concentrations (r = -0.15; p<0.01). Serum
concentrations of PRL averaged 3.91 ± 0.69 ng/ml (range: 0.84-15.8 ng/m1), were
significantly lower during the luteal phase (p<0.0001; t-test), and were positively
correlated with serum concentrations of cortisol (r = 0.14; p<0.05). There was no
significant effect of season on PRL concentrations. One of the elephants appeared to be
hyperthyroid, but since removing her values from the data set did little to affect overall
means, they were included in all of the calculations.
These data suggest that stress may affect secretion of PRL in elephants, and
cortisol and PRL may affect reproductive potential in elephants by altering luteal function.
While this study did not demonstrate a seasonal effect on PRL secretion, it cannot be
concluded that there is none because the reproductive effects of photoperiod are not
always easy to detect. The higher serum concentrations of PRL detected during the
follicular phase suggest that this hormone may play a role in modulating ovarian function
in elephants during this stage of the estrous cycle.
©Copyright by Ursula S. Bechert
January 9, 1998
All Rights Reserved
Factors Affecting Prolactin Secretion in the African Elephant
by
Ursula S. Bechert
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented January 9, 1998
Commencement June 1998
Doctor of Philosophy dissertation of Ursula S. Bechert presented on January 9, 1998
APPROVED:
Redacted for Privacy
Major Professor, representing Animal Sciences
Redacted for Privacy
Interim Head of Dep rtmen of Animal Sciences
Redacted for Privacy
Dean o
I understand that my thesis will become part of the permanent collection of Oregon State
University libraries. My signature below authorizes release of my dissertation to any
reader upon request.
Redacted for Privacy
Dr. Ursula S. Bechert, Author
ACKNOWLEDGEMENTS
I am grateful to Dr. Fredrick Stormshak, my major professor, for taking me on as
his student despite my interests in working with non-domestic species. He has been a
wonderful mentor to me over the years and I value his opinions and friendship. Dr. Jack
Mortenson invited me to do this research at the Wildlife Safari in Winston, Oregon, and
without his efforts this project would not have been possible. The dedication of the
Wildlife Safari elephant keeper staff was tremendous; Chris Morehouse, Clayton Gredig,
Lacey Geary, Glenn Goodman, and Candy Serfling were all invaluable to the project. Dr.
Lloyd Swanson taught me just about everything I know about radioimmunoassays. His
guidance in development of the prolactin assay was essential, and helped foster in me the
ability to derive some pleasure from doing labwork. Now that's something!
I am very grateful to both Dr. David Hess, Oregon Regional Primate Research
Center in Beaverton, and Dr. Samuel Wasser, Center for Wildlife Conservation in Seattle,
Washington, for their efforts in quantifying serum concentrations of progesterone and
cortisol for me, respectively. Dr. Harold Papkoff, University of California, Davis,
generously provided me with purified African elephant prolactin and growth hormone for
development of the prolactin radioimmunoassay. He also graciously offered to edit my
thesis, and has faithfully provided me with humor and encouragement via e-mail for the
past few years. I appreciate the efforts made by Dr. Kathy Ryan, Magee Womens
Research Center in Pittsburgh, PA, Dr. Greg Gerhardt, University of Colorado Medical
Center in Denver, CO, and Dr. Steve Yellon, Loma Linda University in Loma Linda, CA,
in helping me try to establish an assay for analysis of melatonin in elephant serum samples.
Mr. Charles Glaser at the National Weather Service Center in Medford, Oregon kindly
sent monthly weather reports to me, and Drs. Ken Rowe and Dan Schafer gave me advice
regarding statistical analyses. I acknowledge the National Hormone and Pituitary
Program of the NIDDK (Rockville, MD) for providing prolactin antiserum and standard
material for the prolactin assay. Additional financial support for this project was provided
by the Wildlife Safari Foundation and Oregon State University's Lab Animal Resources.
I thank my other Oregon State University graduate committee members (Drs.
Edward Plotka and Jean Hall), departmental staff and office mates (especially Dr. Tim and
Kelly Hazzard, and Shelby Filley) for their inspiration, advice and helpfulness. Their
support and encouragement throughout my sojourn in the Animal Science Department
was deeply felt and appreciated. My family (Dr. Mark and Sean Wolf Hixon, Ilsemarie
and Andrea Bechert) and friends (especially Dr. Susan Chops Libra), who endured me
going through yet another round of graduate school, deserve special recognition. Their
belief in me and my abilities occasionally superceded my own and kept me going.
TABLE OF CONTENTS
Page
REVIEW OF LITERATURE
1
Problem Definition
1
Reproductive Physiology of Elephants
2
Female Elephant Reproductive Anatomy and Puberty
Social Behaviors Related to Reproduction
Estrous Cycle Characteristics
Reproductive Endocrinology
Pregnancy, Parturition and Post-partum Characteristics
Male Reproductive Physiology
Assisted Reproduction and Contraception Techniques
Conclusions
Seasonal Reproduction
Environmental Cues: Photoperiod
Environmental Cues: Temperature
Environmental Cues: Nutrition
Environmental Cues: Pheromones and Estrous Synchronicity
Hormones Affected by Environmental Cues
Melatonin: A Neuroendocrine Transducer of
Photoperiod
Prolactin: Structure and Secretion
Prolactin: Function
Prolactin: Receptors and Mechanism of Action
Seasonal Reproduction in the Tropics
Conclusions
Stress and Cortisol
2
3
5
6
14
19
21
27
29
30
35
37
39
42
42
48
53
59
60
63
64
STATEMENT OF PURPOSE
69
EXPERIMENTAL DESIGN
70
Animals
70
TABLE OF CONTENTS (Continued)
Page
Radioimmunoassays
Prolactin
Progesterone
Cortisol
Statistics
RESULTS
71
71
76
76
77
81
Animals
81
Seasonal Parameters
81
Cortisol
83
Progesterone
83
Prolactin
88
DISCUSSION
93
BIBLIOGRAPHY
101
APPENDICES
129
Appendix A Additional Prolactin Radioimmunoassay
130
Appendix B Melatonin Radioimmunoassay
131
LIST OF FIGURES
Page
Figure
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
Mean (± SE) serum concentrations of progesterone for 103 estrous
cycles from 15 Asian elephants (adapted from Olsen et al., 1994).
7
Mean (± SE) serum concentrations of progesterone during pregnancy
for eight Asian elephants (adapted from Olsen et al., 1994).
12
Serum concentrations of prolactin in an Asian elephant throughout
gestation and after parturition. Day 0 represents time of observed
breeding (adapted from Brown & Lehnhardt, 1997).
16
Time required for sperm to disappear from the testes of adult golden
hamsters when they are exposed to short daylengths, depending on
ambient temperature (adapted from Bronson and Heideman, 1994).
36
Mean (± SE) concentrations of melatonin in plasma of animals held
on (a) simulated natural (SN; n=5) and (b) summer solstice hold
(SSH; n=5) photoperiods with samples taken every 2 h for 26 h on
16-17 December, 1987. The period of darkness (SN 14 h 47 min;
SSH 5 h 44 min) is shown by the hatched bar (adapted from Brinklow
and Loudon, 1993).
44
Fractions from a Sephadex G75-120 column demonstrating a 46.2%
yield in labelled hormone versus free iodine (April 10, 1997).
73
Dose response curves for ePRL standards (ng), serum volume
dilutions (p.1), and eGH (ng) demonstrating parallelism to the standard
curve (y=181.4673-47.8765x; r2=0.996) and lack of cross-reactivity
with eGH.
74
Dose response curve for ePRL after a TRH challenge (633 p.g in 10 ml
saline) in a female African elephant.
75
Dose response curves for cortisol standards (ng) and dichloromethane-extracted serum volume dilutions (µl) demonstrating
parallelism to the standard curve (y---.65.5713-43.0383x; r2=0.980).
78
Dose response curve for cortisol after an ACTH challenge (1500-2000
IU Acthar Gel given im) in two female African elephants.
79
LIST OF FIGURES (Continued)
Page
Figure
11.
12.
13.
14.
15.
16.
17.
18.
19.
Seasonal fluctuations in weekly percentage daylight and temperature
averages over 18 months (July, 1995 through December, 1996).
82
Individual weekly serum cortisol concentration profiles for four female
African elephants over 18 months (July, 1995 through December,
1996); (a) Alice (matriarch); (b) Sneeze; (c) Tiki (potentially
hyperthyroid); (d) Moshi (youngest).
84
Mean (± SE) weekly serum concentrations of cortisol for four female
African elephants over 18 months.
85
Weekly serum concentrations of progesterone in an African elephant
demonstrating cyclicity. All females in this study had similar profiles.
86
Mean (± SE) serum concentrations of progesterone for 20 estrous
cycles from four African elephants.
87
Estrous cycle synchronicity in two pairs of African elephants.
89
Weekly serum concentrations of progesterone and prolactin for three
female African elephants over 18 months, demonstrating a
significant negative correlation (T.:5.9928-0.9670x; r2=0.1249).
90
Individual weekly serum prolactin concentration profiles for four female
African elephants over 18 months (July, 1995 through December,
1996); (a) Alice (matriarch); (b) Sneeze; (c) Tiki (potentially
hyperthyroid); (d) Moshi (youngest).
91
Mean (± SE) weekly serum concentrations of prolactin for four female
African elephants over 18 months.
92
DEDICATION
I would like to dedicate this dissertation to my parents, Wolfgang and Ilsemarie Bechert,
who always told me that I could achieve anything I set my mind to. They gave me the
freedom to dream and the courage to pursue those dreams.
Factors Affecting Prolactin Secretion in the African Elephant
REVIEW OF LITERATURE
Problem Definition
The wild African elephant population was estimated to be less than 650,000 in
1989, compared to 1.3 million in 1979 and Asian elephants number only one tenth of these
estimates. Poaching, habitat loss, and drought are factors that can continue to contribute
to the decline in numbers of this species. African elephants (Loxodonta africana) were
listed as an Appendix 1 endangered species in 1989 by the Convention of International
Trade in Endangered Species, significantly curtailing the trade in ivory and the decline of
wild populations. Recently, however, Zimbabwe, Namibia and Botswana voted to
downlist this species to an Appendix II status (threatened), which would allow regulated
trade by these countries in elephant products. The struggle for habitat preservation
continues as the world human population currently exceeds 5.6 billion and is predicted to
double within the next 30-40 years. National parks and wildlife preserves amount to only
2-3% of the original geographic range of elephants in Africa, and culling animals to keep
populations within the carrying capacities of these preserves has become routine.
However, the potential for population decimation and subsequent loss of genetic diversity
becomes greater when culling, as opposed to reversible contraception, is solely used to
control population numbers.
Reproductive success among captive elephants has been poor. There are
approximately 214 female and 31 male African elephants living in captivity in North
America and, since 1978, there have only been 19 births, and 32% of these calves died
within their first year of life (D. Olson, personal communication). Approximately 16% of
the Asian elephants born in European zoos and circuses arrive stillborn and another 15.7%
are refused or killed by their mothers (Kurt and Mar, 1996). The difficulties of keeping
mature bulls in captivity, the lack of success with artificial insemination trials, and our
2
poor understanding of the needs of mothers and newborn elephants have contributed to
the low birthrate and high neonatal mortality rate.
The estrous cycle of elephants has only been characterized during the last 10-15
years, and as more is learned about these animals it seems that many aspects of elephant
reproductive physiology are unique to these species (reviewed below). Elephants are
generally classified as tropical animals, and as such, are assumed to be reproductively
unresponsive to photoperiod. Yet herds occupy lands ranging over 300 latitude from the
equator in Africa, creating the possibility for a photoperiodic influence on reproductive
cyclicity. Furthermore, the vast majority of African elephants living in captivity in North
America were caught in the wild as infants (1-2 years of age), and it is not known whether
transfer to a more temperate climate might influence an animal's ability to detect and
utilize photoperiodic information to enhance reproductive efforts. Broadening our basic
understanding of the reproductive physiology of African elephants will facilitate
development of assisted reproduction and contraception techniques, which can be used to
improve conception rates in captivity and offer alternatives to culling in the wild. This
thesis reviews both our current understanding of African elephant reproductive physiology
and factors affecting seasonal reproduction, and then describes research which investigates
potential fluctuations in serum prolactin concentrations in female African elephants as
influenced by the effects of season, cortisol concentrations, and stage of the estrous cycle.
Reproductive Physiology of Elephants
Female Elephant Reproductive Anatomy and Puberty
No significant reproductive differences between Asian and African elephants have
been reported in the literature. The reproductive tract of adult female elephants opens
ventrally and is relatively long, averaging 92 cm for the urogenital canal, 28 cm for the
vagina, and 8 cm for the cervix (Balke et al., 1987). In non-pregnant elephants, the
3
urogenital canal makes up half the length of the entire reproductive tract (Balke et al.,
1988b). The vaginal orifice can be as small as 0.7 cm in diameter, and is frequently
reported as a central opening paralleled by two small openings ending as blind canals (1-2
cm in length), which may be remnants of the mesonephric ducts (Balke et al., 1988a).
Tissue folds of residual hymen are broken down during parturition but not necessarily
during copulation (Balke et al., 1988b).
The uterus of the elephant is bicornuate (Sikes, 1971), averaging 23 cm in length
for the uterine body, and 54 cm for the uterine horns (Balke et al., 1987). The oviducts
are relatively short, being approximately 5 cm in length and 0.3 cm in diameter (Perry,
1953). Ovaries measure approximately 6x6x4 cm in diameter, are located medial and
ventral to the kidneys, and are completely surrounded by a well-developed ovarian bursa
(Perry, 1953; Adams et al., 1991). Females have two mammary glands, located ventrally,
between the forelegs.
Delayed breeding can be expected to occur when females have long inter-birth
intervals, long periods of receptivity, and do not have synchronized estrous cycles
(Whitehead, 1994). Sexual maturity in African and Asian female elephants has been
variously reported to occur around 9-12 years of age, as early as 7 (Buss and Savidge,
1966), or as late as 23 years (Basson et al., 1991), and is attained at an earlier age in
captive versus wild elephants (Schmidt, 1993). Maturity is accompanied by a significant
rise in serum progesterone concentrations, which increase from a mean (± SE) of 316 ± 52
pg/ml in African females under 13 years of age to 707 ± 77 pg/ml in those over 13 years of
age (n=15 and 48, respectively; McNeilly et al., 1983).
Social Behaviors Related to Reproduction
Elephants live in herds composed primarily of females and their young, and are led
by a matriarch. Animals over 11 years of age comprise 4.8% of a stable African elephant
population, 55% are juveniles, and the remaining are calves (Calef, 1989). Mature males
4
are excluded from matriarchal families, and move between isolated populations to breed
receptive females (Drysdale and Florkiewicz, 1989). Bulls exhibit the flehmen response
when investigating urine from a cow in estrus (Rasmussen et al., 1982; Schmidt, 1982;
Hess et al., 1983; Diephius, 1993), and the component in the urine which stimulates the
flehmen response in bulls was recently identified as (Z)-7-dodecen-l-y1 acetate
(Rasmussen et al., 1996). Females also utilize the flehmen response when identifying
sexes and sex states, and during mother-young, female-female, and male-female
interactions (Rasmussen, 1988).
Wild female African elephants have been observed to exhibit subtle, behavioral
signs of estrus (Moss, 1983), and mate selectivity, which has been noted in both African
and Asian female elephants (Moss, 1983; Poole, 1989b; Schmidt, 1993). Behavior in
captivity is most likely altered unless family groups are maintained, permitting normal
interactions. The behavior demonstrated by females during estrus attracts attention and
may stimulate male competitiveness (Moss, 1983). Standing estrus usually lasts 2-5 days
(Moss, 1983; Schmidt, 1993), and the presence of large corpora lutea (CL's) does not
prevent cows from entering estrus (Hanks and Short, 1972).
Temporal glands, found in both species and sexes of elephant, produce secretions
which may play a role in chemical communication (Buss et al., 1976), thus influencing, to
some extent, reproductive behavior. They are mixed sebaceo-apocrine skin glands located
between the eyes and ears, over the temporal fossa, and range in weight from 230 to 1590
g in African bulls (Wheeler et al., 1982). The deeper apocrine portion comprises the
majority of the gland, and like mammary glands, temporal glands are modified sweat
glands (Estes and Buss, 1976; Rasmussen et al., 1984). Secretions increase with stress,
similar to other apocrine glands, which typically respond to fear, pain and sex (Hanks,
1973; Buss et al.; 1976; Gorman, 1986). Structurally, temporal glands are the same
between Asian and African elephants (Estes and Buss, 1976), although glandular activity
varies between species and sexes. The temporal glands of African male elephants are
5
typically more active than that of females, but apparently do not secrete on a regular basis
until males are over 35 years of age (Poole, 1989a). Female Asian elephants secrete only
occasionally, usually under stressful conditions (Rasmussen et al., 1984), whereas Asian
males experience consistent cyclic secretions coinciding with bouts of musth (discussed
later).
Precopulatory behavior is similar between Asian and African elephants, except that
Asian elephants often face one another, twining trunks and making mouth-to-mouth
contact (Eisenberg et al., 1971; Moss, 1983). The duration of mounting is not long, with
ejaculation occurring within 30 to 60 seconds after intromission (Schmidt, 1993). The
average number of mounts/ejaculation is 3.7 (Eisenberg et al., 1971).
In the wild, breeding occurs throughout the year (Buss and Smith, 1966), but
usually increases in frequency during the rainy season when the elephants are on a higher
plane of nutrition (Hanks, 1972; Barnes, 1982; Craig, 1984). Hanks (1972) observed a
strong correlation between percentage conceptions and monthly rainfall, with 88% of
African elephant conceptions occurring during the rainy season. Other factors which
could potentially contribute to this observed seasonal effect (e.g., photoperiod,
temperature, and social cues mediated by stress or pheromones) have not been
investigated.
Estrous Cycle Characteristics
Elephants are monovular, polyestrous animals (Short, 1984). Thus far, no
significant differences in estrous cycles between African and Asian elephants have been
observed (Schmidt, 1993). The estrous cycle in Asian elephants was previously believed
to be 18-22 days long, with a 4-day estrus (Jainudeen et al. 1971; Klos and Lang, 1976;
Ramsay et al., 1981; Rueedi and Kuepfer, 1981). These studies were based on vaginal
cytology, behavior, and urinary estrogen (E) levels, which are all considered estrogen-
dependent factors. Plotka et al. (1988) suggest that waves of follicular growth at 3 week
6
intervals could be responsible for these short cycles. Based on serum progesterone (P4)
concentrations, it is now generally accepted that the estrous cycle is actually closer to 1415 weeks in duration (Schmidt, 1982; Hess et al., 1983; Plotka et al., 1988; Cooper, 1989;
Mainka and Lothrop, 1990; Brown et al., 1991; Taya et al., 1991; Olsen et al., 1994).
The estrous cycle averages 15 weeks in length (range: 10-23) for Asian elephants (Olsen
et al., 1994), whereas a possible shorter average of 14 weeks (range: 13-16) is reported
for African elephants (Brannian et al., 1988; Plotka et al., 1988; Roach et al., 1990).
The estrous cycle is divided into two phases (follicular and luteal) based on
fluctuations in circulating concentrations of P4 (Figure 1). Variations in the duration of
each phase as reported in the literature may be due in part to different definitions given for
the luteal phase based on P4 concentrations (Table 1). For Asian elephants the follicular
or inter-luteal phase has a mean (± SE) of 4.6 ± 0.2 weeks in length (n=15; range: 2-10
weeks), and the luteal phase averages 10.5 ± 0.2 weeks (n=15; range: 6-21 weeks; Olsen
et al., 1994). The follicular phase in African elephants averages 4-6 weeks in length, and
the luteal phase averages 9-10 weeks (Brannian et al., 1988; Plotka et al., 1988). During
the follicular phase, bulls demonstrate a higher level of interest in cows (Schmidt, 1982),
and Hess et al. (1983) suggest that this relatively long period of attraction could be
advantageous to reproduction in the wild, where males and females are often separated by
great distances.
Reproductive Endocrinology
Ovulation is thought to occur sometime towards the end of the follicular phase,
although controlled experiments have not yet been done to verify this. Asian elephant
serum luteinizing hormone (LH) concentrations have increased in response to injections of
luteinizing hormone-releasing hormone, but only after 3 days of estrone treatment
(Chappel and Schmidt, 1979). Surges of LH in Asian and African elephants have been
associated with consequential increases in P4, but not consistently (Hess et al., 1983;
7
500
N = 103 Estrous Cycle
400 -
300Estrus
200 -
Nonluteal
Phase
100 Luteal Phase
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
Weeks
Figure 1. Mean (± SE) serum concentrations of progesterone for 103 estrous cycles from
15 Asian elephants (adapted from Olsen et al., 1994).
TABLE 1. Length of estrous cycle, and luteal and follicular phases based on serum progesterone (P4) concentrations.
na
Study durationb Estrous cycles
Elephas maximus:
Follicular phases
Luteal phases
Definition of luteal phase
Source
P4 consistently >40 pg/ml
for 2 consecutive wks
na
P4 >mean plus 2 SD of
remaining values
P4 >100 pg/ml
P4 >100 pg/ml for 2
consecutive wks
point at which P4 inc by
50 pg/ml & stayed
elevated for 2 wks after
dec <200 pg/ml
na
Olsen, JH et al., 1994
point at which P4 inc by
50 pg/ml & stayed
elevated for 2 wks after
dec <200 pg/ml
P4 values >40 pg/ml,
based on basal values <40
pg/ml
na
Plotka, ED et al., 1988
15
45
15.1 ± 0.3 (10-23)
4.6 ± 0.2 (2-10)
10.5 ± 0.2 (6-21)
2
32
4
6-18e
16.8 ± 0.6
13.2 ± 0.7
ndd
3.6 ± 0.6
nd
9.8 ± 0.7
6
2
7-30
84 & 132
5.1 ± 0.4
nd
2
24
16.3 ± 0.4
14.9 ± 0.3 &
15.6 ± 0.2, resp.
14.7 ± 0.5
4.2 ± 0.5
10.5 ± 0.3 (8-12)
9.1 ± 0.4 & nd,
resp.
10.6 ± 0.6 (9-13)
2
24
16.1 ± 2.1
nd
(8-12)
Loxodonta africana:
4
18 & 36
15.9 ± 0.6
5.9 ± 0.6
10.0 ± 0.3 (8-14)
3
15-18
13.3 ± 1.3
4.2 ± 0.6
9.1 ± 1.1
6
29
13.1
nd
nd
Taya, K et al., 1991
Brown, JL et al., 1991
Hess, DL et al., 1983
Cooper, KA, 1989
Plotka, ED et al., 1988
Mainka, SA and CD
Lothrop, 1990
Brannian, JD et al.,
1988
Roach, R et al., 1990
an: number of animals in the study; bduration of study (mos) based on samples taken weekly unless otherwise noted; edata (wks) presented as: mean
± SE (range); dnd/na: not done/not applicable; esamples were collected 1-3 times weekly
co
9
Brannian et al., 1988; Plotka et al., 1988; Brown et al., 1991). Fifty percent of the LH
surges in Asian elephants reported by Hess et al. (1983) were unrelated to P4 increases,
and, in another study (Brown et al., 1991), second surges in LH were occasionally
observed 2-3 weeks into the luteal phase. Multiple peaks of LH during the follicular phase
have been reported (Brannian et al., 1988; Plotka et al., 1988), and it was originally
suggested that these peaks serve to cause eventual luteinization of additional follicles to
form secondary corpora lutea (Plotka et al., 1988). A recent study by Kapustin et al.
(1996), utilizing four captive African elephants, confirmed the occurrence of two LH
peaks per estrous cycle. The assay utilized ovine standards and detected peaks of LH,
both approximately 3.3 ng/ml in magnitude occurring 19-22 days apart. Only one of these
peaks resulted in sustained P4 elevations, and was classified as an ovulatory LH (ovLH)
peak. As the active luteal phase terminated and serum P4 concentrations dropped below
80 pg/ml, an anovulatory LH peak (anLH) occurred, followed by sustained low levels of
P4. Approximately 3 weeks later an ovLH peak occurred, preceded by 2-3 days of
gradually increasing progesterone concentrations. There are many questions that remain
unanswered regarding the purpose of the anLH peaks, as well as how the duration of the
resulting follicular phases can be precisely regulated when they do not appear to be
controlled by luteal life span as usually occurs in other species.
The CL of African and Asian elephants can persist from one cycle to the next
(Hanks and Short, 1972; Smith and Buss, 1975), unlike most other mammals with the
exception of the horse (Stabenfeldt et al., 1975). Additionally, accessory CL's can form
(originating from unovulated follicles) during the luteal phase when higher levels of P4
may prevent estradiol-induced LH surges and ovulation (Plotka et al., 1988). Elephant
luteal cells have been described as containing peripherally distributed organelles,
mitochondria with fewer cristae, and less extensive Golgi complexes (Ogle et al., 1973).
These findings suggest less steroidogenic capacity.
10
The concentration of P4 in the serum of elephants is comparatively lower than that
of other mammals. The rate at which the elephant CL converted pregnenolone to P4 is
2.4% per hour, compared to 74% per hour for pigs (Smith et al., 1969). However, recent
studies (Heistermann et al., 1994; Schwarzenberger et al., 1996) suggest that high
amounts of 5a-reduced progesterone metabolites (specifically 5a-pregnane-3,20-dione
and 5a-pregnane-3a-o1-20-one) are produced by the CL in quantities 3-4 times that of
progesterone. Concentrations of P4 and these 5a-reduced progesterone metabolites were
highly correlated (i =0.80; p<0.001) in a parallel pattern (Schwarzenberger et al., 1996). A
wide range of serum concentrations of P4 have been reported in the literature for
elephants. This high degree of variability may in part be explained by varying degrees of
cross-reactivity with P4 metabolites through the use of different assays. Pregnane
compounds have generally been considered to be inactive metabolites of progesterone,
possessing no biological function. However, they can bind the progesterone receptor with
10-90% the affinity of progesterone, depending on the species, so their function in
elephants remains undefined (Schwarzenberger et al., 1996).
Patterns of serum P4 concentration fluctuations are unique to and consistent for
each female (Schmidt, 1993), although there is some inconclusive evidence suggesting that
synchrony of estrous cycles occurs among elephants housed together in captivity
(Turczynski et al., 1992). In non-pregnant Asian cows, P4 concentrations during the
follicular phase ranged from 30 pg/ml (Hess et al., 1983) to 150 pg/ml (Brown et al.,
1991). Progesterone secretion often increases prior to the LH surge that presumably
causes ovulation. It has been suggested that the follicle itself may be responsible for this
initial rise in serum P4 concentration (P. Malven, personal communication), or
alternatively, perhaps CL's held over from previous cycles become more functional at this
time. During the luteal phase in Asian elephants, P4 concentrations averaged 456 ± 23
pg/ml (n=2; Plotka et al., 1988) and ranged from 150 pg/ml (Mainka and Lothrop, 1990)
to 700 pg/ml (Hess et al., 1983). During pregnancy, P4 concentrations overlapped with
11
concentrations found during the luteal phase, with a mean (± SE) of 555 ± 16 pg/ml (n=8;
range: 40-2110 pg/ml; Olsen et al., 1994). In general, concentrations of P4 are higher
during the first half of gestation, dropping to lower concentrations during the latter half of
gestation (Figure 2). Similarly, in African elephants during pregnancy, P4 concentrations
rose slightly above those found during the luteal phase to a mean of 831 ± 299 pg/ml
during the first half of gestation, decreasing to 498 ± 297 pg/ml during the latter half of
gestation (n=44; DeVilliers et al., 1989). In non-pregnant African elephants, serum P4
concentrations averaged 707 ± 77 pg/ml (n=63; range: 92-1495 pg/ml; McNeilly et al.,
1983).
Non-invasive monitoring of the estrous cycle in wild or captive elephants becomes
possible by measuring P4 metabolites in urine or feces. Niemuller et al. (1993) reported
positive correlations between P4 and both urinary 5f3-pregnanetriol and 17ahydroxyprogesterone metabolites (r =-0.98; p<0.01 and r=0.95; p<0.01, respectively).
Maximum concentrations of 513-pregnanetriol during the luteal phase averaged 237 ng/mg
creatinine (range: 75-802 ng/mg), and dropped to an average of 45 ng/mg creatinine
(range: 5-172 ng/mg) during the follicular phase. Wasser et al. (1996) demonstrated that
fecal progestin concentrations >1500 ng/g seem to characterize the luteal phase of the
estrous cycle in a group of five female African elephants. Parallelism between serum P4
and fecal progestins was demonstrated (r =0.81; range: 0.61-0.92) when fecal samples
were collected 2 days after serum samples.
There are conflicting reports regarding serum concentrations of estrogen (E) in
elephants. Total urinary E concentrations rise just prior to increases in serum P4, and
bulls have flehmen-like responses to female urine at this time (Mainka and Lothrop, 1990).
Peaks in E secretion occurred at 3-week intervals throughout the luteal phase, but these
waves were not typically associated with development of ovulatory follicles because P4
concentrations were high enough at this time to interfere with ovulation (Brown et al.,
1991). Perhaps these fluctuations in serum concentrations of E correspond to waves of
12
1400
1300-
N = 8 Pregnancies
1200 -
11001000-
Parturition
900 800 700
600 -
500-
400 300
200 -
1000
10
20
30
40
50
60
70
80
90
100
Weeks
Figure 2. Mean (± SE) serum concentrations of progesterone during pregnancy for eight
Asian elephants (adapted from Olsen et al., 1994).
13
follicular growth similar to those found in cattle (Sirois and Fortune, 1988).
Concentrations of serum E have been reported to vary continuously throughout the
estrous cycles of African and Asian elephants, with no apparent pattern (Plotka et al.,
1975; Hess et al., 1983; Wasser et al., 1996), although one study found a negative
correlation between serum estradiol and P4 concentrations in two Asian elephants (Taya
et al., 1991). Plasma concentrations of E, as well as urinary concentrations of estrone and
estradiol, are lower in elephants than those found in other species (McNeilly et al., 1983).
Serum estradiol concentrations ranged from nondetectable to 13.6 pg/ml in one African
elephant sampled seven times (Plotka et al., 1988), and estrogens ranged between 9-37
pg/ml for 9 Asian and African elephants sampled once (Plotka et al., 1975). Serum
estrone concentrations reported in the literature are highly variable, averaging 8.3 ± 0.6
pg/ml (n=6; Hess et al., 1983) or ranging between 160-290 pg/ml (Hodges et al., 1983) in
non-pregnant elephants, and increasing to an average of 22.2 ± 1.1 pg/ml (n=2; Hess et
al., 1983) or a range of 161-594 pg/ml (Hodges et al., 1983) during pregnancy. Some of
the discrepancy in reported estrogen concentrations may be partially due to estrogen
conjugates (primarily estradiol), which can be twice the concentration of the free form
(Ramsay et al., 1981; Czekala et al., 1992).
Elephant gonadotropins appear to be similar in their chemical and physical
properties to those of other species (McFarlane et al., 1990). Follicle stimulating hormone
(FSH) and inhibin concentrations fluctuate in 12-14 week cycles and are inversely
correlated with one another, as they are in most domestic species (Brown et al., 1991).
Prolonged secretion of FSH into the elephant's luteal phase may serve to recruit follicles
for the next ovulatory cycle, as can occur in other mammalian species such as the primate
(Greenwald and Roy, 1994). African elephant mean (± SE) serum FSH concentration was
95 ± 9 ng/ml (n=63; range: 30-256 ng/ml; McNeilly et al., 1983), while a lower range of
5-59 ng/ml was reported for Asian elephants (Brown et al., 1991). Inhibin is primarily
secreted by rat follicular granulosa cells early in the cycle (Erickson and Hsueh, 1978;
14
Woodruff et al., 1988), while luteal cells can produce inhibin after ovulation in other
species (Davis et al., 1987; Rodgers et al., 1989; Basseti et al., 1990). Concentrations of
inhibin reportedly range between 0.1-1.2 ng/ml in Asian elephants (Brown et al., 1991).
Serum prolactin (PRL) concentrations did not appear to differ between captive
elephant species, and did not vary between seasons or with estrous cycle stage and
lactational status in one study (Brown and Lehnhardt, 1997), although changes in
concentration do occur during gestation and are discussed below. Prolactin
concentrations in non-pregnant females averaged 4-5 ng/ml and ranged between 0.5-14.7
ng/ml (Brown and Lehnhardt, 1997).
Serum concentration of testosterone fluctuations were positively correlated with
concentrations of serum P4 (r=0.458; p<0.01) in 2 ten-year old Asian females (Taya et al.,
1991). Testosterone concentrations averaged 0.08 ng/ml (n=4; range: 0.05-0.11 ng/ml) in
Asian females, while African female elephants had higher concentrations, averaging 0.25
ng/ml (n=5; range: 0.14-0.46 ng/ml; Rasmussen et al., 1984).
Pregnancy. Parturition and Post-partum Characteristics
An elephant's first pregnancy usually occurs between 13-17 years of age (Laws,
1967), with peak fertility suggested to occur in 18-19 year old cows (Hanks, 1972).
Transuterine horn migrations are probably rare, because primary CL's have usually been
observed to be ipsilateral to the embryo (Hanks, 1972). Based on placental scars in the
uterus, the position of implantation is usually two-thirds along the cornu from the tubal
end (Laws, 1967). Twins are relatively uncommon, reported at a rate of 1.35% in African
elephants (Abeyratne, 1982). Placentation is zonary, and in African elephants, chorionic
villi are found only in the equatorial region of the blastocyst and are never diffuse
(reviewed by Renfree, 1982). It is thought that most other species with zonary
placentation develop their placenta from diffuse chorionic villi that regress at the polar
areas of the chorionic sac.
15
Gestation is normally 21-22 months in duration (Laws, 1967; Maeberry, 1972;
Klos and Lang, 1976; Schmidt, 1993). Pregnancy usually has been diagnosed by
measuring weekly serum concentrations of P4 and is considered positive if concentrations
do not decline at the end of the luteal phase, approximately 16 weeks after mating
(Schmidt, 1993). However, because a luteal phase of 21 weeks duration has been
reported in an Asian elephant (Olsen et al., 1994), consistently high concentrations of P4
should be measured for 22 weeks to confirm pregnancy with confidence. A significant rise
in serum PRL concentration occurs after 6-7 months gestation (McNeilly et al., 1983;
Brown and Lehnhardt, 1997), and often only a single sample is required because
concentrations in non-pregnant elephants do not overlap those found in pregnant animals.
Assays in both studies utilized ovine standards. Prolactin concentrations in non-pregnant
African elephants have a mean of 6.9 ± 0.7 ng/ml (n=63; range: 0.8-22 ng/ml) and during
pregnancy increase to 50 ± 7 ng/ml (n=63; range: 3.5-212 ng/ml; McNeilly et al., 1983).
A recent study by Brown and Lehnhardt (1997) divided gestation into early, mid and late
periods and reported Asian elephant mean PRL concentrations of 21.4 ± 4.6 ng/ml (n=4;
range: 1.2-50.2 ng/ml), 468.2 ± 121.8 ng/ml (n=4; range: 50.1-1064.2 ng/ml), and 525.1 ±
80.3 ng/ml (n=3; range: 152.3-1038.3 ng/ml) for these periods, respectively (Figure 3).
Serum concentrations were slightly lower overall for African elephants sampled similarly.
Cross-reacting placental lactogens may contribute to the elevated gestational PRL
concentrations described, but this has not been investigated.
Dates of conception for culled, pregnant elephants can be calculated utilizing
formulas based on fetal weight (Perry, 1953). The most recent formula proposed is: T =
106 w1/3 + 138, where T is the age of the fetus in days and w is the fetal mass in kg
(Craig, 1984). Perhaps as rectal ultrasound techniques for elephants improve (Hildebrandt
et al., 1996), estimates of fetal age and condition will become a possibility for animals kept
in captivity.
16
1000 -
750 Parturition
500 -
1
250 -
0
0
120
240
360
480
600
720
840
Days from Breeding
Figure 3. Serum concentrations of prolactin in an Asian elephant throughout gestation
and after parturition. Day 0 represents time of observed breeding (adapted from Brown
and Lehnhardt, 1997).
17
During pregnancy, serum P4 concentrations increase but not to the degree found in
other species. As discussed previously, individual CL's secrete comparatively little P4 (4.1
ng/mg; Ogle et al., 1973), so it was proposed that a critical mass of luteal tissue must be
attained for pregnancy to occur or continue (Hanks and Short, 1972; Smith and Buss,
1975). Corpora lutea have been shown to persist into subsequent cycles and have long life
spans (Hanks and Short, 1972), being maintained up to 2 months postpartum (Buss and
Smith, 1966). Uterine, placental and follicular sources of P4 may also contribute to
circulating blood levels of P4 (DeVilliers et al., 1989). During the 22 months gestation,
there is an enormous variation in CL mass (7-56 g) and number (2-42) between elephants
(Hanks and Short, 1972). McNeilly et al. (1983) found that non-pregnant African females
typically have 1-6 CL's while pregnant females have 12-14 CL's. Individual luteal cells
increase in size and secrete more P4, which peaks in concentration approximately 3
months into gestation, declining to lower levels the latter half of gestation (Figure 2;
DeVilliers et al., 1989; Olsen et al., 1994).
Estrogen concentrations are increased during pregnancy in elephants, and are
consistently higher after the 6th month of gestation than concentrations found in nonpregnant elephants, despite considerable variability among individuals (Hodges et al.,
1983, 1987). Asian elephant urinary total E concentrations increased from 50 pg/pg
creatinine to 400-3000 pg/p.g creatinine within the first year of pregnancy and remained
elevated, declining sharply at parturition (Mainka and Lothrop, 1990). Fetal viability can
be ascertained to an extent by the continued high levels of serum estrogen, because this
requires a functional placenta (Bamberg et al., 1991).
Pregnancy significantly inhibited Graafian follicle development (Smith and Buss,
1975), especially between 5-16 months gestation (Hodges et al., 1983). Compared to
non-pregnant cows, blood concentrations of LH were significantly lower between 9-16
months gestation, and concentrations of FSH decreased after 2 months of pregnancy to
18
reach significantly lower concentrations during 9-12 months gestation (McNeil ly et al.,
1983).
About 2-4 days prior to parturition, maternal P4 concentrations declined
precipitously to <40 pg/ml in Asian elephants (Olsen et al., 1994), and daily monitoring of
serum P4 concentrations can be useful for predicting parturition (Brown and Lehnhardt,
1995). Cows often pass cervical mucus 12-24 hours prior to delivery (Schmidt, 1986),
although a 5 day interval between passage of mucus and delivery of a healthy calf has
occurred (Schmidt, 1993). As parturition progresses, cows appear restless and a
noticeable swelling of the urogenital canal occurs beneath the base of the tail. Labor is
normally completed within 1 hour after contractions begin, but can range from 20 minutes
to several days (Schmidt, 1993). The placenta is usually discharged within 1-3 hours after
birth (Maeberry, 1972). Healthy newborns should be up and walking within 15-60
minutes.
Only 34% of those calves delivered in captivity survive to subadult ages, versus
52% in wild populations (Kurt, 1970). It has been suggested that reproductive success in
captivity would improve if older cows would be permitted to interact with first-time
mothers and their calves as part of a breeding group (Kurt, 1970; Schmidt, 1993). The
natural mortality rate among wild, mature African elephants is 2-3% per year, 2% for
those between 1-5 years of age, and increases to around 10% for elephants in their first
year of life (Pilgram and Western, 1986), although mortality rates as high as 20% for the
first 2 years have been reported (Lee, 1987).
Calves weigh an average of 107 kg at birth, ranging between 50-170 kg (Klos and
Lang, 1976). They are completely dependent on their mothers for food during the first 3
months, gradually adding forage to their diets, and are usually weaned by 2 years of age
(Kurt, 1970; Lee, 1987). Calves have been observed to suckle intermittently for an
additional 3-4 years, and stay with the mother for 10 years (Lee, 1987). Females can
nurse 2 calves, but it is rare for both to survive (Lee, 1987). Allomaternal caretaking
19
occurs commonly among African and Asian elephants, especially by young, nulliparous
females (Buss and Smith, 1966; Dublin, 1983; Lee, 1987; Rapaport and Haight, 1987).
While all mothers will protect one another's calves, nursing another cow's calf is usually
only permitted if the calf is related (e.g., daughter's calf) (Rapaport and Haight, 1987).
Potential benefits to allomaternal behavior include enhancement of inclusive fitness and
greater access to resources for the caretaker of a dominant cow's calf (Dublin, 1983).
The length of post-partum anestrus can be influenced by variations in habitat
conditions (Barnes, 1982). A period of lactational anestrus of up to 2 years has been
reported in African elephants (Laws, 1967; Hanks, 1972). In contrast, a 45-46 week
lactational anestrus period was reported for six Asian elephants (Olsen et al., 1994). The
calving interval in wild populations of African elephants averages 4 years (Kurt, 1970)
(range: 3-9 years; Barnes, 1982), while the reported interval from parturition to
conception is estimated to be between 9 months to 4 years (Buss and Savidge, 1966;
Laws, 1967). Females will usually have given birth to 3-9 calves by the time they are 50
years old (Laws, 1967).
Male Reproductive Physiology
The basic anatomy and physiology of the male elephant reproductive system has
been well described (Short et al., 1967; Sikes, 1971; Hanks, 1972). Due to the internal
location of the testes caudal and ventral to the kidneys, the elephant has no inguinal canal,
cremaster muscle, or pampiniform plexus (Short et al., 1967). The size of the testicles of
an elephant are relatively small when compared to other mammals using a body weight
ratio (Johnson and Buss, 1967). The testicles of 5 year-old Asian males range from 6.37.5 cm in diameter, increasing to 20x13 cm in size in 11 year-old bulls (Olsen and Byron,
1993). Elephants have no distinct epididymis, but instead have a highly convoluted caudal
vas deferens (approximately 160 cm in length, unstraightened) with longitudinal folds of
epithelium that hold the majority of live spermatozoa (Short et al., 1967). The penis is not
20
ossified and has a sigmoid flexure during erection (Short et al., 1967), with large levator
penis muscles that allow for a great deal of voluntary control (Eisenberg et al., 1971).
Accessory organs and testicles grow throughout the animal's life (Hanks, 1973; Smith et
al., 1987), and spermatogenesis begins at about 15 years of age (Hanks, 1973). The
means of spermatocrit, Na, K and protein concentrations of fluids in the rete testis are
similar to those found in the ram (Jones, 1980), and the shape and size of spermatozoa
resemble those of the horse (Short et al., 1967).
Puberty is reached between 7-18 years of age in African male elephants (Johnson
and Buss, 1967), and 7 years of age is the earliest recorded onset of sexual maturity in
Asian bulls (Schmidt, 1993). Body length, weight, plane of nutrition and genetics are
other variables that have been more closely correlated with the onset of puberty than age
(Johnson and Buss, 1967).
Individual reproductive cyclicity for males is likely because there are changes in the
mean seminiferous tubule diameter (Laws, 1967), broad ranges of testosterone production
(3-490 lig/10 g testis; Short et al., 1967), and periodic bouts of musth (Buss and Johnson,
1967). Musth has been compared to rut, which some seasonally breeding animals
experience (Poole, 1987), although in the elephant it may be induced by sexual activity
(Jainudeen et al., 1972). During musth males typically become aggressive, temporal
glands enlarge and produce a discharge, there may be a continuous dribble of urine, eating
and resting activities are minimized, testosterone levels rise, (Barnes, 1982; Poole, 1987;
Rasmussen et al., 1984; Schmidt, 1993), and water consumption increases, perhaps to
compensate for urine marking (Poole, 1989a). The physical and behavioral characteristics
of musth are similar for African and Asian bulls (Poole, 1987). Bouts occur on individual
cycles in 90% of the post-pubertal Asian male elephant population annually, start between
10-15 years of age, and usually last 2-3 months (Jainudeen et al., 1972; Schmidt, 1993).
Musth in African bulls has been observed to occur sporadically in young males and with
greater intensity and regularity in bulls over 35 years of age (Poole, 1989a). The potential
21
role musth plays remains speculative, but it has been suggested that reproductive success
increases during musth (Hall-Martin, 1987; Poole, 1987, 1989a, b). Increased serum
testosterone concentrations have been positively correlated with increased
spermatogenesis in some species (e.g., Lincoln, 1971), and a positive correlation between
the intensity of musth and the serum testosterone concentration in Asian bulls has been
observed (Niemuller and Liptrap, 1991). On the other hand, semen characteristics do not
appear to vary with season or temporal gland activity in African elephants, and
testosterone concentration was not found to be correlated with either sperm motility or
concentration (Howard et al., 1984). Additionally, it has been suggested that bulls in
musth have a decreased ability to breed due to overaggressiveness or even a lack of
interest in mating (Schmidt, 1993).
Temporal gland secretion ratios of dihydrotestosterone: testosterone change
during musth, to favor the latter (Rasmussen et al., 1990), and may affect social
interactions through chemical cues. Cholesterol (Buss et al., 1976), cresol (Wheeler et al.,
1982) and other volatile substances such as ketones, which predominate at the peak of
musth (Rasmussen et al., 1990), have also been examined as other temporal gland
secretion variables that may serve as chemical cues. A combination of both the potential
effects of increased serum concentrations of testosterone, and altered temporal gland
secretions, affecting chemical cues and social interactions, is likely to be involved in
altering male reproductive potential during musth.
Assisted Reproduction and Contraception Techniques
Some broad benefits of assisted reproduction include the maintenance of genetic
diversity and a decreased need to house and ship animals for breeding purposes. This is of
particular interest for captive elephant populations because mature bulls can be unsafe to
house in captivity. Embryo sex preselection, manipulation, cryopreservation and transfer,
in vitro maturation and fertilization of oocytes, manipulation of estrous cycles to induce
22
ovulation, collection and storage of spermatozoa, and artificial insemination programs are
techniques useful for the preservation of endangered species (Hearn, 1986). Investigative
efforts in these areas of study with elephants have met with limited success, in part
because so many basic reproductive physiology questions remain unanswered, but also
because opportunities for research have often been limiting due to small sample sizes or
lack of experimental controls to validate findings.
In vitro maturation and fertilization of oocytes have been described for a number
of species, but there has only been one report of attempts to mature and fertilize African
and Asian elephant oocytes in the literature (Christensen et al., 1993). In that study,
oocytes were cultured in Talp media with 20% fetal calf serum, and five metaphase I
oocytes were produced but fertilization trials were not successful. Techniques for embryo
sex preselection, manipulation, cryopreservation and transfer, and manipulation of estrous
cycles to induce ovulation applied to elephants have not been published.
Advantages of developing a successful artificial insemination (AI) technique for
elephants are numerous and include: decreasing the number and transport of bulls kept in
captivity for breeding purposes, minimizing the potential for sexually transmitted diseases,
eliminating mate incompatibility problems, and increasing genetic diversity through the use
of semen collected from wild bulls. Artificial insemination has been performed
successfully in a diversity of exotic species (Seager, 1983), and the first documented AI
attempt with an African elephant was made by Nevill et al. (1976). No pregnancies have
been reported from AI of elephants to-date.
Using a combination of transrectal ultrasound to image follicular progression
(Adams et al., 1991; Griffin and Ginther, 1992; Hildebrandt et al., 1996) and hormonal
analyses to monitor stage of the estrous cycle and, more definitively, LH surges, it has
become possible to predict the time of ovulation with greater accuracy. Current
challenges for AI in elephants include determining the optimal anatomical site for
insemination, preserving semen quality during transport, and providing more AI
23
opportunities to include those females that have been assessed as good reproductive
candidates but are not currently involved in a reproductive program. Different species
require different anatomical sites for insemination if Al is to be successful. For domestic
cattle, AI is most effective when semen is deposited in or through the cervix (Knight et al.,
1951), whereas intrauterine insemination is required for red deer (Fennesy et al., 1990),
and vaginal insemination is adequate for dogs (Seager and Fletcher, 1973). Based on
elephant reproductive anatomy, penetration by the bull is probably limited to the upper
one third of the urogenital canal (Balke et al., 1988a). Ejaculation during natural mating
appears to occur in the urogenital canal, but the volume of semen and mechanical action
provided by the penis would be difficult to duplicate with AI, therefore it is believed that
insemination would be more successful if semen was deposited in the cervix or uterus
(Balke et al., 1988b). The long urogenital canal, constriction and potential hymen at the
urogenital-vaginal junction, and tight cervix necessitate the use of a fiberoptic scope, at
least 120 cm in length, and specialized insemination catheters to overcome these obstacles
during AI attempts. These expenses, along with the costs of semen collection,
preservation and transport, transrectal ultrasound equipment, and hormonal analyses can
make AI trials unfeasible for many institutions. Development of a cooperative AI program
among institutions housing females that have been identified as good reproductive
candidates would not only improve the odds of developing a successful AI procedure, but
would also synergistically increase elephant conservation efforts among participating
institutions.
Collection of semen from bull elephants has been done by either utilizing artificial
vaginas (with varying success), or rectal-probe electroejaculation. A 9 cm diameter probe
with electrodes concentrated in only 25% of the circumference, which focuses stimulation
on the accessory glands, has been successfully used to collect semen when delivering 10-
30 V in a repetitious manner (Howard et al., 1984, 1985). Typical ejaculate samples from
African elephants have means of 93.3 ± 48.4 ml in volume (range: 20-425 ml), contain
24
2409 ± 521 x 106 sperm/ml (range: 88-4760 x 106 sperm/m1), have 70 ± 6% sperm
motility (range: 60-95%), and pH of 7.4 ± 0.1 (n=9; Howard, 1984). For an Asian bull,
the ejaculate volume is usually 200-300 ml (Schmidt, 1993). Spermatozoa appear to have
greater post-thaw motility and longevity in vitro when derived from less viscous semen
(Howard et al., 1986). Variation in accessory gland stimulation during electroejaculation
probably accounts for differences in seminal viscosity (Howard et al., 1986), with the
bulbourethral glands secreting a more viscous fluid as compared to the watery secretions
of the seminal vesicles (Short et al., 1967).
African elephant sperm motility decreases by 50% within 3-5 hours post-collection
(Howard et al., 1984), and 80% of the sperm have vigorous motility 1 hour after
collection when stored at 300 C (Ruedi and Kuepfer, 1981). Semen extenders are
available for storage at either room temperature or 4 °C (Schmidt, 1993). Techniques for
the cryopreservation of sperm are still being perfected. Studies with African elephant
semen by Howard et al. (1985, 1986) report varying results using different diluent
extenders, yet later studies showed no significant differences. Pelleted semen resulted in
significantly higher post-thaw spermatozoa) motility than straw-frozen semen (46 versus
17%, respectively; p<0.01), acrosomal integrity was better maintained when the initial
cooling rate was faster (6.5 °C /minute versus 1.5°C/minute), duration of post-thaw
motility in vitro was greater when physiological saline, rather than tissue culture solution,
was used as the thawing medium, and longevity of motility was longer for post-thaw
spermatozoa maintained at 21 °C rather than 37 °C (p<0.001; Howard et al., 1986).
Semen cryopreservation techniques warrant further investigation because, even given
reported optimal conditions, post-thaw sperm motility does not exceed 40-60% and
rapidly declines to 20-40% or less within the first 3 hours (Howard et al., 1986).
Many factors are involved in the natural control of reproduction and should be
considered when devising a plan of contraception for animals. Age, health status, level of
nutrition, and social effects, such as pheromonal stimulation of estrus, and delayed
25
breeding due to stress or competition, as well as environmental factors, such as seasonal
influences, all contribute to variations in fertility (Seal, 1991). Goals for the development
of a wildlife contraceptive agent should include reversibility to maintain the gene pool, a
method for remote delivery of contraceptives, minimal effects on social behavior and
structure, lack of toxicity to pregnant animals and to other animals through the food chain,
low cost, and high level of effectiveness (Kirkpatrick and Turner, 1991). Culling is often
an integral component of a contraceptive program, initially bringing population sizes to
within the carrying capacities of parks. Potential disadvantages of culling include
disruption of social structure, induction of a population growth response, permanent loss
of individual genomes, and public protest in regards to the morality of culling practices.
For wild elephants, translocations of entire family groups have been accomplished, but are
costly and only provide a short-term solution to overpopulation due to limited habitat
availability. Contraception offers a viable alternative to costly translocations and culling,
and methods pursued for use in elephants have included castration, gonadal injections of
caustic agents, immunocontraception, and hormonal analogues.
Castration of elephants is done primarily to increase docility of bulls kept in
captivity. A variety of techniques and equipment used for castration has been reported in
the literature (Fowler and Hart, 1973; Flanagan and Flanagan, 1983; Olsen and Byron,
1993; Foerner et al., 1994). It is generally recommended that males less than 5 years of
age be castrated because both testicles can then be reached through a single abdominal
incision (Olsen and Byron, 1993; Foerner et al., 1994). The best approach seems to be
through a left flank incision with the uppermost rear leg extended posteriorly, to remove
the contralateral right testicle first since it has a longer spermatic cord and tunic than the
left testicle (Olsen and Byron, 1993). Difficulties encountered during the surgery include
coping with large amounts of fat and areolar tissue beneath musculature (Fowler and Hart,
1973; Olsen and Byron, 1993), ligating vessels prior to removal of the testicle due to a
short stalk (Flanagan and Flanagan, 1983), dealing with highly mobile, abundant greater
26
omentum, and getting through the tough peritoneum (Olsen and Byron, 1993; Foerner et
al., 1994). Testicular injections of caustic agents have not been successful (Olsen and
Byron, 1993; Foerner et al., 1994). Other techniques to control or prevent musth in bulls
include injections of Lupron (a GnRH agonist), reduced rations (Schmidt, 1993), and
chaining (Kurt, 1970). Lupron can decrease serum concentrations of testosterone to those
of castrated bulls in about 3 weeks, but the effects on spermatogenesis are not known
(Schmidt, 1993).
Working with the immune system to inhibit reproduction requires a multifaceted
approach because responses are affected by stress, nutrition and hormone interactions
(Hunter, 1989). The porcine zona pellucida (PZP) has been the most studied because it is
readily available from abattoirs. The PZP vaccine may work by inhibiting fertilization
(e.g., blocking sperm receptor sites), or possibly preventing implantation (Turner et al.,
1992). Among 35 species immunized with PZP vaccine, contraceptive efficacy ranged
from 77-100% (Kirkpatrick et al., 1993). A recent study with six wild elephants in Kenya
demonstrated that significant levels of antibodies were produced in response to PZP
vaccinations (Schwoebel et al., 1997), and trials are currently underway in Kruger
National Park to investigate the effectiveness of the PZP vaccine as a contraceptive agent
when administered to feral elephants. Unfortunately, these vaccines have caused ovarian
histopathology and dysfunction in several mammalian species (Tesarik et al., 1990),
because the zonae antibodies bind to developing follicular ZP, as well as to zona of
ovulated ova (Kirkpatrick et al., 1992). It has been suggested that directing antibodies
against cumulus antigens may reduce negative effects on the ovary because these antigens
appear late in preovulatory development, and therefore antibodies would primarily focus
on mature follicles (Tesarik et al., 1990). It is not yet known if the PZP vaccine will serve
as an effective, long-term, reversible contraceptive agent for elephants. If it does prove to
be efficacious, a future challenge will be the development of an effective remote-delivery
system since elephants are currently being anesthetized for vaccine administration.
27
Exogenous hormonal analogues have been used as reversible contraceptive agents
for quite some time. Gonadotropin-releasing hormone (GnRH) analogues have been used
in free-ranging African bull elephants, but with mixed results (Brown et al., 1993).
Gonadotropin-releasing hormone agonists cause a decrease in gonadotropin secretion by
hyperstimulating the pituitary until desensitization occurs, but temporary increases in
gonadotropin concentrations are side effects. When Lupron was administered to feral
African male elephants, partial desensitization of the pituitary occurred with lower LH
release, yet the testicles had increased levels of testosterone production (Brown et al.,
1993). Antagonists to GnRH compete for binding sites and cause an immediate
suppression of gonadotropin production, but higher and more frequent doses are usually
required compared to agonists. Lower LH and testosterone concentrations (p<0.05) were
detected in bull elephants given 4 or 12 mg Detirelix (a GnRH antagonist) i.m., followed 2
days later by a GnRH injection, as compared to saline-injected controls (Brown et al.,
1993). Antigens for LH, FSH, luteinizing hormone-releasing hormone (LHRH; also
referred to as GnRH), and prostaglandin F2a (PGF2a) can also be targeted to decrease
ovulation or spermatogenesis (Reeves et al., 1989), but, to my knowledge, work in this
area has not yet been attempted with elephants. The potential contraceptive effect of
exogenous estrogens, delivered through implants, is currently being investigated in a
group of feral elephants located in Kruger National Park. Further work will determine the
potential role hormonal analogues will play as contraceptive agents for wild elephants.
Conclusions
New questions and research needs have been created by recent intriguing findings
regarding the female elephant's estrous cycle, hormonal profiles, and initial research
conducted to develop assisted reproduction techniques and contraceptive agents. For
example, what is the purpose of the anovulatory LH surges that occur so consistently, and
how is their timing regulated? What are the effects on reproductive cyclicity and
28
performance when tropical elephant species are moved to temperate climates, if any?
What portion of the measured increase in serum concentrations of PRL during pregnancy
is due to cross-reacting placental lactogens, and what role do placental lactogens
potentially play during pregnancy? A standardized progesterone metabolite assay would
make uniform comparisons between studies possible, and continued development of
hormonal assays, designed to non-invasively monitor endocrine profiles in elephants,
would expand our database. Potential pheromonal and social-behavioral effects on
elephant reproduction merits investigation. Why do only 34% of the calves born in
captivity survive to subadult ages? Further development of assisted reproduction
techniques, as well as reversible contraceptive alternatives, will enable us to maintain
genetic diversity among captive populations of elephants, and keep wild populations
within the carrying capacities of reserves. Learning more about the unique, basic aspects
of elephant reproduction will be challenging and will guide our efforts in the development
of assisted reproduction and contraceptive techniques.
29
Seasonal Reproduction
Natural selection favors adaptations that cause reproduction to occur in harmony
with environmental variations. Seasonal dietary and physical factors, such as photoperiod,
food availability, rainfall, humidity, and temperature, can affect reproductive potential and
strategy, as can the energetics of reproduction (e.g., body size, life span, length of
gestation), social cues transmitted by dominance behaviors and/or pheromones, and
various physiologic and reproductive adaptations, like delayed implantation and
hibernation. Whether these factors are utilized by each animal to influence reproductive
effort is highly species specific. Some species have evolved as long-day breeders (e.g.,
mink, horses and hamsters), whereby increasing hours of daylight during spring months
induce cycling. Other species, like sheep, evolved as short-day breeders and mate in the
fall. For both groups, due to variations in reproductive biology, births usually occur in the
spring or summer so that young can take advantage of plentiful food resources and have
time to prepare for harsher winter conditions.
Many examples serve to illustrate the diversity of reproductive strategies which
can result in aseasonal, or seasonally facultative, obligatory and opportunistic breeders.
For example, the effects of photoperiod, inseparable from the effects of food availabililty
and temperature, can be seen when comparing the temperate versus tropical breeding
seasons of three lagomorph species. The arctic hare, geographically limited to areas north
of 700 latitude, can produce only 1 litter per year, whereas the snowshoe hare, between
40-700 latitude, typically produces 2-3 litters per year over a 5-6 month breeding season,
and the cottontail rabbit is able to breed year-round below 300 latitude (reviewed by
Bronson and Heideman, 1994). This shift in seasonality occurs further north in smaller
animals due to shorter life expectancy and higher reproductive rates. If similar
reproductive cycle comparisons were made for different ungulate species, aseasonal
reproduction would become more obvious below 100 latitude (Bronson and Heideman,
1994).
30
An animal's breeding period may fall within a very narrow window of opportunity.
As an obligatory seasonal breeder, the brown marsupial mouse (Antechinus stuartii) of
Australia mates only during a 2-week period once a year so that lactation is timed to
coincide with the peak in abundance of their primary food source, insects (Bronson and
Heideman, 1994). Occasionally, only one of the sexes may have a seasonal reproductive
cycle (e.g., female pygmy bats; Haplonycteris fischeri), or females, having to invest much
energy into pregnancy, birth and lactation, may demonstrate a stronger seasonal
reproductive cycle than males (Heideman, 1989). Seasonal patterns of reproduction are
slightly different for ewes and rams, suggesting that the mechanisms influencing these
rhythms are sexually differentiated (Lubbers and Jackson, 1993). Australian sea lions, have
a reproductive cycle that averages 17.5 months (reviewed by Bronson and Heideman,
1994), making it clear that the mechanisms underlying seasonal reproductive patterns in
animals are complex and not yet well understood.
This section will review how certain environmental cues, such as photoperiod,
temperature, and nutritional state, appear to influence seasonal reproduction, and the roles
melatonin, prolactin and cortisol have in influencing the cyclicity of reproduction. The
interdependence of these factors make cause and effect relationships difficult to define. A
brief review of the contribution each factor is thought to make in influencing seasonal
reproduction will hopefully facilitate our understanding of how the neuroendocrine system
translates environmental signals to link together photoperiodic, nutritional and social cues
with the reproductive system.
Environmental Cues: Photoperiod
Photoperiod has been linked to seasonality of reproduction in numerous temperate
species. Long life spans and living at higher latitudes generally promote dependence on
photoperiodic cuing (Bronson and Heideman, 1994), and animals that rely on seasonal
food sources are more likely to have their reproductive cycles controlled by photoperiod
31
(Menaker, 1971). Evolutionarily, the same neuroendocrine pathway, common to all
photoperiodic species, is used in different ways to adapt the reproductive process of a
given species to a particular ecological situation. Experiments conducted with males to
investigate testicular growth cycles best illustrate this difference. As short-day breeders,
ram testicular weights increase during winter months (Lincoln and Peet, 1977), in contrast
to Syrian hamster testicular weights, which increase during summer months from 2-5 to
20-25 mg/g body weight (reviewed by Bronson and Heideman, 1994). Seasonal changes
in spermatogenesis and testicular steroidogenesis have recently been documented in black
bears as well, and implicate a role for estrogen in re-initiating spermatogenesis during
gonadal recrudescence (Tsubota et al., 1997). Thus, photoperiod acts as a trigger to set in
motion a cascade of neuroendocrine events that orchestrate seasonal reproductive
cyclicity.
Experiments involving transfer of temperate species across the equator such that
photoperiod and other environmental factors were shifted by 6 months, resulted in
eventual adjustments in the annual reproductive cycle and further illustrate the importance
of photoperiod in timing reproductive cyclicity (reviewed by Karsch et al., 1984).
Subsequent similar studies demonstrated that light was the primary environmental cue
responsible for these changes which, along with the influence of olfactory cues (at least in
sheep), affected the LH pulse generator and ovulation (reviewed by Karsch et al., 1984).
An endogenous rhythm of reproduction exists in many species and photoperiod
serves to synchronize this rhythm to season (reviewed by Reiter, 1974; Karsch et al.,
1989; Brinklow and Loudon, 1993; Kaplan and Katz, 1994). Blinded ewes, kept separate
from rams, still maintain a circannual reproductive rhythm, lending credence to this belief
(Karsch et al., 1984). This endogenous rhythm is maintained for several breeding seasons
or up to 3 years following pinealectomy, superior cervical ganglionectomy, or exposure to
constant photoperiod (reviewed by Henniawati et al., 1995).
32
The importance of photoperiodic history in determining the reproductive response
to a photoperiod challenge was underscored in a recent study in which ewes were
challenged by exposure to long- followed by short-days at different times during the year,
and were also given a challenge after having their photoperiodic history normalized
(Sweeney et al., 1997). The reproductive responses were indeed different during different
times of the year, and, while the majority of ewes with normalized photoperiodic histories
responded similarly, there were slight differences in the duration of reproductive activity
based on the time of year the challenge was given, indicating that other factors (perhaps
the phase of the endogenous rhythm) may influence reproductive activity (Sweeney et al.,
1997). Photoperiodic history can actually develop in utero. Melatonin receptors have
been identified in the brain and pituitary of fetal lambs and deer (Helliwell and Williams,
1990; Helliwell et al., 1991, respectively), and melatonin can cross the blood brain barrier
(Kopin et al., 1961). Melatonin can also cross the ovine placenta (Yellon and Longo,
1987), but prolactin cannot (Thomas et al., 1975). Based on this research, postnatal
prolactin secretion was measured and it was found that fetal and neonatal deer can
respond to photoperiodic information received as melatonin signals from the mother in
utero, and that this photoperiodic history influences postnatal responses to photoperiod
(Adam et al., 1992).
Photorefractoriness can occur and has been demonstrated in several species
(Nicholls et al., 1988). Male starlings (long-day breeders) treated with prolonged
exposure to long days required several weeks of decreasing daylength or exposure to
short days for photoresponsiveness to recur (Boulakoud and Goldsmith, 1995).
Photorefractoriness has also been demonstrated in goats exposed to short daylengths more
than 7 months (Kaplan and Katz, 1994).
Photoperiod has been shown to affect the onset of puberty in cattle (Peters et al.,
1978; Petitclerc et al., 1983a, b), sheep (Parkinson et al., 1995), and ferrets (Ryan and
Volk, 1995). Supplemental lighting appears to increase circulating concentrations of PRL
33
and progesterone in water buffalo heifers, suggesting that photoperiod plays a role in
initiating reproductive cyclicity in this species (Perera et al., 1989), and photosensitivity is
required in male starlings as well, for attainment of sexual maturity in response to
increasing daylength (Boulakoud and Goldsmith, 1995). Using sheep as a model,
photoperiod is believed to regulate onset of puberty by changing hypothalamohypophyseal sensitivity to estradiol inhibition of LH secretion (reviewed by Stormshak,
1997), and insensitivity to estradiol's inhibitory effect occurs earlier in males than females
(reviewed by Foster, 1994). Thus, the timing of puberty in sheep is differentially regulated
by sex, and is believed to be based on androgenization of the system that relays
photoperiodic information through melatonin to affect GnRH secretion (Foster, 1994).
Male lambs are either less sensitive to photoperiodic cues, or utilize them differently than
females, and this is a post-pineal difference because patterns of melatonin secretion do not
vary between the sexes (Foster, 1994). Additionally, the attainment of puberty is a
thyroid-dependent event in males, whereby thyroidectomy hastens the onset of puberty for
male lambs and of the breeding season for rams (Parkinson et al., 1995). However,
puberty is not differentially regulated by sex in all species. For some long-day breeders,
short-lived species, or species that mature over a long period of time, the advantage of
early puberty in the male is lost, so puberty is attained at more similar ages for both sexes
(Foster, 1994).
Thyroid hormones were shown to be required for the induction of anestrus in ewes
through experiments with thyroidectomized animals, which did not respond to long day
photoperiod exposure with decreasing concentrations of LH, as did intact animals (Dahl et
al., 1994). There were no differences in serum concentrations of PRL or melatonin
between control and thyroidectomized ewes, suggesting that the thyroid can somehow
uncouple the reproductive neuroendocrine axis from photoperiodic influence (Dahl et al.,
1994). The role of thyroid hormones during induction of the breeding season is less clear.
Thyroidectomizing males of certain species during the non-breeding season caused a
34
premature increase in testicular weight gain (Parkinson and Follett, 1994; Thrun et al.,
1997). However, a recent study demonstrated no role for thyroid hormones in the onset
of the breeding season for ewes ( Thrun et al., 1997), suggesting that the effect that thyroid
hormones have on reproductive activity is dependent on sex.
Steroid hormones are also involved in affecting annual reproductive cycles. For
example, estradiol can slow the LH pulse generator of ewes in the summer but not in the
winter months (Karsch et al., 1984). It is not yet known how steroids interact with
photoperiod, and olfactory and pheromonal cues to affect changes in LH secretion. It has
been proposed that pituitary sensitivity to the negative feedback of estradiol is influenced
by photoperiod (Karsch et al., 1984), while another study suggests that the positive
estradiol feedback mechanism, responsible for perpetuating the pre-ovulatory LH surge,
may be dependent on a photoperiodic signal for activation (Henniawati et al., 1995).
Photoperiod may also inhibit LH secretion independent of steroid feedback (Robinson et
al., 1985; Curlewis, 1992). Additionally, the synaptic input to preoptic GnRH neurons in
ewes changes seasonally, with a twofold decrease in synaptic density during anestrus
compared to the breeding season (Thrun et al., 1997; Xiong et al., 1997). A recent
investigation used male deer mice (Peromyscus maniculatus) as a model to investigate the
effects of photoperiod on GnRH secretion (Korytko et al., 1997). All mice exposed to a
short day photoperiod had a significant increase in total brain GnRH content as compared
to those exposed to a long-day photoperiod (p<0.02); however, LH and testosterone
concentrations varied within the group of mice exposed to a short day photoperiod.
These differences could be based on differential pituitary sensitivity to GnRH stimulation,
a possible discrepancy in equating hypothalamic GnRH content with actual GnRH
secretion into the hypothalamic-hypophyseal portal circulation, or differences in either
dopamine or norepinephrine turnover (Korytko et al., 1997).
Sexual behavior in small ruminants, induced by steroid treatments, is also
dependent on season. Following administration of 25-300 .ig estradiol, female goats
35
exposed to short photoperiods were significantly more receptive to males than females
exposed to long photoperiods (Kaplan and Katz, 1994).
Abrupt changes in daylength have mixed results on estrous cyclicity in pigs
(Prunier et al., 1996), as well as other species, and the varying effects of endogenous
rhythm, photoperiodic history, and time of year may be the cause of these inconsistencies.
Additionally, age-related changes have been detected in Siberian hamsters, whereby they
become less responsive to the inhibitory effects of short days regardless of photoperiodic
history (Bernard et al., 1997). This variable could contribute to inconsistent results seen
in response to photoperiod in other animals, since an alteration in circadian rhythmicity
occurs in advanced age in several species (Bernard et al., 1997).
Environmental Cues: Temperature
Ambient temperature can augment the effects of photoperiod on the reproductive
system or, in some cases, even dominate them. Figure 4 depicts a synergistic effect of
short day photoperiod and colder temperature on diminished testicular sperm
concentrations in adult golden hamsters (Bronson and Heideman, 1994). In domestic
pigs, variation in ambient temperature has been shown to have a more significant effect
(p=0.013) on the return to estrus after weaning than the variation in light duration
(p=0.65; Prunier et al., 1996), with higher temperatures increasing serum PRL
concentrations (Griffith and Minton, 1992).
Temperatures at either extreme can inhibit reproduction. A negative correlation
between high summer temperatures and conception rates in humans has been observed
(reviewed by Short, 1984). Conversely, ovulation can be inhibited when temperatures
become too low. Body size, fat reserves and food intake are variables that need to be
considered when evaluating the effect of temperature on reproduction (reviewed by
Bronson and Heideman, 1994). Small mammals are especially susceptible to low
temperature because of their large surface-to-volume ratio, which results in a rapid loss of
36
4
2
20
40
60
80
Days Exposed
Figure 4. Time required for sperm to disappear from the testes of adult golden hamsters
when they are exposed to short daylengths, depending on ambient temperature (adapted
from Bronson and Heideman, 1994).
37
body heat to the surroundings. Also, the mass of young born to small female mammals is
relatively great in relation to their own size, so the energetic costs of reproduction are
greater as compared to larger mammals.
Environmental Cues: Nutrition
Nutritional status is linked to reproductive potential, and there have been several
studies demonstrating circannual cycles of growth in various species due to
photoresponsive changes in voluntary feed intakes (sheep, cattle and deer: Forbes, 1982,
rams: Kay and Suttie, 1980, cattle: Peters et al., 1980, goats: Ash, 1986; Henniawati et al.,
1995). Surprisingly, increases in body weight have been observed in prairie voles without
changes in food intake, when they were exposed to short photoperiods, suggesting a
direct effect of photoperiod on metabolic pathways (Kriegsfeld and Nelson, 1996).
Gonadal steroids seem to be, in part, responsible for mediating these effects of
photoperiod (Kriegsfeld and Nelson, 1996). Photoinducible rhythms in appetite can
persist for up to 3 years following a pinealectomy or superior cervical ganglionectomy
(Bittman, 1985), again supporting a role for an endogenous rhythm, this time influencing
seasonal weight changes.
Nutritional status has long been recognized as an important factor affecting
reproductive success (Rhind et al., 1985), but insights regarding potential mechanisms
linking nutritional status with the neuroendocrine-reproductive axis have only recently
been made. Neuropeptide-Y (NPY) can decrease sexual behavior in rats (Clark et al.,
1985; White and Martin, 1997), and is dependent on circulating glucocorticoids for degree
of activity. Because NPY concentrations in the suprachiasmatic nucleus vary with
changes in the light:dark cycle, NPY may have a role in photic entrainment of circadian
rhythms (White and Martin, 1997). Corticosterone appears to stimulate, while insulin and
leptin inhibit NPY secretion (White and Martin, 1997).
38
Postbreeding nutritional status of beef heifers has been shown to significantly
affect pituitary responsiveness to GnRH (Leers-Sucheta et al., 1994). Leptin has been
shown to stimulate the reproductive endocrine system of rats by either activating or
serving as a permissive signal to the reproductive axis (Barash et al., 1996). Leptin relays
information about the body's fat content to the brain, decreases food intake, and increases
body temperature (Pellymounter et al., 1995). Additionally, leptin decreases
glucocorticoid-stimulated hypothalamic NPY release (Stephens et al., 1995), reduces
circulating corticosterone concentrations (Steiner, 1996), possibly inhibits NPY synthesis
and release (White and Martin, 1997), and has a permissive role in the onset of puberty in
rats (Cheung et al., 1997).
Increased concentrations of PRL have been positively correlated with food intake
and body weight in several species (Ryg and Jacobsen, 1982; Eisemann et al., 1984a;
Gerardo-Gettens et al., 1989), although the mechanism of action is not clear.
Bromocriptine (a dopamine agonist) has been shown to decrease the appetite and growth
rate in red deer, and it has been suggested that dopamine plays a role in this suppression
(McMahon et al., 1997). Melatonin and gonadal steriods are also likely to have effects on
body mass regulation (Kriegsfeld and Nelson, 1996), and conversely, melatonin secretion
can be altered by food restriction (reviewed by Bronson and Heideman, 1994). Estrogens
increase the number of PRL receptors in rat livers (Posner, 1976; Eisemann et al., 1984b),
and administration of diethylstilbestrol to ruminants increases PRL, growth hormone and
thyroid-stimulating hormone concentrations, and enhances accretion of lean body mass
(Eisemann et al., 1984b). Penguins abandon reproductive activity when body protein
stores are mobilized, and during this time they have increased concentrations of
progesterone and decreased concentrations of prolactin and testosterone (Cherel et al.,
1994). Finally, the effects that the corticotropin-releasing factor (CRF)adrenocorticotropin-adrenal steroid axis can have on reproductive potential are magnified
during food restriction, since this hyperactivates the axis (Bronson and Heideman, 1990).
39
Environmental Cues: Pheromones and Estrous Synchronicity
Pheromones are known to have profound effects on the reproductive system of
many species, mediating the onset of puberty in females, ovulation, and estrous cycle
synchronicity (Keverne, 1983), as well as playing a role in mate selection and individual
recognition (reviewed by Vandenbergh, 1994). Well-known are the Lee-Boot and
Whitten Effects, first documented in mice in the late 1950's (reviewed by Short, 1984).
The odor of a male mouse can reinstate cyclicity in a group of females and is known as the
Whitten Effect. Similarly, the presence or smell of a ram can increase the frequency of LH
pulsatile secretion in ewes, decreasing the time to ovulation (Martin, 1984). The LeeBoot Effect is characterized by estrous suppression among females, the strength of
suppression being positively correlated with population density. This dose-dependent
relationship suggests an important role for the female's social environment (McClintock,
1981) and potential influence of the adrenal gland and stress mechanisms (Marchlewska-
Koj, 1983). However, in other species, like rats and humans, ovarian cycles can be
synchronized and enhanced rather than suppressed by the effects of female social groups
(McClintock 1971, 1978).
There are numerous other effects pheromones can have on the reproductive
system. Releasing a chemosignal around the time of ovulation is adaptive for females, to
ensure the timely presence of a male when conception is possible (Vandenbergh, 1994).
Inbreeding has been shown to be minimized through the sharing of chemosensory
information present in mouse urine, which enables individuals to recognize one another
(Vandenbergh, 1994). Priming pheromones have been shown to affect the timing of
puberty by either accelerating or inhibiting its onset in several species, and androgens can
increase, while adrenal hormones decrease, the production of these pheromones (reveiwed
by Vandenbergh, 1994). Primer pheromones appear to decrease PRL secretion (probably
as a result of increased dopamine release), and increase LH concentrations (Keverne,
1983). Pregnancy can be blocked in mice by pheromonal stimuli from a foreign male,
40
which act through decreased PRL concentrations to prevent corpus luteum formation and
subsequent implantation (Marchlewska-Koj, 1983).
Urine, feces, saliva and the skin with its glands can all serve as sources of
chemosignals (Vandenbergh, 1994). Gonadal steroids can influence both the emission and
reception of chemosignals, and sexual experience or learning may also play a role in the
efficacy of pheromonal transmission (reviewed by Vandenbergh, 1994). Hormonal status
of female elephants appears to affect their receptivity to chemosignals from both males and
other females (R. Schulte and L.E. Rasmussen, personal communication).
Elephants utilize chemosensory signals during mate selection (L.E. Rasmussen,
personal communication). Females have been able to distinguish between musth and nonmusth urine and their level of interest appeared to be related to stage of estrous cycle
(L.E. Rasmussen, personal communication). Several compounds have been identified as
chemosignals in elephants: (1) cyclohexanone appears to be a male-to-female signal
present in temporal gland secretions during musth, (2) (Z)-7-dodecen-1-y1 acetate
(Rasmussen et al., 1997), present in female preovulatory urine, heightens sexual behavior
and flehmen responses in primarily dominant bulls, and (3) volatile signals from
respiratory, urinary, and temporal gland secretions can be released by dominant males in
musth and may negatively affect the duration of musth experienced by subordinate males
(L.E. Rasmussen, personal communication). It is interesting to note that the sex
pheromone, (Z)-7-dodecen-1-y1 acetate, is the same compound that is found in over 100
species of Lepidoptera where it serves a similar function, relaying female-to-male
chemosensory information (L.E. Rasmussen, personal communication). The social status
of bull elephants appears to affect their behavioral response to (Z)-7-dodecen-1-y1 acetate,
because younger, subdominant males would either ignore or back away from samples
containing the pheromone (Rasmussen et al., 1997). Hormonal and social status,
therefore, both appear to modulate chemosensory communication among elephants.
41
Seasonal reproduction can result from ecological and social factor interactions
(McClinctock, 1981; Clarke et al., 1992), potentially coordinating and fine-tuning
reproductive efforts in species not dependent primarily on environmental cues for cyclicity.
Synchronicity may only exist locally, for example within a pride of lions (Bertram, 1975),
a group of captive lion-tail macaques (Clarke et al., 1992), or a clutch of North Atlantic
gannets (McClintock, 1981), and when entire populations are examined, reproductive
cycles may appear asynchronous. For sheep, members of the same flock can influence
onset of the breeding season (O'Callaghan et al., 1994), even to the point of synchronizing
the reproductive neuroendocrine response to photoperiod in pinealectomized ewes, which
are not capable of responding to photoperiodic cues on their own (Wayne et al., 1989).
Synchronous estrous cycles among females are more likely to occur in established
all-female groups, and are achieved through auditory, visual, olfactory or body contact
cues. In general, it is not yet known if synchrony results from signals originating from a
leader within the group or from interactions among equipotent individuals, although
evidence suggests the possibility of the former being true for some species (McClintock,
1981). If one female is more responsive to environmental cues, she could serve as the
leader, integrating this information into the group through social interactions and chemical
cues (McClintock, 1981). For female sable antelope, both the frequency of flehmen
responses and the ability to time births synchronously with herdmates were highly
correlated with dominance rank (Thompson, 1995).
The advantages to birth synchrony and reproductive seasonality have been
documented in numerous species. Synchronous births could minimize predation through
group protection of young (Estes, 1976), increase gregarious behavior in females, ensure
calves are of the same age so they can move with the herd (Dauphine and McClure, 1974),
maximize feeding efficiency through social foraging (Emlen and Demong, 1975), and
facilitate socialization of similarly-aged young (Clarke et al., 1992). Prolactin-dependent
42
pheromones may lengthen estrous cycles in rats, permitting lactating females to cycle and
conceive again with their cohort (McClintock, 1983).
Clearly, an understanding of the ecology, social structure, mating system, and
seasonal or other reproductive strategy of a species is useful when interpreting the
potential effects pheromones might have on the reproductive dynamics of individual
animals.
Hormones Affected by Environmental Cues
Melatonin: A Neuroendocrine Transducer of Photoperiod
It is known that most classes of vertebrates (birds, reptiles and fish) are able to
respond to changes in photoperiod by utilizing extraretinal photoreceptors located deep in
the brain, and normal daylight has even been shown to activate photosensitive probes
implanted surgically in various regions of the brain, including the hypothalamus, of sheep
(reviewed by Karsch et al., 1984). Subsequent experiments, however, have demonstated
that retinal photoreceptors are required for timing the annual reproductive cycle in the
ewe.
The light signal is received by photosensitive retinal cells and is then transferred
along retinal hypothalamic tracts to the suprachiasmatic nucleus, through the medial
forebrain bundle, along the thoracic spinal cord, continues through preganglionic
sympathetic nerves to synapse in the superior cervical ganglion, and finally ends in the
inhibition of norepinephrine release from postganglionic nerves synapsing on the pineal
gland (reviewed by Illnerova, 1991). Any lesion between the suprachiasmatic nucleus
(SCN) and the pineal gland can alter rhythms induced by the light-dark cycle. It is
possible that the SCN is responsible for perpetuating an endogenous rhythm of
reproductive cyclicity when animals are kept under constant photoperiod, hence the
nickname "biological clock".
43
Pineal glands tend to be larger in animals living in arctic or antarctic regions than
those occupying temperate or tropical regions (Reiter, 1974), and the pineal gland of the
elephant is relatively small (Lincoln, 1984). The pinealocyte functions through protein
kinase A (PKA) to increase the activity of the enzymes, N-acetyltransferase (Borjigin et
al., 1995) and hydroxyindole-O-methyl-transferase ( HIOMT), which convert serotonin to
melatonin, an indoleamine (Kopin et al., 1961). Melatonin's half-life is slightly greater in
cerebrospinal fluid (CSF) than in blood (40 versus 30 minutes; Reppert et al., 1979), and
approximately 70% of the melatonin in plasma is bound to albumin; however, none is
bound to proteins in CSF (Cardinali et al., 1972). Melatonin is metabolized within the
body by 6-hydroxylation followed by conjugation, mainly with sulfate, and remaining
metabolites are excreted primarily in the urine (Kopin, et al., 1961; Kveder and Mclsaac,
1961).
It has been shown that the duration of the noctoral rise in melatonin is more
important than the amplitude in affecting a response to photoperiod, and that melatonin
can determine the reproductive rhythm, and does not merely play a permissive role
(reviewed by Karsch et al., 1984). During winter months, the diurnal pattern of melatonin
secretion is altered so that the animal is exposed to nightly concentrations of melatonin for
a greater amount of time (Figure 5). Additionally, melatonin receptor densities in the
anterior pituitary have been shown to decrease in the mornings in rats maintained on 50:50
light:dark cycle (Vanecek et al., 1990), concurrent with an observed decrease in sensitivity
to melatonin early in the day. There are conflicting reports regarding potential feedback
control exerted by target organs on pineal function. In rats, sheep, horses, and humans
HIOMT activity was found to vary during the estrous cycle (reviewed by Cardinali, 1981);
however, a more recent study by Zarazaga et al. (1996) observed that diurnal patterns of
melatonin secretion in ewes were not altered by steroid treatments or by phase of estrous
cycle. Receptors for estradiol, testosterone, 5a-dihydrotestosterone, progesterone, and
44
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as
E
cn
co
30
cr.
20-
10
0 II
09.00
13.00
_
17.00
21.00
-
01.00
Time (h)
05.00
09.00
Figure 5. Mean (± SE) concentrations of melatonin in plasma of animals held on (a)
simulated natural (SN; n=5) and (b) summer solstice hold (SSH; n=5) photoperiods with
samples taken every 2 h for 26 h on 16-17 December, 1987. The period of darkness (SN
14 h 47 min; SSH 5 h 44 min) is shown by the hatched bar (adapted from Brinklow and
Loudon, 1993).
45
PRL have been found in pineal glands of various species (Cardinali, 1981), so steroidal
modulation of pineal function is plausible. Moreover, experiments support the theory that
the activity of afferent sympathetic neurons controls the sensitivity of the pineal gland to
circulating hormones (Cardinali, 1981).
Patterns of melatonin secretion are not always been easy to detect in some species.
For example, diurnal patterns of melatonin secretion are not always observed in domestic
pigs as they are in European wild pigs (reviewed by Griffith and Minton, 1992). In
contrast to domestic breeds, male and female European wild pigs are true seasonal
breeders, and do exhibit a circadian rhythm of melatonin secretion as well as seasonal PRL
cycles (Mauget, 1982; Ravault et al., 1982). However, greater intensity lighting (e.g.,
approximately 1,783 versus 113 lx), as well as increased duration of lighting, has been
shown to influence melatonin and PRL concentrations in domestic pigs (Prunier et al.,
1996). Regardless, nocturnal increases in serum concentrations of melatonin are not
necessary for gilts to attain puberty (Bollinger et al., 1997; Diekman et al., 1997; Diekman
and Green, 1997).
As mentioned previously, maternal melatonin, but not PRL, can cross the ovine
placenta to enter the fetal circulatory system (Yellon and Longo, 1987). Newborn lambs
may require 2-3 weeks to develop their own diurnal melatonin rhythm, yet changes in
photoperiod within the first week of life quickly result in changes in serum concentrations
of PRL , suggesting that melatonin may still be passed to the lamb through colostrum and
milk from the ewes (Bassett, 1992).
Melatonin is primarily known for its role as the neuroendocrine transducer of
photoperiod to influence reproduction and other rhythmic biological functions.
Subcutaneous implants of melatonin have advanced the breeding season and enhanced
ovulation rates of ewes (Forcad et al., 1995). Melatonin receptors have been identified in
the gonads of various mammalian and avian species, and an in vitro study suggests a direct
46
action of melatonin to lower responsiveness of avian ovarian granulosa cells to LH for
progesterone production (Murayama et al., 1997).
G-protein-coupled melatonin receptors have been identified in various regions of
the brain of domestic dogs, including the pars tuberalis, pars distalis, the SCN, and
olfactory epithelium suggesting potential roles for melatonin in translating photoperiodic
signals, modulating gonadotropin secretion, and processing olfactory information
(Stankov et al., 1994). Actual site(s) of melatonin's action as a messenger of photoperiod
in the brain are not known. A prominent population of melatonin binding sites in the SCN
has been found in several species, including various rodents and humans (reviewed by
Bittman and Weaver, 1990; Maywood et al., 1996). However, this may not be the case
for all species because melatonin receptors have been primarily localized to the pars
tuberalis in other animals (Bittman and Weaver, 1990; Rozell and Mead, 1993; Skinner
and Robinson, 1995). The function of the pars tuberalis is not known, and cell
identification has been difficult. It appears that the majority of cells in this region do not
consist of gonadotrophs (Morgan et al., 1994), and that gonadotrophs that are present
have migrated from the pars distalis (Everett, 1994), leaving 90-95% of the parenchymal
cells in the pars tuberalis uncharacterized (Bittman and Weaver, 1990). Results of recent
studies indicate that cells in the pars tuberalis produce an unidentified peptide(s) called
tuberalin, which can promote PRL secretion in lactotrophs (Hazlerigg et al., 1996;
Morgan et al., 1996). Perhaps melatonin acts in the pars tuberalis to influence the
secretion of tuberalin, although a study by Malpaux et al. (1994), utilizing melatonin
implants around the pituitary stalk, suggests that melatonin acting in the pars tuberalis is
not involved in the control of PRL secretion.
Melatonin implants in the mediobasal hypothalamus have caused increased
secretion of LH (Lincoln and Maeda, 1992; Malpaux et al., 1993). In support of this
earlier research, Lincoln and Clarke (1997) utilized hypothalamo-pituitary disconnected
rams to demonstrate multiple sites of action for melatonin in the brain. They
47
demonstrated that melatonin acts primarily in the pars tuberalis to affect PRL secretion,
and in the mediobasal hypothalamus to regulate GnRH-induced LH secretion. An
extrahypothalamic region, the bed nucleus of the stria terminalis (BNST) has also been
shown to affect GnRH secretion in Syrian hamsters (Raitiere et al., 1997). Anterior
BNST subnuclei responded only to short-day photoperiodic cues by transmitting an
inhibitory signal to GnRH cells in the mediobasal hypothalamus in these animals.
Additionally, melatonin can directly inhibit the GnRH-stimulated release of LH from
gonadotrophs cultured in vitro (Venecek et al., 1990), suggesting that some melatonin
receptors are present on these cells as well. A high concentration of melatonin binding
sites has been identified in the hippocampus, where estrogen receptors can also be found,
suggesting a potential role for melatonin in mediating some sexual behaviors through
hippocampal projections into septal and preoptic areas (Bittman and Weaver, 1990).
Melatonin receptors have also been found in a wide array of red deer fetal tissues,
suggesting possible involvement of melatonin in early development (Williams et al., 1997),
in addition to acquisition of photoperiodic history which can influence postnatal responses
to photoperiod (Adam et al., 1992).
Precisely how melatonin is able to regulate PRL and GnRH-induced LH secretion
is not known, but diurnal fluctations in systemic melatonin concentrations and receptor
populations, coordinated with steroid feedback cycles, and input from other
neuroendocrine compounds (e.g., tuberalin) probably enable melatonin to have both direct
and indirect effects on the secretion of these peptide hormones. In male Syrian hamsters,
melatonin appears to affect GnRH release from cells found in the mediobasal
hypothalamus, specifically the dorsomedial nucleus, most likely through an indirect effect
through either dopamine, gamma amino butyric acid (GABA), glutamate, NPY, or (3-
endorphin in conjunction with steroid hormones (Maywood et al., 1996). It is likely that
the differences noted between species in response to photoperiodic cues arise from
differences in the way melatonin is able to act within the brain. There have been
48
discrepancies in identification of melatonin binding sites in various parts of the brain of
different animals. The complexity involved in the neuroendocrine transduction of
photoperiod is summarized by Karsch et al. (1984) who state: "What a truly remarkable
system it is that enables a circannual rhythm of reproduction to be timed by a circadian
rhythm of melatonin, which acts via a circhoral rhythm of hypothalamo-hypophyseal
activity to regulate a 16 -day estrous cycle of the ewe."
Prolactin: Structure and Secretion
Prolactin and growth hormone are thought to have diverged from a common
ancestral gene more than 4 x 108 years ago (Nicoll et al., 1986). In comparison to
prolactin (PRL), growth hormone (GH) has only one tryptophan residue and only two
disulfide bonds; a generalization that does not hold true for teleosts where PRL and GH
are more similar (Bern, 1983). In most mammalian species, with the exception of rats and
mice, PRL is a 199 amino acid polypeptide hormone with a molecular weight of 23,000-
24,000, is produced primarily by acidophilic lactotropes in the anterior lobe of the
pituitary, and is variably glycosylated, depending on the species (Neill and Nagy, 1994).
Similar to other species, elephant PRL contains three disulfide bonds and two tryptophan
residues, but has only three methionine residues compared with that of ovine PRL, which
has seven (Li et al., 1987). Elephant PRL is 72% homologous to the primary structure of
ovine PRL (Li et al., 1989).
Serum concentrations of PRL are usually inversely related to melatonin
concentrations, and secretion of PRL can be inhibited by: (1) dopamine (PRL inhibiting
factor; PIF), which travels from the arcuate-periventricular nucleus in the hypothalamus
through hypophyseal portal blood (Thomas et al., 1989), (2) GABA, from the median
eminence, (3) acetylcholine, and (4) glucocorticoids (reviewed by Neill and Nagy, 1994).
Additionally, somatostatin has been shown to decrease PRL secretion in teleosts and
amphibians (Bern, 1983) as well as rats (Jahn et al., 1993), and endothelin (ET)-1 and ET-
49
3 can inhibit PRL secretion in rats (Domae et al., 1992; Chao et al., 1994). Pituitary PRL
is stored in granules, secretion appears to depend on calcium, and release can be
stimulated by: (1) nursing, (2) stress, (3) ovarian hormones, especially estrogen (Jahn et
al., 1993), (4) thyroid-releasing hormone (TRH), (5) vasoactive intestinal peptide (VIP)
via a para and autocrine effect (Neill and Nagy, 1994), and (6) certain immunoreactive
factors (e.g., interleukins (IL)-1 and IL-6, interferon gamma) (Draca, 1995). In bullfrogs
and rats, serotonin has also been shown to increase both synthesis and secretion of PRL
(Bern, 1983; reviewed by Jahn et al., 1993).
A short-loop feedback control mechanism for prolactin secretion may exist
whereby high PRL concentrations increase dopamine turnover in the mediobasal
hypothalamus, and the higher levels of dopamine in turn suppress further PRL secretion
(reviewed by Curlewis, 1992). During exposure to an increased photoperiod, there is an
increased turnover of dopamine yet PRL levels increase (Curlewis, 1992), suggesting a
potential decrease of lactotrope sensitivity to dopamine at this time or, alternatively, direct
control of PRL secretion by PRL releasing factors (e.g., tuberalin, as previously
described).
Endogenous opioids can inhibit PRL secretion differentially in stallions, geldings
and mares, suggesting a role for gonadal steroids or products (Aurich et al., 1996).
Similarly, endogenous kappa opioids can differentially inhibit tuberoinfundibular dopamine
neurons in the median eminence to tonically stimulate basal secretion of PRL in male, but
not female, rats (Manzanares et al., 1993). Serum concentrations of LH and PRL in the
ewe increase in response to luteal regression, ovariectomy, or administration of estradiol1713 (reviewed by Deaver and Dailey, 1983). How estrogens regulate PRL secretion is
not yet clear, and most likely varies between species. For example, pituitary LH secretion
in seasonally breeding wallabyes is not directly modulated by estradiol feedback as it is in
eutherian mammals, but instead estrous cyclicity is probably controlled by the corpus
luteum held in a quiescent state by the action of PRL (Brinklow and Loudon, 1993). Both
50
natural and synthetic estrogens can increase pituitary and serum PRL concentrations, but
pituitary mRNA and PRL content in rats peaked after serum PRL concentrations peaked
(Lawson et al., 1993), indicating that secretion of stored PRL is the first response to
estrogen stimulation by lactotropes.
Thyroid-releasing hormone injections stimulated PRL release from lactotropes as
an immediate response that was not dose-related in one study by Convey et al. (1972);
however, other studies found responses to be dose-related (reviewed by Neill and Nagy,
1994). In pseudopregnant rats, TRH can only stimulate PRL secretion when dopamine
concentrations are low, suggesting that either TRH acts as a dopamine antagonist at the
level of the pituitary or that dopamine controls the responsiveness of lactotropes to TRH
(Schuiling et al., 1993; reviewed by Neill and Nagy, 1994).
Stress can affect the secretion of PRL and gonadotropins through the actions of
serotonin, and thyroid hormones, as well as corticotropin-releasing factor (CRF) and
glucocorticoids (reviewed later). Serotonin can either facilitate or inhibit LH secretion,
depending on the steroid environment, but during severe stress, serotonin is involved in
inhibition of GnRH secretion (Weiner et al., 1988). Central nervous system neurons have
high levels of nuclear thyroid hormone binding capacity, and thyroidectomy has been
shown to block the serotonin-induced increase in PRL secretion in response to stress in
rats (Ramalho et al., 1990). A recent study demonstrated that hyperthyroid rats had a
significantly diminished response to stress by secreting less PRL due to a disruption in
endogenous serotoninergic brain input (Ramalho et al., 1995). Dysthyroid states are
known to affect the expression of serotonin receptors in the brain (Mason et al., 1987),
and it appears that normal thyroid function is necessary to allow PRL release during stress
(Ramalho et al., 1995).
High ambient temperatures can elevate serum concentrations of PRL in pigs
(Becker et al., 1985; Klemcke et al., 1989; Minton et al., 1989), and dairy cows (Marcek
and Swanson, 1984). In fact, low temperatures (-00C) overrode any potential increases
51
in PRL secretion in response to photoperiod in dairy cows, and in pigs, elevated
temperatures were more highly correlated with increased PRL concentration than was
photoperiod.
There are many other factors that can influence PRL secretion, some perhaps
unique only to certain species. Teleost lactotropes increase secretion of PRL with a
decrease in osmotic pressure, and this has also been shown to occur for mammalian
lactotropes in vitro (Bern, 1983). Positive correlations between increased dietary
saturated fatty acid intake and systemic PRL concentrations in humans have been made
(Baghurst et al., 1992), and PRL concentrations declined when bull calves were fasted
(Kazmer et al., 1992). Pregnancy has long-term effects on PRL secretion (Musey et al.,
1987), and pregnant rats appear to have a decreased ability to respond to stress, which
would normally increase PRL levels in non-pregnant rats (Jahn et al., 1993). The
glutamatergic agonist, N-methyl-DL-aspartic acid, suppressed PRL secretion in mares
with differential effects depending on the stage of the cycle (Fitzgerald and Davison,
1997), indicating that stage of estrous cycle can also affect PRL secretion. Serum
prolactin, along with GH, adrenocorticotropin hormone (ACTH), and 0-endorphin,
consistently increase in concentration with exercise, while gonadotropins, especially LH,
often decline in concentration after exercise (Elias et al., 1997). Manipulations of blood
pH resulted in equivocal alterations of GH and PRL concentrations, suggesting that other
mechanisms are involved in altering hormone concentrations in association with exercise;
possibly changes in intracellular calcium concentrations (Elias et al., 1997).
Mechanisms for regulation of hormone secretion can include changes in
membrane channel permeability, electrical activity and intracellular chemical messenger
activation. Some research has been conducted to elucidate dopamine's mechanism of
action to inhibit PRL synthesis and secretion. Dopamine binds lactotrope D2 receptors to
tonically inhibit PRL secretion from the anterior pituitary by reducing adenylyl cyclase
activity, inhibiting calcium influx, and hyperpolarizing the lactotrope membrane through
52
activation of K+ conductance (reviewed by Gregerson et al., 1994a). Hyperpolarization,
followed by spontaneous depolarization, may allow for an influx of calcium ions
associated with an observed rebound effect of increased PRL secretion in response to
dopamine withdrawal (Gregerson et al., 1994b).
Circadian PRL rhythms have been described in cattle, but without a consistent
pattern. Peaks of PRL secretion were found in the afternoon (Koprowski et al., 1972;
Mollet and Malven, 1982), in the evening or early morning (Bines et al., 1983; Zinn et al.,
1986), in the morning (n=2) and late afternoon (n=4) within the same group of cows
(Lefcourt et al., 1994), or not at all (Fulkerson et al., 1980). No relationship between
milking and PRL secretion has been found (Mollet and Malven, 1982; Lefcourt et al.,
1994), although changes in photoperiod and PRL concentrations have been reported to
influence mammary development and milk secretion, but with inconsistent results (Peters
et al., 1981; Marcek and Swanson, 1984; Bassett, 1992). Midday diurnal surges in PRL
secretion were observed consistently in mares, regardless of the stage of the estrous cycle
(Rorer et al., 1987).
In summary, factors that affect PRL synthesis and secretion are numerous, and
probably reflect the need for a diverse array of control mechanisms that enable different
species and sexes to further translate photoperiod into neuroendocrine responses adapted
to their unique reproductive biology. Not all of the factors involved in regulation of PRL
secretion are likely to be of equal importance for all species, and they may vary in
effectiveness, depending on the reproductive and physiological status of the individual. In
general, blood concentrations of PRL increase during long-day photoperiods, are
positively correlated with temperature, and secretion is stimulated by nursing, acute stress,
exercise, ovarian hormones, TRH, and VIP. Systemic concentrations of PRL can
decrease in response to dopamine, GABA, acetylcholine, melatonin, glucocorticoids (in
association with chronic stress) and somatostatin. Additionally, the interrelationship
between the neuroendocrine and immune systems is just beginning to be defined, and
53
creates many more opportunities for dual modulation of PRL and immunoreactive product
secretion.
Prolactin: Function
Prolactin has functions affecting many different physiological characteristics such
as growth and development, including metamorphosis in amphibians (Hondo et al., 1995),
osmoregulation, reproduction and immunomodulation (Adler, 1991). The diversity of
effects PRL has on different systems is truly fascinating. For example, PRL can function
as a fresh-water adapting hormone in some teleosts by decreasing water reabsorption from
the bladder (reviewed by Bern, 1983). The variety of effects may in part be mediated
through changes in receptor function. Prolactin receptors in different organs within the
same species may not be identical and may become altered, depending on the physiological
state of the animal (e.g., pregancy; Nicoll et al., 1986). This section primarily reviews
PRL's actions that could potentially be linked to seasonal reproduction.
It is difficult to derive a single model to describe PRL's role in seasonal
reproduction, given that some species breed in the summer when PRL concentrations are
high while others breed in the winter when PRL concentrations are low. For example,
testicular weights in male hamsters increase in response to increasing concentrations of
PRL, whereas rams have a similar gonadal response but in association with declining
concentrations of PRL in the winter (reviewed by Curlewis, 1992). Additionally,
establishing a clear, functional link between PRL and gonadal responses has not been easy.
Melatonin administered at the onset of long days in goats, can prolong ovarian cyclicity,
but PRL suppression during anestrus does not result in an early resumption of cyclicity in
sheep (reviewed by von Brackel-Bodenhausen et al., 1994). Perhaps the stimulus for
testicular recrudescence stems from differences in translation and subsequent
interpretation of the photoperiodic message by the neuroendocrine system at the level of
54
melatonin. Differential responses to melatonin and PRL administration or suppression
indicate other factors are involved in the regulation of estrous cyclicity.
In the rat brain, some of the neurochemical effects of PRL have included: increased
GABA, 13-endorphin, and oxytocin release, and decreased LHRH release from the
hypothalamus (reviewed by Buntin, 1993). Sexual and parental behaviors, as well as food
intake, are influenced by PRL. For example, systemic PRL concentrations increase in
penguins during incubation, brooding and chick feeding (altricial young) periods (Cherel et
al., 1994), suggesting a role for PRL in affecting parental behavior. Specific binding sites
for prolactin have been identified in the brain of the ring dove (Streptopelia risoria), with
dense numbers found within the hypothalamus (preoptic area, suprachiasmatic nucleus,
paraventricular nucleus, ventromedial nucleus, and infundibular regions) (Buntin et al.,
1988). Prolactin may exert the majority of its neuroendocrine effects through direct action
on the brain. Although the blood-brain barrier precludes direct access of circulating PRL
to many CNS binding sites, a blood-to-cerebrospinal fluid transport may occur through the
epithelial cells of the choroid plexus, or alternatively, the brain may synthesize its own
PRL-like molecules (Buntin, 1993). Approximately 30-40% of immunoreactive PRL
neurons in the rat medial basal hypothalamus accumulate estradiol, suggesting that sex
steroids are likely important modulators of the brain prolactinergic system (Buntin, 1993).
Dopamine plays a role in regulation of seasonal reproduction, and whether this
effect is mediated through PRL as influenced by nutrition, temperature and photoperiod is
not yet known. In mares, administration of a dopamine D2 antagonist significantly
advances the time of first ovulation (Besognet et al., 1996). In this species, which breeds
during the early spring, PRL concentrations increase during estrus, and a dopamine
antagonist may mimic this seasonal effect. In ewes, there is evidence for dopaminergic
inhibition of GnRH neurons, which has been suggested to be the mechanism of estradiol
negative feedback on LH secretion during anestrus (Havern et al., 1994; Besognet et al.,
1996).
55
There is some evidence supporting a role for PRL in attainment of puberty. Both
PRL and GH contribute to ovarian maturation at puberty by facilitating the effects of FSH
and LH, and PRL may decrease the secretion of 3a-androstanediol in the rat during the
ovarian transition to proestrus at puberty; 3a-androstanediol may cause delayed onset of
puberty (reviewed by Stormshak, 1997). Additionally, treatment of prepubertal gilts with
PRL stimulated LH release from the pituitary and slightly increased the number of
LH/hCG receptors in the ovaries as compared with control animals (Jana et al., 1996).
Prolactin can affect ovarian physiology at several stages, including follicular
maturation, ovulation, steroidogenesis, luteinization and CL function, and often does so in
conjunction with the immune system (discussed later). Prolactin may play a role in
inhibiting folliculogenesis and ovulation. High systemic concentrations of PRL during
anestrus may inhibit GnRH secretion in ewes, as in rats, although the mechanism is not
clear (reviewed by Curlewis, 1992). Results of an in vitro study showed that PRL can
directly inhibit GnRH secretion from GT1 neuronal cells in a dose-dependent manner
(Milenkovic et al., 1994). Additionally, PRL may inhibit ovulation in humans by inhibiting
estrogen secretion from granulosa cells through interference of androgen aromatization, or
by inhibiting LH activity (McNeilly et al., 1982). Receptors for PRL have been identified
on theca interna cells, and in vitro studies have demonstrated a rapid and irreversible
inhibition of LH-stimulated steroid biosynthesis when PRL was added to the culture
(reviewed by Magoffin and Erickson, 1994). Prolactin can further inhibit ovulation by
decreasing follicular plasmin generation in red deer (reviewed by Clarke et al., 1997), and
by stimulating metalloproteinase inhibitor activity in rat granulosa cells, especially in cases
of hyperprolactinemia where its effect cannot be overriden by increasing gonadotropin
concentrations (Murray et al., 1996). Hyperprolactinemia was associated with
reproductive acyclicity in an African elephant (Brown and Lehnhardt, 1997), and
iatrogenic hyperprolactinemia in ring doves resulted in significant, dose-related decreases
in plasma LH concentrations as well as decreased testicular weights (Buntin et al., 1988).
56
During lactation or hyperprolactinemia (when systemic PRL concentrations are
increased), folliculogenesis is inhibited and estrogen biosynthesis is suppressed (McNeil ly
et al., 1982; Magoffin and Erickson, 1994). Spotted hyaenas are unique in that PRL
concentrations in lactating females decrease significantly (p<0.05) after birth, due to an
infrequent suckling pattern, allowing females to resume breeding shortly after the loss of a
litter, or even during lactation (van Jarrsveld et al., 1992). For other species, the role of
PRL in lactational anestrus is thought to be initiated by a nursing stimulus that inhibits
dopamine release, resulting in increased PRL secretion from the anterior pituitary. It has
been proposed that PRL can have a positive effect on f3-endorphin secretion, and 13-
endorphin in turn suppresses GnRH secretion, decreasing LH release, thus inhibiting
ovulation (reviewed by Short, 1984). However, the definite mechanisms that link the
suckling stimulus with the GnRH pulse generator have not yet been proven (McNeilly,
1994).
While PRL may inhibit LH release from the pituitary in certain circumstances, it
has also been shown to enhance LH receptor expression in granulosa, luteal and Leydig
cells of several species, and can facilitate follicular remodeling changes at the time of
ovulation by inducing a2-macroglobulin synthesis in granulosa cells (reviewed by Clarke
et al., 1997). In mice, rats and hamsters, coitus, which results in increased PRL secretion,
is required for development of fully functional CL's (Short, 1984). Both serum PRL and
pituitary PRL mRNA tended to increase in concentration at the time of and just prior to
ovulation in ewes, but due to individual variations, this difference was not found to be
significant even though serum PRL concentrations were approximately 50% less during
luteal phases (Landefeld et al., 1991). Prolactin can also stimulate progesterone
production in ewes by inhibiting expression of 20a-hydroxyprogesterone dehydrogenase
(thereby reducing progesterone transformation into 20a-hydroxyprogesterone), and by
causing dephosphorylation of elongation factor 2, which stimulates large luteal cell
hypertrophy and protein synthesis (reviewed by Clarke et al., 1997).
57
Prolactin can serve as either a luteotropic, luteolytic or luteostatic agent,
depending on the species and reproductive status, and has been shown to play a role in
terminating embryonic diapause in several species (Rose et al., 1983; Berria et al., 1989;
reviewed by Curlewis, 1992; Bernard and Bojarski, 1994). Treatment with PRL increases
progesterone levels and advances the time of implantation in mink, but progesterone
administration alone does not terminate embryonic diapause (Rose et al., 1983). Further
studies suggest that a high systemic ratio of progesterone to estradiol is required to
increase the uterine PRL receptor population (Rose et al., 1996), although the mechanism
whereby PRL initiates implantation is not yet known. In other species, like wallabyes,
PRL is responsible for the seasonal inhibition of CL function, thereby maintaining seasonal
quiescence in response to photoperiod (Hinds, 1989).
Because the immune system is intimately associated with many reproductive
events, the relationship between photoperiod, melatonin, PRL and the immune system
seems worthy of discussion. There is evidence for a circannual fluctuation in immune
function correlated with photoperiod, temperature and melatonin secretion, and this was
further demonstrated in laboratory studies, which showed that short daylengths enhance
certain aspects of immune function (reviewed by Nelson et al., 1995). Field trials
occasionally report compromised immune function during autumn and winter months;
however, additional environmental stressors, such as restricted food and low
temperatures, could counteract the immunostimulatory effects of short daylength (Nelson
et al., 1995). Melatonin administration can increase the weight of lymphatic tissues,
counteract the immunosuppressive effects of glucocorticoids, and has immunostimulatory
effects by modulating antibody response, tumorigenesis, and gonadal regression in some
species, thereby reducing the immunosuppressive effects due to testosterone (Nelson et
al., 1995). It is not clear whether melatonin's effects on the immune system are by a direct
mechanism or are indirectly mediated through PRL.
58
Prolactin receptors are ubiquitously expressed by cells in the immune system, and
certain subpopulations of lymphocytes can secrete PRL (Sabharwal et al., 1992; Draca,
1995; Royster et al., 1995), suggesting that PRL could have a role as an autocrine or
paracrine regulator of immune function (Yu-Lee, 1997). The effect PRL can have on the
immune system varies depending on the concentration, since hyperprolactinemic states
have been associated with immunological diseases while hypoprolactinemia can result in
suppressed immune function (Nelson et al., 1995; Yu-Lee, 1997). Prolactin stimulates
phagocytosis by macrophages, induces lymphocyte proliferation (Hartman et al., 1989;
Clevenger et al., 1990), enhances response of natural killer cells, T-cells, and B-cells to
mitogenic signals (Nelson et al., 1995), and inhibits glucocorticoid-induced apoptosis in T
cells (Yu-Lee, 1997). Immunostimulation by PRL may occur directly through induction
of growth-related genes, or indirectly by upregulation of cytokine receptor expression
(e.g., IL-2) and its own receptors on T cells (Yu-Lee, 1997). By regulating the expression
of a multifunctional transcription gene factor, like interferon regulatory factor-1, PRL
could affect immune function in numerous ways, depending on the cell type and its stage
of differentiation and development (Yu-Lee, 1997).
The dual role of PRL as a luteolytic and luteotropic hormone in the rat appears to
be mediated through the immune system, whereby PRL increases the proliferative activity
of vascular cells in the newly forming CL, while simultaneously inducing expression of
monocyte chemoattractant protein-1 (Bowen et al., 1996) which results in an invasion of
macrophages (Gaytan et al., 1997). Macrophages in the rat have been reported to
stimulate growth of granulosa cells in maturing follicles, release mitogenic factors,
enhance PRL-induced progesterone secretion from mature granulosa cells, and are
thought to play an important role in the process of luteolysis (Gaytan et al., 1997).
Prolactin modulates reproductive functions in males as well. Hyper- and
hypoprolactinemic states were shown to disrupt testicular endocrine function in boars, as
well as alter the process of spermatogenesis to lower fertility (Jedlinska et al., 1995).
59
Annual changes in serum concentrations of PRL in black bears were positively correlated
with testosterone secretion, with a peak in PRL concentration occurring several weeks
prior to the annual peak in testosterone (Tsubota et al., 1995). Testicular function appears
to be affected by PRL because receptors for this hormone have been detected on the
surface of Leydig cells, Sertoli cells, all phases of spermatogonia and spermatocytes, as
well as spermatozoa (reviewed by Hondo et al., 1995). Serum concentrations of PRL are
elevated during the sexually inactive period in rams, and it has been suggested that PRL
may act to maintain functional integrity of the testes, allowing for a rapid response to
increasing levels of gonadotropins as hours of daylight decrease in the fall (Lincoln and
Clarke, 1997). Some studies suggest PRL may accelerate the process of capacitation and
acrosomal reaction of spermatozoa (reviewed by Hondo et al., 1995), activate 313- and 17
f3-hydroxysteroid dehydrogenase to affect testosterone synthesis (reviewed by Jedlinska et
al., 1995), and enhance FSH binding to Sertoli cells (Guillaumot et al., 1996).
Prolactin: Receptors and Mechanism of Action
The PRL receptor (PRL-R) is part of the cytokine/GH/PRL receptor superfamily
which activates JAK-2 and STAT transcriptional factors once dimerization has occurred
(Jahn et al., 1997; Yu-Lee, 1997). There are two (or perhaps three; reviewed by
Camarillo and Rillema, 1997) forms of PRL-R in the rat, which vary in length of the
cytoplasmic domain, bind PRL with equal affinity, but mediate different biological effects
(Jahn et al., 1997). The short form (291 amino acids) was found in the liver, and the long
form (591 amino acids) was found in mammary tissue (Jahn et al., 1997).
As expected, given the diversity of functions PRL has in numerous systems, PRL
binding sites have been identified in many tissues. Prolactin binding sites have been found
in the uterus and kidney of the mink, as well as the uterus of the rat, rabbit and ewe
(reviewed by Rose et al., 1983). Utilizing the techniques of in situ hybridization,
immunohistochemistry, and radioligand binding, distribution of the PRL-R in fetal
60
Sprague-Dawley rats was examined (Royster et al., 1995). Messenger RNA, encoding for
the two isoforms of PRL-R, was expressed in all three germ layers, including: olfactory
neuronal epithelium, olfactory bulb, trigeminal and dorsal root ganglia, cochlear duct,
brown adipose tissue, submandibular glands, whisker follicles, tooth primordia,
chondrocytes of developing bones, adrenal cortex, gastrointestinal and bronchial mucosa,
renal tubular epithelia, choroid plexus, thymus, liver, pancreas, and epidermis (Royster et
al., 1995). Some of these tissues are involved in odor detection, facial sensation, taste and
the neonatal suckling reflex.
Prolactin receptor expression has been shown to change in relation to stage of
estrous cycle in some species. Ovarian PRL-R mRNA expression changed throughout the
reproductive cycle in the rat, initially being localized to the theca layer and then increasing
to a peak at proestrus when it was strongly expressed in the granulosa layer (Clarke et al.,
1993). However, in red deer there were no differences in expression of PRL-R mRNA
during the estrous cycle and pregnancy (Clarke et al., 1997).
Several kinases and signaling molecules are activated in response to PRL,
including: protein kinase C, RAF-1, members of the s6 kinase family, MAP kinases, JAK
kinases, src kinases, SHC, GRB, SOS, vav and pim (reviewed by Camarillo and Rillema,
1997). Additionally, the ras signaling pathway was recently shown to be involved in
PRL's effects on pre-T lymphoma cell lines (Nb2) and mouse mammary gland explants
(Camarillo and Rillema, 1997). By administrating lovastatin (a specific inhibitor of
farnesylation), ras was not able to anchor into the cell membrane, and this abolished PRL's
effects on the cell (Camarillo and Rillema, 1997).
Seasonal Reproduction in the Tropics
Evolutionary pressure favoring reproductive photoresponsiveness may diminish at
lower latitudes in smaller mammals that might breed more opportunistically, perhaps
matching reproductive efforts with nutritional conditions (reviewed by Bernard and Hall,
61
1995). Seasonal reproduction is not uncommon in the tropics, and has usually been linked
to rainfall patterns that can have a major impact on food availability. Because factors
relating to nutrition are believed to override any potential influence photoperiod could
have on seasonal reproduction in tropical climates, few studies have investigated how
neuroendocrine transduction of photoperiod might affect reproduction. Four-fifths of the
Earth's mammals live in the tropics, but most of what we know about mammalian
reproduction has been learned by studying the relatively few species that inhabit the
temperate zone (Bronson and Heideman, 1994). This could potentially bias our
understanding of photoperiodic signalling, especially because recent studies have
demonstrated a great deal of species diversity regarding melatonin's site and mechanism of
action in the brain.
Reproductive effects of photoperiod are not always easy to detect nor are they
consistent. An animal's age, its endocrine and nutritional status, and other factors, like
pheromonal influences, can all affect or perhaps mask photoperiodic responses (reviewed
by Heideman and Sylvester, 1997). A keen sensitivity to photoperiod may potentiate the
effects limited food resources have on reproductive activity, as recently demonstrated in
rats, which are normally considered to be reproductively unresponsive to photoperiod
(reviewed by Heideman and Sylvester, 1997). Several studies have unmasked a robust
reproductive response to short-day exposure in the laboratory rat (Raffia norvegicus)
after treatment with either an olfactory bulbectomy, chronic food deprivation, or neonatal
androgen treatment (Heideman and Sylvester, 1997). It is possible that the photoperiodic
neuroendocrine pathway exists in these animals, but is only functional under certain
conditions (e.g., short days only inhibit reproduction when food is limiting) (Heideman
and Sylvester, 1997). It could also be that animals are photoresponsive only at certain
times in their reproductive life, for example, age of puberty (Bernard and Hall, 1995).
Selection for continuous breeding in certain domestic species has not always been
matched by the annual PRL profile. For example, cattle have marked seasonal changes in
62
serum PRL concentration while domestic pigs have minimal variation in PRL levels, yet
both are capable of breeding throughout the year (reviewed by Curlewis, 1992). There is
evidence that domestication of some mammals (e.g., pigs) decreases their sensitivity to
short photoperiod (Spears and Clark, 1987; Griffith and Minton, 1992). Additionally,
photoperiod may influence melatonin secretion patterns, but be uncoupled from or not
affect reproductive cyclicity. For example, studies involving transfer of tropical cane mice
to artificial short- or long-day photoperiods, demonstrated daily melatonin patterns
similiar to temperate species; however, the reproductive axis was not influenced by these
changes (Heideman and Bronson, 1990).
Assuming that the size of the pineal gland is directly correlated to functional
secretion can be misleading. The pineal gland of a tropical bat (Anoura geoffroyi) is
relatively small, yet nocturnal serum melatonin levels increased to 50-152 pg/ml,
comparable to nocturnal concentrations of melatonin typically found in mammals
inhabiting temperate zones (Heideman et al., 1996). The pineal gland in these bats is in
intimate contact with the great cerebral vein, so that at times only the endothelium and
connective tissue components separate the gland from the lumen of the vein.
In the tropics, more seasonal and predictable environments would likely favor the
evolution of photoresponsiveness (Bronson and Heideman, 1994). Syrian hamsters are
able to respond to subtle changes in photoperiod typical of 100 and 50 latitude, indicating
that the sensitivity required to utilize this information exists (Heideman and Bronson,
1993), and reproductively photoresponsive rodents and shrews have been found below
200 latitude (Bronson and Heideman, 1994). As we broaden our understanding of how
the neuroendocrine system translates environmental signals to link together photoperiodic,
nutritional, and social cues with the reproductive system, we may be able to improve our
ability to detect the effects of photoperiod in tropical species.
63
Conclusions
The study of seasonal reproduction is complex because there are numerous
environmental and physical signals involved, which often interact with one another and are
dependent upon the hormonal milieu of the animal. Olfactory and pheromonal, GH, PRL,
cortisol and thyroid hormones can all influence the reproductive axis (Prunier et al., 1996).
Research in this field requires an integrative approach to account for the possible effects of
endogenous rhythm, photoperiodic history, thyroid and gonadal hormones, temperature,
stress level, plane of nutrition, social status, and potential pheromonal input. It is likely
that all animals have a functional photoperiodic neuroendocrine pathway that is utilized to
varying degrees depending on the sex and the current physical and environmental
circumstances.
Many interesting questions remain unanswered. Precisely how and where
melatonin and PRL have their effect in the brain and other systems to influence
reproduction has not yet been determined, although variations among species do seem to
occur. Is photoperiod influencing reproductive potential on some level in most tropical
species? Are animals able to adapt to a different climate by adjusting how much they rely
on photoperiod to time reproductive cyclicity? To what extent can alterations in steroidal
hormone ratios explain PRL's unique role as a luteotropic, luteolytic, and luteostatic
agent? How is temperature able to influence PRL secretion? Exploration of additional
roles melatonin and PRL play in fetal development, male reproduction, and immune
function will be interesting. Further characterization of tuberalin and the cells of the pars
tuberalis will contribute to understanding the control of PRL secretion from the anterior
pituitary.
The diverse array of control mechanisms that enable different species and sexes to
translate environmental signals into neuroendocrine responses will continue to pose a
challenge to the formulation of a model capable of illustrating the mechanics underlying
seasonal reproduction. Perhaps as we attempt to unmask reproductive responses to
64
photoperiod in species not traditionally thought to be reproductively photoresponsive, we
will expand our understanding of the control mechanisms involved in the neuroendocrine
interpretation of this type of signal.
Stress and Cortisol
Serum concentrations of cortisol fluctuate in circannual as well as diurnal patterns
in many species, peaking during morning waking hours (reviewed by Turek and Canter,
1994), and these rhythms are entrained by photoperiod (Minton et al., 1989). Because
serum concentrations of cortisol usually increase in response to either real or perceived
stresses, cortisol has frequently been termed the "stress hormone".
"Stress" is defined as physical or emotional stimuli of varying length and intensity,
and has long been known to increase adrenocortical activity and decrease reproductive
success (reviewed by Wingfield et al., 1994). One of the earliest studies to examine the
potential effects of stress on reproduction, investigated the relationship between
population density and reproductive performance in rodents (Christian, 1971). Since that
time, research has focused on identifying the hormones and mechanisms through which
stress mediates its effects on reproduction. Stress-related hormones can influence the
reproductive system at all three levels of the hypothalamic-pituitary-gonadal (HPG) axis
(Rivier and Rivest, 1991), and these effects include: delayed puberty, elongated or
anovulatory cycles, implantation failure, spontaneous abortion, or high infant mortality
(Wingfield et al., 1994). The degree of severity varies among species, resulting in absolute
reproductive failure in some while merely decreasing the rate of success in others
(Wingfield et al., 1994).
In certain situations, adrenal hormones can serve as a survival mechanism. For
example, corticosterone opposed PRL's effects on parental behavior in penguins by
redirecting activities towards foraging (Cherel et al., 1994), and testosterone levels in rats
have been shown to increase under "fight-flight" types of stimuli, but decrease during
65
stresses accompanied by depression (Collu et al., 1984).
Different types and intensities
of stress elicit different neuroendocrine responses (Schedlowski et al., 1992), and this may
in part be mediated by activation of different endogenous opiate ligands and/or receptor
subtypes (Pau et al., 1996). There appear to be at least two distinctly different responses
to stress, depending on the type, severity, and duration of the stress. Acute stress can
result in small and temporary increases in plasma LH and testosterone concentrations,
most likely mediated through central nervous system (CNS) mechanisms; whereas chronic
stress consistently inhibits LH release, with pituitary and gonadal hormones additionally
modulating CNS effects (reviewed by Rivier and Rivest, 1991).
Corticotropin-releasing factor (CRF), proopiomelanocortin (POMC)-derived
peptides (e.g., ACTH and (3-endorphin), catecholamines, adrenal glucocorticoids, and
gonadal hormones have all be identified as potential mediators of stress-related
reproductive suppression (Rivier and Rivest, 1991). Glucocorticoids affect virtually every
organ system in the body (Herman et al., 1995), appear to inhibit PRL secretion, as well as
ACTH release, through negative feedback effects (Neill and Nagy, 1994; Taylor et al.,
1995), and function in immunomodulation (e.g., IL-1 increases glucocorticoid levels,
while glucocorticoids inhibit production of IL-1 and IL-2; Khansari et al., 1990; Draca,
1995). Cortisol concentrations were positively correlated with PRL concentrations only
during periods of acute stress in male cotton-top tamarins (Saguinus oedipus), suggesting
that different neural pathways are involved in PRL release during chronic versus acute
stress (Ziegler et al., 1996).
Lipocortin-1 (LC1) is a Ca2+ and phospholipid binding protein that has been
shown to: (1) mediate some of the anti-inflammatory, anti-pyretic and anti-proliferative
actions of glucocorticoids, (2) contribute to the regulatory effects steroids have on the
neuroendocrine system, (3) facilitate the early inhibitory actions glucocorticoids have on
CRF and ACTH release in the brain, and (4) control PRL release from human decidual
tissue (Taylor et al., 1995). Recent research suggests that LC1 synthesis in the anterior
66
pituitary is regulated by glucocorticoids, and that LC1 plays a critical role in the
glucocorticoid-induced suppression of PRL secretion by acting within the cAMPdependent signal transduction sequence, augmenting dopamine's inhibitory influence
(Taylor et al., 1995). Thyroid-releasing hormone acts through phospholipase C, so
glucocorticoid/LC1-induced suppression of PRL release would most likely not be able to
overcome stimulation of PRL release by TRH (Taylor et al., 1995).
Primary mediators of the stress-response in the CNS appear to be the CRF
neurons, located mainly in the medial parvocellular paraventricular nucleus, and they are
sensitive to modulation by glucocorticoids (Herman et al., 1995), catecholamines,
acetylcholine, and serotonin (Khansari et al., 1990). Mechanisms by which CRF
influences GnRH secretion are not fully understood, and most likely vary between species,
as evidenced by differential responses to intraventricular injections of CRF in rodents and
primates (Rivier and Rivest, 1991). A direct effect is possible because there are
anatomical connections between dendrites of GnRH-secreting neurons and CRF axon
terminals in the preoptic area (Maclusky et al., 1988). Corticotropin-releasing factor also
regulates the synthesis of POMC-derived peptides, and while endogenous opioids appear
to generally exert a tonic inhibitory influence on the HPG axis of rats by acting directly on
GnRH neurons in the mediobasal hypothalamus (River and Rivest, 1991), studies with
primates conclude that this action seems to be steroid-dependent (Pau et al., 1996).
During the luteal phase, when both estrogen and progesterone are present, the opioidinhibition of GnRH release is enhanced (Pau et al., 1996). Thus, CRF and endogenous
opioids could act synergistically or additively, in conjunction with gonadal steroids, to
affect changes in GnRH secretion in response to stress. Additionally, CRF can directly
activate catecholaminergic neurons in the locus coeruleus (Butler et al., 1990; Rivier and
Rivest, 1991), but there have been conflicting reports regarding the action of
catecholamines on the hypothalamic-pituitary axis. These inconsistent results probably
stem from the observation that the effects seem to be estrogen-dependent, thereby
67
resulting in stimulation of GnRH neurons by norepinephrine (NE) when estrogen is
present, and inhibition of these neurons in its absence (Rivier and Rivest, 1991).
Serum PRL concentrations increase markedly, within minutes, in response to acute
stress, and could reflect important roles for PRL in maintaining integrity of the immune
system as well as mediating behavioral or emotional responses (Fujikawa et al., 1995).
Suppression of gonadotropin secretion in response to hyperprolactinemia appears to be
mediated by the actions of CRF, because antagonism of CRF reverses this suppression
(reviewed by Akema et al., 1995). Corticotropin releasing factor appears to play a
permissive role in the stress-induced release of PRL as well, because an
intracerebroventricular injection of a-helical CRF(9-41), a CRF antagonist, suppressed
PRL release; however, CRF administration during non-stressful conditions did not
increase PRL secretion, but did increase ACTH secretion while decreasing LH release,
indicating that the dose was sufficient (Akema et al., 1995). It is not yet clear whether or
how PRL may act on the CNS during stressful conditions, and PRL cannot cross the
blood-brain barrier. Stress has been shown to induce gene expression of the long form of
PRL receptors in the rat choroid plexus, raising the possibility that these receptors serve as
part of a receptor-mediated transport mechanism, bringing serum PRL into the
cerebrospinal fluid (Fujikawa et al., 1995). Prolactin has been shown to induce expression
of its own receptors in many tissue types, with steroid hormones playing a regulatory role;
perhaps the stress-induced elevation in serum PRL concentration serves to stimulate
expression of PRL-R's in the choroid plexus as well (Fujikawa et al., 1995).
Receptors and mRNAs for CRF, POMC-derived peptides as well as receptors for
adrenal corticosteroids have been identified in rodent gonads, enabling stress-related
hormones to exert direct effects on gonadal function by decreasing sensitivity to
gonadotropins, and inhibiting steroidogenic activity (Rivier and Rivest, 1991). A study
investigating the etiology of gestation failure in foxes (Vulpes vulpes) found that females,
which did not successfully whelp at term, had decreased levels of progesterone during the
68
period of implantation, yet concentrations of PRL and LH (providing luteotropic support)
were not lower at this time (Hartley et al., 1994). Low progesterone concentrations were
believed to be induced by a stress-related mechanism, because increased concentrations of
cortisol were observed between 3-7 weeks post-ovulation only in these females (Hartley et
al., 1994). In another study with baboons, systemic levels of progesterone were tied to
stresses originating from social ranking, and/or long-term environmental cues that
appeared to determine the amount of progesterone required for successful implantation
(Wasser, 1996). Females of low dominance rank or experiencing suboptimal seasonal
conditions appeared to require higher concentrations of progesterone for implantation, and
it was suggested that these stresses may therefore act as a reproductive filter, allowing
conception to occur only in those females experiencing low levels of stress related to
social and environmental influences (Wasser, 1996).
Because the regulation of GnRH neurons is complex and the number of factors
involved in transducing a stress signal are numerous, a good understanding of the control
mechanisms that mediate stress effects on the HPG axis is still lacking. Often dual forces
appear to act in opposition, depending on the current hormonal environment. For
example, acute stress appears to increase serum levels of PRL through actions mediated
by the CNS, while glucocorticoids inhibit PRL secretion through a negative feedback
mechanism under conditions of chronic stress. In summary, reproductive responses to
stress appear to be dependent on species, type and chronicity of stress experienced, and
current reproductive status that can alter steroidal modulation of CNS activity.
69
STATEMENT OF PURPOSE
The African elephant (Loxodonta africana) is an endangered species, plagued by
habitat loss and poaching in the wild, and by low birthrate coupled with high neonatal
mortality rate in captivity. Broadening our understanding of the basic reproductive
physiology of elephants will facilitate development of assisted reproduction and reversible
contraception techniques, which can be used to improve conception rates in captivity and
offer alternatives to culling in the wild.
Prolactin is a peptide hormone which is involved in a number of diverse
physiologic roles, particularly with respect to reproduction, including modulation of
ovarian function and regulation of seasonal reproduction. This research will examine
seasonal changes in prolactin secretion in non-pregnant female African elephants, as well
as potential functional interrelationships between secretions of prolactin, cortisol, and
progesterone. This is of particular interest in elephants because little is known about the
control of corpora lutea function and life span, folliculogenesis, ovulation, and the effects
of stress on the reproductive axis. As an observational study with a limited sample size,
no causal inferences can be made regarding hormonal function in African elephants;
however, results will direct future research designed to investigate factors that control
prolactin secretion and function.
70
EXPERIMENTAL DESIGN
Animals
Four female African elephants, housed at the Wildlife Safari in Winston, Oregon
(430 latitude), were sampled for this study. Three of the females were wild caught,
transferred to the U.S. at approximately 1 yr of age and have been at the Wildlife Safari
since 1972. The fourth female was caught in the wild at 2 yr of age and was transferred to
the Wildlife Safari in 1979. All of these elephants were nulliparous and have never been
involved in a breeding program. Two other African elephants (a male and female) were
also housed with these four elephants.
Management of elephants was done by free contact. Animals were allowed to
roam in an outdoor dirt enclosure during the day and were chained indoors at night.
During winter months, when outside temperatures did not exceed 40C, they were kept
inside under natural and artifical lighting conditions which matched the environmental
photoperiod. Elephants were fed 4.5 kg Mazuri elephant supplement (Purina Mills Inc.
Feeds, St. Louis, MO) mixed with 28 g sheep salt, 56 kg coastal grass hay, and
approximately 4.5 kg of produce each day, with water available ad libitum.
Weekly 15 ml blood samples were taken from ear veins for 18 months.
Collections were made on the same day each week between 0900-1000 h prior to
releasing elephants into the outdoor enclosure. The blood was allowed to clot at room
temperature for 45-60 min prior to centrifugation and serum separation. Serum from each
elephant was divided into three aliquots for future prolactin, progesterone and cortisol
analyses, and was stored at -200C until assayed. Additional blood samples, for melatonin
analysis, were taken during the winter and summer solstice every 3 h for 48 h on
December 21-23, 1995, June 19-21, 1996 and December 15-17, 1996. Samples were also
collected 30 min prior to and after each sunrise and sunset. Blood was allowed to clot at
room temperature for 1-3 h prior to centrifugation and serum separation. Samples were
71
stored at -200C until assayed. However, analyses of samples for melatonin by
radioimmunoassay (RIA) were unsuccessful (see Appendix B).
Monthly weather reports, recording daily maximum and minimum temperatures,
rainfall and percentage cloud cover for the Winston area, were collected for 18 months
and processed by the National Weather Service Center in Medford, Oregon for this study.
Radioimmunoassays
Prolactin
Serum prolactin was measured by heterologous RIA using an anti-human prolactin
antisera (NIDDK-anti-hPRL-3), iodinated ovine prolactin (NIDDK-oPRL-I-2; Brown and
Lehnhardt, 1995), and African elephant standards (elephant PRL [G100, 2.1] purified by
C.H. Li, UC, San Francisco). For each iodination, 5 p.g prolactin (in 25 pl of 0.01 M
NaHCO3 buffer) were incubated with 1 mCi carrier-free Na125I (ICN Biochemicals,
Costa Mesa, CA) and 5 .tg chloramine-T (in 10 pl 0.05 M phosphate buffer; pH 7.5) for 1
min. The reaction was stopped by adding 20 pg sodium metabisulfite (in 20 pl 0.05 M
phosphate buffer; pH 7.5). Labelled hormone was separated from the free Na125I using
gel filtration chromatography (Sephadex G-75-120; Sigma, St. Louis, MO). Each column
was prepared using a 0.8 x 19 cm 10 ml disposable pipette fitted with a 4 mm glass ball.
Approximately 0.60 g of G-75-120 Sephadex was allowed to soak overnight in 100 ml
double-distilled water, and roughly half of the supernatant was discarded prior to pouring
the column to 10 ml. The column was conditioned by running 4-5 ml Tris-HCI with 0.1%
BSA (room temperature) through it. The iodination reaction mixture was placed onto the
column and eluted with Tris-HC1 with 0.1% BSA. Thirty collection tubes, each
containing 500 p.1 Tris-HC1 with 0.1% BSA, were used to collect 500 111 fractions of the
elution. Tubes were vortexed and 10 IA aliquots were counted. Results were graphed and
the iodination fraction immediately following the labelled hormone peak was used in the
72
assay (Figure 6). Dilutions to 22,000 cpm were made with 0.01 M phosphate buffered
saline (PBS) with 0.1% BSA (pH 7.0). Iodinated fractions used 1 wk or longer after
iodination were cleaned to separate labelled hormone from free Na1251 using gel filtration
chromatography (Sephadex G-50-150) as described above, and the labelled hormone peak
fraction was used in the assay.
Each assay was incubated at 4 °C for 5 days in a total volume of 1 ml, using
polypropylene test tubes. Samples (100-200 pl) were adjusted up to 500 IA with
phosphate buffer (0.01 PBS, 0.1% BSA, 0.8% NaC1, 0.01% merthiolate, pH 7.0). These
samples and standards (500 .d; 0.1-20 ng/tube) were incubated with antibody (1:10,000 in
200 ill) for 24 h, followed by the addition of 125I-prolactin (22,000 cpm in 100 lap and
incubation for another 24 h. On Day 3, antibody-bound complexes were precipitated by
incubation for 72 h with 200 µl sheep anti-rabbit gamma globulin (1:40). On Day 6, 3 ml
0.01 phosphate buffer (0.01 PBS, pH 7.0) were added to assay tubes prior to
centrifugation for 30 min at 1,364xg and 4 °C. Tubes were drained for 10-15 min and
counted for 1 min each on a Beckman Gamma 5500 Counter.
The antibody bound 25-30% of the 125I-prolactin with 2-3% nonspecific binding.
Serial dilutions of elephant serum pools were parallel to the standard curve (Figure 7).
Upon addition of 0.5, 1.0, 5.0, and 10.0 ng elephant prolactin to 50 Ill elephant serum,
0.5, 0.9, 4.2, and 11.2 ng were recovered after subtraction of endogenous hormone (y =
0.483 + 0.913x; R2 = 0.998). Assay sensitivity was 0.5 ng/tube (p<0.0005). The
intraassay and interassay coefficients of variation were 8.2 and 13.2%, respectively. A
biological response to TRH challenge was demonstrated (Figure 8), and cross-reactivity
with African elephant growth hormone (elephant GH[HPLC] purified by C.H. Li, UC, San
Francisco) was insignificant (Figure 7). A challenge dose of TRH for administration to
elephants was derived from calculations based on metabolic scaling (633 pg) as well as
suggestions that 3-4 times the human dose (200 lig) be utilized. Two doses were
prepared by dissolving 1.266 mg TRH in 20 ml sterile saline and cold-sterilizing the
73
12
labeled fraction used
10
8
0
0
6
E, 4X
2
0
I
1
0
5
1
10
-1
15
20
25
30
35
Fraction
Figure 6. Fractions from a Sephadex G 75-120 column demonstrating a 46.2% yield in
labelled hormone versus free iodine (April 10, 1997).
74
* ePRL standards
a elephant serum volumes
4-- elephant serum volumes + 6 ng/ml ePRL
v eGH standards
0.1
1
10
100
1000
10000
log dose, ng or ul
Figure 7. Dose response curves for ePRL standards (ng), serum volume dilutions (il),
and eGH (ng) demonstrating parallelism to the standard curve (y=181.4673-47.8765x;
r2=0.996) and lack of cross-reactivity with eGH.
75
20
0
111111 ill
15
30
45
60
75
90
105
120
135
Minutes post-TRH injection
Figure 8. Dose response curve for ePRL after a TRH challenge (633 ug in 10 ml saline)
in a female African elephant.
76
solution by filtration through a 0.2 um acrodisc (Millipore, Marlborough, MA). The
preparation was stored at 40C until injected intravenously into an ear vein. Blood samples
were collected at -30, -15, 0, 5, 10, 15, 30, 60, and 120 min after injection of 633 .tg of
TRH. The challenge was repeated in the same animal 2 mo after the initial challenge was
made.
Progesterone
Serum progesterone was analyzed by RIA as described by Hess et al. (1983), using
antisera Surve #1. Briefly, 1 ml aliquots of serum were extracted with 6 ml of diethyl
ether and purified on Sephadex LH-20 columns, using hexane:benzene:methanol
(62:20:13, v:v) as the eluting solvent. One-ml water blanks were assayed and averaged
16.6 pg. Serum concentrations were calculated after subtracting blank values and
correcting for recovery losses. Sensitivity ranged between 3-5 pg/tube (90% binding),
mean percentage recovery with chromatography was 87.2%, and the intra- and inter-assay
coefficients of variation were 9.1 and 13.3%, respectively.
Cortisol
Serum concentrations of cortisol were measured by S.K. Wasser at the Center for
Wildlife Conservation, Seattle, WA. One ml chilled dichloromethane (DCM) was added
to 0.2 ml serum in 12 x 75 mm glass test tubes kept in an ice bath and then vortexed
thoroughly for 15-20 sec. To facilitate separation of organic and aqueous phases, samples
were centrifuged for 10 min at 721xg, and 0.1 ml of the DCM layer was removed and
evaporated to dryness in a separate test tube. One ml PBS gel was added to the dry
extract and vortexed thoroughly. For the RIA, 0.3 ml of this solution (6 ul serum
equivalent) was used.
Serum cortisol was measured by RIA using an R1222 anti-cortisol-3-BSA
antibody (G. Niswender, Colorado State University, Fort Collins, CO) at a 1:3000
77
dilution, [1,2,6,7- 3H(N)]- Cortisol (NET-396, New England Nuclear, Boston, MA) at
approximately 13,000 dpm/tube, and cortisol standards diluted from 100 p.g/ml (ICN
Biochemicals, Costa Mesa, CA). To 0.3 ml serum extract, 0.1 ml antibody and 0.1 ml
labelled cortisol were added and allowed to incubate at 4°C for 16-24 h. Separation of
bound and free hormone was achieved by incubation with 0.5 ml 0.5% charcoal
suspension with 0.05% dextran at 4°C for 12 min followed by centrifugation at 4°C and
1,130xg for 20 min. The supernatant was poured into 7 ml liquid scintillation vials
containing 4.5 ml Ultima Gold Cocktail (Packard Instruments, Meriden, CT), and counted
in a Beckman LS6500 Liquid Scintillation Counter.
The antibody bound 35-40% of the tracer at the 1:3000 dilution. Assay sensitivity
was 0.19 ng/tube, with the standard curve ranging from 0.2-40 ng/ml. Mean percentage
recovery with extraction was 85%, and the intra- and inter-assay coefficients of variation
were 2.7 and 6.9%, respectively. Serial dilutions of elephant serum pools were parallel to
the standard curve (Figure 9), and a biological response to adrenocorticotropin hormone
(ACTH) challenge was demonstrated (Figure 10). The ACTH used was formulated as a
long-acting gel (Acthar Gel, Armour Pharmaceutical, Collegeville, PA) in a 500 IU/ml
concentration. Dosages (approximately 250 IU/500 kg) were based on responses to
previous ACTH challenge trials in other species, including one African elephant. In this
study, 3 ml (1500 IU) were injected into one elephant and 4 ml (2000 IU) were given to
another in two deep intramuscular injections, using an 18 g, 3" spinal needle. Blood
samples were taken at -15, 60 min and then hourly for up to 8 h after initial ACTH
injection.
Statistics
Serum concentrations of prolactin, progesterone and cortisol were graphed
through time for both individual elephants and group means to detect cyclicity or patterns
of secretion. In an effort to define "season" more precisely, a weather database was
78
-0 cortisol standards
is elephant serum volumes
log dose, ng or ul
Figure 9. Dose response curves for cortisol standards (ng) and dichloromethane-extracted
serum volume dilutions (W) demonstrating parallelism to the standard curve (y= 65.571343.0383x; r2=0.980).
79
100
80
60
40
--0 Moshi
--II Sneeze
20
0
i
0
2
4
6
8
Hours post-ACTH injection
Figure 10. Dose response curve for cortisol after an ACTH challenge (1500-2000 IU
Acthar Gel given im) in two female African elephants.
10
80
created to examine cyclical changes in average temperature and percentage daylength.
One-way ANOVA and a Tukey pairwise multiple comparison procedure was used to test
for variance in mean temperatures between seasons, and a Kruskal-Wallis one-way
ANOVA on ranks followed by a Dunn's pairwise multiple comparison procedure was used
to test for variance in percentage daylight among seasons.
Correlations between hormones within individual elephants were calculated
initially by using regression plots, and a LOG scale was used when appropriate.
Relationships between hormones were examined by calculating correlation coefficients
achieved by adding together variances and covariances to adjust for variations between
individual means. Variation in serum concentration of cortisol among elephants was
assessed by one-way ANOVA.
By aligning and averaging serum concentrations of progesterone for 20 estrous
cycles, mean ± SE for a composite estrous cycle was constructed. Serum concentrations
of progesterone consistently below 200 pg/ml defined the follicular phase, and
concentrations consistently above 200 pg/ml defined the luteal phase of the estrous cycle.
To detect potential differences in prolactin secretion during the estrous cycle, a correlation
coefficient for PRL and progesterone was calculated, and a two-sample t-test (a=0.05)
was performed to assess differences in serum concentrations of prolactin between luteal
and follicular phases.
Potential seasonal effects on serum concentrations of prolactin were tested by
using a repeated measures ANOVA F-test (a=0.05), blocking for individuals and
calculating separate concentration means for fall, winter, spring and summer seasons.
81
RESULTS
Animals
One of the elephants in the study appeared to be hyperthyroid, because her
thyroxine (T4 ) levels averaged 21.6 ± 1.79 µg/dl (Oregon Medical Laboratories, Eugene,
OR) for six blood samples taken over the course of a year (October, 1996 through
September, 1997), compared with T4 levels in five other Wildlife Safari elephants sampled
in August and October, 1996, which averaged 13.2 ± 2.84 µg/dl. Normal T4
concentrations in wild African elephants have been reported to range between 9.2-13.6
p.g/dl (n=51), and concentrations tended to be higher in stressed animals (Mikota et al.,
1994). Interestingly, mean serum concentrations of progesterone, prolactin and cortisol in
this potentially hyperthyroid individual were all slightly increased over those of the other
elephants in the study. However, because deleting the data of this animal from the data set
did little to affect overall means, they were included in all of the statistical calculations.
Seasonal Parameters
To construct parameters with which to examine potential seasonal effects on
prolactin (PRL) secretion, percentage daylight and average temperatures were averaged
on a weekly basis (Figure 11). At 43° latitude, it was assumed that the seasons would be
well characterized by these parameters (e.g., the light:dark cycles were approximately
12:12 [50% daylight] during the spring and fall equinox, and 63 and 38% daylight during
the summer and winter solstice, respectively). To confirm these seasonal differences in
percentage daylight and mean temperature, values for each season were compared
statistically. There were significant differences in percentage daylight and mean
temperature between seasons (p<0.001 for both; one-way ANOVA), except between fallspring comparisons. Summer was defined as the period between June 21-September 20,
82
90
Y-
80
2
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30
20
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i
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10
20
30
40
50
60
70
80
Weeks (July 13, 1995 to December 26, 1996)
Figure 11. Seasonal fluctuations in weekly percentage daylight and temperature averages
over 18 months (July, 1995 through December, 1996).
83
fall (September 21-December 20), winter (December 21-March 20), and spring (March
21-June 20).
Cortisol
The overall mean (± SE) serum concentration of cortisol was 5.7 ± 1.3 ng/ml
(range: 1.4-19.3). There was significant variation in serum concentrations of cortisol
among elephants (p<0.0001; one-way ANOVA), creating unique secretion profiles for
each animal (Figure 12). Mean serum cortisol levels appeared to peak on three separate
occasions (Figure 13), but only the first peak can be attributed to a known outside source
of stress. There were no apparent relationships between serum concentrations of cortisol
and seasonal parameters, with the exception of a mild negative correlation between
cortisol concentration and percentage daylight in one elephant, the matriarch (r=0.23;
p<0.05).
Progesterone
All elephants in this study showed a cyclical pattern of progesterone (P4) secretion
(Figure 14). Based on changing serum concentrations of P4, the estrous cycle was 14.1 ±
0.9 wk in length (range: 9-17 wk). Mean serum P4 concentration for 20 estrous cycles
was 516.3 ± 211.8 pg/ml (range: 46-2223 pg/ml). The luteal phase was defined by P4
concentrations consistently above 200 pg/ml, and averaged 8.9 ± 0.7 wk in length (range:
5-12 wk), with a mean serum P4 concentration of 750.3 ± 171.9 pg/ml (range: 201-2223
pg/ml). The follicular phase was defined by P4 concentrations consistently below 200
pg/ml, averaged 5.1 ± 0.4 wk in length (range: 4-8 wk), and had a mean serum P4
concentration of 103.1 ± 17.5 pg/ml (range: 46-192 pg/ml). Interestingly, the days that
keepers noticed females passing vulvar mucous were also the days of lowest serum P4
concentration during the follicular phase. Figure 15 illustrates the mean (± SE) serum P4
concentrations for all of the elephants calculated over 20 estrous cycles after
84
(b)
(a)
20 18 16 -
rl
4
2
0
0
10
20
30
40
50
60
70
80
0
I
I
I
I
I
10
20
30
40
50
60
70
80
Weeks
Weeks
(c)
(d)
22
20 18
18 -
16 -
16 -
ti
14 12 10 -
84
0
10
20
30
40
Weeks
50
60
70
80
I-111111
10
20
30
40
50
60
70
80
Weeks
Figure 12. Individual weekly serum cortisol concentration profiles for four female African
elephants over 18 months (July, 1995 through December, 1996); (a) Alice (matriarch); (b)
Sneeze; (c) Tiki (potentially hyperthyroid); (d) Moshi (youngest).
85
20
18
16
14
E 12
cn
U.c0
10
8
6
4
2
00 i4
8 12 16 20 24 28 32 36 40 4448 52 56 60 64 68 72 76 80
Weeks
Figure 13. Mean (± SE) weekly serum concentrations of cortisol for four female African
elephants over 18 months.
86
1800
1600
1400
E 1200
ea
0.
. 1000
c
2
a)
ca
a)
cn
a_
800
600
400
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0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 64 68 72 76 80
Weeks
Figure 14. Weekly serum concentrations of progesterone in an African elephant
demonstrating cyclicity. All females in this study had similar profiles.
87
1200
1000
800
I
600
I
1
400
Luteal Phase
200
Follicu ar Phase
0
0
1
1
1
1
5
10
15
20
25
Weeks
Figure 15. Mean (± SE) serum concentrations of progesterone for 20 estrous cycles from
four African elephants.
88
synchronizing luteal and follicular phases. Some evidence of natural synchronicity in
cycling was apparent between two pairs of elephants (Figure 16). There was no
correlation between serum P4 concentration and seasonal parameters. There was a
tendency for serum P4 concentrations to increase as cortisol levels decreased (r=-0.15;
p<0.01).
Prolactin
The overall serum PRL concentration was 3.91 ± 0.69 ng/ml (mean ± SE; range:
0.84-15.8 ng/ml). Serum concentrations for the samples analyzed ranged between 0.8415.8 ng/ml. Although individual means for serum PRL concentrations over the 18 month
study period only ranged between 2.58-4.93 ng/ml, there was significant variation among
elephants (p<0.0001; one-way ANOVA).
There was a significant negative correlation (r= -0.41; p<0.01) between serum PRL
and P4 concentration for all of the elephants, illustrated graphically in Figure 17 for three
of the animals. During the luteal phase, PRL concentrations were lower with a mean (±
SE) of 3.47 ± 0.09 ng/ml (range: 0.84-11.03) as compared with follicular phase
concentrations of 4.69 ± 0.20 ng/ml (range: 1.23-15.8) (p<0.0001; two-sample t-test).
There was a significant positive correlation between serum PRL and cortisol
concentrations (r =0.14; p<0.05).
Individual serum concentrations of PRL plotted against time appeared to fluctuate
randomly (Figure 18). The repeated measures ANOVA test for a seasonal effect on PRL
secretion was not significant (p>0.05, Figure 19).
89
1600
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1800 1600 1400
1200
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600
400 200
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Weeks
Figure 16. Estrous cycle synchronicity in two pairs of African elephants.
90
9
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7-
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Servals
4,
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1000
100
LOG progesterone (pg/ml)
Figure 17. Weekly serum concentrations of progesterone and prolactin for three female
African elephants over 18 months, demonstrating a significant negative correlation
(3r=5.9928-0.9670x; r2=0.1249).
91
(a)
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Weeks
Figure 18. Individual weekly serum prolactin concentration profiles for four female
African elephants over 18 months (July, 1995 through December, 1996); (a) Alice
(matriarch); (b) Sneeze; (c) Tiki (potentially hyperthyroid); (d) Moshi (youngest).
92
12
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Figure 19. Mean (± SE) weekly serum concentrations of prolactin for four female African
elephants over 18 months.
93
DISCUSSION
In this study there was no significant effect of season on serum concentrations of
PRL, but at this latitude, several wild and domestic species do utilize photoperiod to
adjust their reproductive cycles to season. An endogenous rhythm of reproduction exists
in many species and photoperiod serves to synchronize this rhythm to season (Reiter,
1974; Karsch et al., 1989; Brinklow and Loudon, 1993; Kaplan and Katz, 1994). It is not
known yet whether transfer of an animal from a tropical to a more temperate climate
might influence the ability to detect and utilize photoperiodic information to enhance
reproductive efforts, because few studies have been conducted on this subject (Heideman
and Bronson, 1990). Many of the elephants held in captivity today live in temperate
climates, so assessment of a potential response to photoperiod is of interest. Experiments
involving transfer of temperate species, like sheep, across the equator such that
photoperiod and other environmental factors were shifted by 6 months, resulted in
eventual adjustments in the annual reproductive cycle (Karsch et al., 1984). A study by
Hanks (1972) detected a strong correlation between season and pregnancy rate in wild
African elephants, with 88% of conceptions occurring during the rainy season. Factors
other than nutrition, which could potentially contribute to such an observed seasonal effect
(e.g., photoperiod, temperature, and social cues mediated by stress or pheromones), have
not been investigated.
Ambient temperature has been shown to augment the effects of photoperiod on the
reproductive system in several species (reviewed by Bronson and Heideman, 1994), and in
pigs, temperature can even have more of an effect on return to estrus after weaning than
photoperiod (Prunier et al., 1996). Higher temperatures have been shown to increase PRL
secretion in pigs and cattle (Marcek and Swanson, 1984; Becker et al., 1985; Klemcke et
al., 1989; Minton et al., 1989; Griffith and Minton, 1992); however in this study, no
significant effect of temperature on PRL secretion was observed.
94
Recent research with rats (animals normally considered to be reproductively
unresponsive to photoperiod) unmasked reproductive responses to changing photoperiod
once adjusted for variables such as hormonal influences, nutritional status, and
pheromonal cues (Heideman and Sylvester, 1997). Therefore, it is conceivable that the
potential influence photoperiod and/or ambient temperature may have on PRL secretion in
these elephants could be masked by the effects of hormonal fluctuations in relation to the
estrous cycle, cortisol concentrations, pheromonal influences, and potentially other factors
that influence or regulate secretion of these hormones. Additionally, it is likely that these
elephants were not exposed to the full effect of the winter season because they were
housed indoors under artificial lighting when outside temperatures did not exceed 40C.
Increased serum concentrations of PRL have been positively correlated with food
intake and body weight in several species (Ryg and Jacobsen, 1982; Eisemann et al.,
1984a; Gerardo-Gettens et al., 1989), although the mechanism of action is not clear.
Nutritional status has long been recognized as an important factor affecting reproductive
success (Rhind et al., 1985), but insights regarding potential mechanisms linking
nutritional status with the neuroendocrine-reproductive axis have only been made in recent
years (e.g., neuropeptide-Y). In this present study, the elephants were fed a similar diet
throughout the year, so the impact that nutrition might have on reproductive potential and
PRL secretion was minimal.
The mean estrous cycle length of 14 weeks, average 9-week luteal phase and 5week follicular phase, agrees with previously published characteristics of the estrous cycle
for African elephants (Brannian et al., 1988; Plotka et al., 1988; Roach et al., 1990), and
indicates that the elephants were cycling normally throughout the study. The "follicular
phase" is purportedly the time of follicular growth and development, culminating in
ovulation during the nadir of P4 secretion. The higher serum concentrations of PRL
detected during the follicular phase suggest that this hormone may play a role in
modulating ovarian function in elephants. In support of this, a number of recent studies
95
have demonstrated involvement of PRL in regulation of ovarian processes in various
species (Curlewis, 1992; Magoffin and Erickson, 1994; Bowen et al., 1996; Murray et al.,
1996; Clarke et al , 1997; Gaytan et al., 1997). Additionally, ovarian PRL receptor
expression has been shown to vary throughout the reproductive cycle in some species, like
the rat (Clarke et al., 1993), but not in others (e.g., red deer; Clarke et al., 1997).
Prolactin can stimulate LH release from the pituitary in pigs (Jana et al., 1996), and
enhance LH receptor expression in red deer granulosa and luteal cells, as well as facilitate
follicular remodeling changes during ovulation (Clarke et al., 1997). Conversely, PRL
has been shown to play a role in inhibiting folliculogenesis and ovulation in humans by
inhibiting LH-stimulated steroid biosynthesis (McNeilly et al., 1982; Magoffin and
Erickson, 1994), decreasing follicular plasmin generation in red deer (Clarke et al., 1997),
inhibiting estrogen secretion from human granulosa cells (McNeilly et al., 1982), and
stimulating metalloproteinase inhibitor activity in rat granulosa cells (Murray et al., 1996).
The follicular phase, as defined by fluctuations in serum concentrations of P4, in the
elephant is comparatively long in duration, occupying approximately one-third of the
length of the entire estrous cycle. Mares are in estrus for roughly 7 days out of a 28 day
cycle, while estrus in cows and ewes lasts a matter of hours (Hafez, 1993). Perhaps PRL
is involved in the regulation of ovulation in elephants, contributing to the extension of the
length of the follicular phase to an average of 5 weeks. Research designed to follow
folliculogenesis through the combined use of ultrasound (Hildebrandt et al., 1996) and
endocrine profiles to measure LH, follicle-stimulating hormone (FSH), estrogen, P4 and
prolactin (PRL), would provide insight regarding potential hormonal interactions timing
estrous cycle events and ovulation.
It is conceivable that the observed increase in systemic concentrations of PRL
during the follicular phase simply reflect the positive feedback effects of follicular
estrogens. Estrogen has been shown to increase PRL secretion in some species (Lawson
et al., 1993), and concentrations of this gonadal steroid typically increase during the
96
follicular phase, perhaps stimulating PRL secretion from the anterior pituitary in these
elephants. Further research to elucidate PRL's potential role in modulating ovarian
function in the African elephant is required to give meaning to the observed increase in
serum concentrations of PRL during the follicular phase that were likely influenced by
positive feedback by estrogen.
This research demonstrated a significant negative correlation between serum
concentrations of P4 and PRL, in contrast to a recent study by Brown and Lehnhardt
(1997) in which no significant differences in serum concentrations of PRL were observed
in elephants based on estrous cycle stage. The African elephants in this latter study were
living in a similar temperate climate just below 400 latitude, and the reason for the
difference in results is not known. The radioimmunoassays used to quantify serum
concentrations of P4 and PRL were different and could potentially be the basis for the
conflicting results, although ranges for serum concentrations of PRL were similar.
The broad range in duration of the luteal phase (5-12 weeks) as defined by P4
concentrations for these elephants is interesting, and has been a consistent finding for both
African and Asian elephant species in numerous studies (Hess et al., 1983; Plotka et al.,
1988; Mainka and Lothrop, 1990; Olsen et al., 1994). Lack of a standardized P4 assay,
use of different definitions for "luteal phase" (see Table 1), and relatively low serum
concentrations of P4 in elephants most likely contribute to the reported variations in
length. Concentrations of P4 in elephants typically range between 30 and 700 pg/ml (Hess
et al., 1983) compared to 1.5 and 3 ng/ml in sheep (Goodman, 1994); however,
Heistermann et al. (1994) and Schwarzenberger et al. (1996) recently found that 5apregnane-3,20-dione and 5a-pregnane-3a-o1-20-one are secreted in greater quantities
from the elephant corpus luteum (CL) than is P4. Even after accounting for these
variables, the obvious question remains: What controls the life span of the CL in the
elephant? The answer will likely be complex because, unlike most other mammals,
elephants can have secondary and accessory CL (Plotka et al., 1988), and they are able to
97
maintain CL through one or two additional cycles (Hanks and Short, 1972; Smith and
Buss, 1975). The endocrine capability and physiological significance of these variously
derived and aged CL remains a mystery. Presumably an overall decline in CL function
allows cycling to continue. Prolactin can serve as either a luteotropic, luteolytic or
luteostatic agent, depending on the species and reproductive status (Rose et al., 1983;
Berria et al., 1989; reviewed by Curlewis, 1992; Bernard and Bojarski, 1994; Clarke et al.,
1997). Research with rats have demonstrated that some of these actions are mediated
through the immune system (Bowen et al., 1996; Gaytan et al., 1997). Perhaps in
normally cycling elephants, PRL plays a luteotropic role during the follicular phase by
contributing to the maintenance of CL retained from previous cycles. Alternatively, PRL
may participate in the process of luteolysis or suppress the function of those CL being
maintained into the next cycle. A dual role for PRL as a luteolytic and luteotropic
hormone has been identified in the rat and appears to be mediated through the immune
system, whereby PRL increases the proliferative activity of vascular cells in the newly
forming CL, while simultaneously inducing expression of monocyte chemoattractant
protein-1 (Bowen et al., 1996) which results in an invasion of luteolytic macrophages
(Gaytan et al., 1997). It certainly seems plausible that PRL plays a role in modulating the
function and life span of the elephant CL.
There was some evidence of estrous cycle synchronicity within two pairs of
elephants. Synchronous estrous cycles have been observed among elephants in captivity,
but reports have been anecdotal and few have been published (Turczynski et al., 1992).
The difficulty in validating potential cases of estrous cycle synchronicity among captive
elephants stems from inadequate sample sizes, so that the possibility that the synchronous
estrous cycles observed are merely coincidental cannot be excluded. However,
pheromones, which mediate estrous cycle synchronicity, are known to have profound
effects on the reproductive system of many species, in that they mediate both the onset of
puberty in females and ovulation (Keverne, 1983), as well as play a role in mate selection
98
and individual recognition which can potentially minimize inbreeding (Vandenbergh,
1994). The pheromone present in female Asian elephant preovulatory urine has recently
been identified as (Z)-7-dodecen-l-y1 acetate by Rasmussen et al. (1997). Perhaps
exposure of the females to this pheromone accounts for the synchronicity of cycles.
Variations in weekly serum cortisol concentration profiles between elephants in
this study were observed, and to some extent, probably reflect differences in individual
ability to handle various stresses (Schledlowski et al., 1992). Stress is defined as physical
or emotional stimuli of varying length and intensity, and has long been known to increase
adrenocortical activity and decrease reproductive success (reviewed by Wingfield et al.,
1994). Cortisol secretion can increase in response to either real or perceived stress (Rivier
and Rivest, 1991), so it is difficult to determine the cause of cortisol fluctuations in these
elephants.
There was a significant positive correlation between serum concentrations of PRL
and cortisol in the African elephants in this study. This detected relationship may be
coincidental or reflect the ability of stress to alter secretion of PRL. Different types and
intensities of stress elicit different neuroendocrine responses (Schedlowksi et al., 1992),
and this may in part be mediated by activation of different endogenous opiate ligands
and/or receptor subtypes (Pau et al., 1996). Acute stress has been shown to increase
serum concentrations of PRL through a CNS mechanism (Fujikawa et al., 1995; Ziegler et
al., 1996) in which CRF plays a permissive role (Akema et al., 1995), while
glucocorticoids inhibit PRL secretion in rats through a negative feedback mechanism
under conditions of chronic stress (reviewed by Rivier and Rivest, 1991; Taylor et al.,
1995). Thus, it is likely that PRL secretion in elephants as affected by stress depends on
the type (acute or chronic) and intensity of stress experienced, as well as modulation by
steroidal hormones, and this could reflect important functions for PRL in mediating
immune and reproductive responses to stress.
99
Serum concentrations of P4 were negatively correlated with cortisol
concentrations in these elephants, and in several studies with various species, suppressed
P4 secretion was believed to be induced by a stress-related mechanism (Rivier and Rivest,
1991; Hartley et al., 1994; Wasser et al., 1996). Receptors for PRL, CRF, POMCderived peptides, and cortisol have been identified in rodent gonads, enabling stressrelated hormones to exert direct effects on gonadal function by decreasing sensitivity to
gonadotropins and inhibiting steroidogenic activity (reviewed by River and Rivest, 1991).
Because a simultaneous increase in secretion of both cortisol and PRL occurs in response
to acute stress, both hormones may contribute to observed decreases in progesterone
secretion. This study tends to support this hypothesis. While the mechanisms that
mediate the stress response through the hypothalamo-pituitary-gonadal axis are not yet
entirely understood, it seems prudent to assume that stressful conditions will have negative
effects on an elephant's reproductive potential, as can occur in other species.
Hyperthyroidism has not yet been reported in elephants, although Sikes (1971)
found a thyroid gland in an adult bull elephant that weighed 471 g, compared to weights in
other adults ranging from 200-340 g, and she suggested the higher weight was pathologic.
Common clinical signs of hyperthyroidism in small animals include weight loss,
hyperexcitability, increased appetite, more frequent or abnormal urination, as well as
gastrointestinal signs such as diarrhea and increased volume of feces (Fraser, 1991). Tiki,
the elephant suspected of being hyperthyroid, did have symptoms of weight loss,
frequently urinates with her legs crossed, has a "hay belly" or enlarged abdomen and her
stools were comparably softer in consistency. She also had occasional bouts of ventral
abdominal edema, and the cause had not been determined. Her mean serum P4, cortisol
and PRL concentrations were slightly elevated over those of the other elephants in this
study, and possibly reflected the general increase in her metabolic rate.
In conclusion, no effect of season on serum concentrations of PRL was observed
in this study. The higher concentrations of serum PRL detected during the follicular phase
100
suggest that PRL plays a role in modulating ovarian function in elephants. Stress may
affect secretion of PRL in elephants, and cortisol and PRL may diminish reproductive
potential in elephants by altering luteal function. Further studies are needed to
demonstrate the specific roles PRL plays in modulating ovarian function in elephants, and
how environmental and physiological cues are transduced to affect PRL secretion and
action. Research in this area requires an integrative approach, because many factors
contribute to the control of the secretion and function of PRL
.
101
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APPENDICES
130
Appendix A Additional Prolactin Radioimmunoassay
A radioimmunoassay (RIA) was recently validated using
125
I labelled ovine
prolactin (NIDDK-oPRL-I-2), African elephant standards (elephant PRL[G100, 2.1]
purified by C.H. Li, UC San Francisco), and anti-elephant prolactin antisera raised in
rabbits instead of anti-human prolactin antisera (NIDDK-anti-hPRL-3). This assay has not
yet been utilized for serum analyses. At a 1:5000 dilution, the antibody bound 30% of the
125I-prolactin with 3% nonspecific binding. Serial dilutions of elephant serum pools were
parallel to the standard curve, and cross-reactivity with African elephant growth hormone
was insignificant. Upon addition of 0.5, 1.0, 5.0, and 10.0 ng elephant prolactin to 50 41
elephant serum, 0.3, 1.1, 3.6, and 7.8 ng were recovered after subtraction of endogenous
hormone (y = 0.0.018 + 0.8823x; R2 = 0.962).
African elephant prolactin iodinations were not successful because of low and
variable binding and inconsistent recoveries. Various manipulations were made utilizing
lactoperoxidase and chloramine-T iodination techniques, trying different iodination
fractions, and using different antibody dilution combinations. Because the prolactin was
prepared over 10 years ago, molecular instability resulting from the period of long storage
could be a contributing factor to the iodination problems observed (H. Papkoff, personal
communication).
131
Appendix B Melatonin Radioimmunoassay
Attempts were made to quantify African elephant serum melatonin concentrations
using a MA with various extraction techniques, gas chromatography, and high
performance liquid chromatography (HPLC) methods. The RIA utilized G/S/704-6483
anti-melatonin Guildhay antibody (Stockgrand Ltd., Guildford, Surrey, UK), 3H-
melatonin (NET-801, New England Nuclear, Boston, MA) diluted to approximately 6,000
cpm/tube, and melatonin standards (Sigma, St. Louis, MO). The sheep anti-melatonin
antibody was diluted 1:6000 in Tricine-buffered saline (TBS; pH 5.5) with 25% charcoalstripped elephant serum. Initially, extractions were performed using 1 ml chloroform
(Photrex; J.T. Baker, Phillipsburg, NJ) added to 350 p.1 night serum samples or 2 ml
chloroform added to 700 p.1 day serum samples using a Hamilton Digital Diluter. After
vortexing for 30 sec, 800 p.1 of the chloroform phase was removed and evaporated to
dryness in a separate test tube. Residues were resuspended in 500 µ1 TBS, vortexed
thoroughly, and incubated with 200 p.1 antibody for 30 min at room temperature. Then
100 pi 3H-melatonin was added to each sample, tubes were vortexed briefly and left to
incubate at 40C for 19-21 h. Separation of bound and free hormone was achieved by
incubation with 0.5 mL 2% charcoal suspension with 0.02% dextran at 40C for 15 min
followed by centrifugation at 40C and 1315xg for 15 min. Supernatant was poured into
20 ml vials containing 6 ml scintillation cocktail (Ecolume; ICN Biochemicals, Costa
Mesa, CA) and counted on a Beckman LS6500 Liquid Scintillation Counter for 2 min
each.
The antibody bound 40% of the tracer at the 1:6000 dilution with 3% nonspecific
binding, and assay sensitivity was estimated to be 2 pg/tube, with the standard curve
ranging from 0.1-500 pg/tube. Extraction efficiency calculations ranged between 85-90%,
yet upon addition of 10, 50 and 200 pg melatonin to 700 pi elephant serum, only 4.6, 18.8
and 74.6 pg were recovered after subtraction of endogenous hormone (y = 0.627 +
132
0.686x; R2 = 0.746). Recoveries made by adding exogenous melatonin to samples after
the extraction procedure were much better (y = 1.73 + 0.926x; r2 = 0.991), indicating that
the problem existed in the extraction procedure. Quantities of melatonin detected in
African elephant day serum samples ranged between nondetectable to <10 pg/ml, not
differing significantly from night serum samples. The intraassay coefficient of variation
was high, ranging between 14-30%.
Believing the extraction problem to be based on a substance in the elephant serum
that could bind melatonin, serum pH was adjusted to 8.5. This tended to simultaneously
increase the concentration of melatonin measured in control sera (means of 9.2 versus 5.2
pg/ml) and decrease intraassay variability (14.7% versus 28.5%), but not significantly.
The pH change had no effect on recovery rates with extraction: after addition of 10, 50
and 200 pg melatonin, 5.5, 20.8 and 70.28 pg were recovered after subtraction of
endogenous hormone (y = 0.845 + 0.673x; R2 = 0.724). Dichloromethaneextractions
were attempted in another laboratory, but recovery rates were also poor (S. Yellon, Loma
Linda University, Loma Linda, CA). Solid phase extraction, using Prep-Sep C-18
columns (Ryan and Volk, 1995) again yielded low recovery rates and low or nondetectable levels of melatonin in serum (K. Ryan, Magee Womens Research Institute,
Pittsburgh, PA). Gas chromatography gave unreliable results (D. Holtan, Oregon State
University, Corvallis,OR), and serum melatonin was not detectable by HPLC (G.
Gerhardt, Colorado State University, Ft. Collins, CO).
Quantities of melatonin detected in African elephant day serum samples ranged
between nondetectable to <10 pg/ml, and did not differ significantly from night serum
concentrations of melatonin. It is possible that African elephants have naturally low
melatonin concentrations which do not fluctuate on a diurnal rhythm as observed in other
species; however, recovery rates were poor when exogenous sources of melatonin were
added to elephant serum samples, suggesting that the problem stems from inadequate
sensitivity or detection capabililty in the RIA utilized.