Laura Graham | Toronto Metropolitan University (original) (raw)

Papers by Laura Graham

Research paper thumbnail of A two-stage method to approach weaning stress in horses using a physical barrier to prevent nursing

Applied Animal Behaviour Science

Research paper thumbnail of Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus)

Theriogenology, 2002

P < 0:05) in the summer. Ejaculates were collected throughout the year; however, peaks in average... more P < 0:05) in the summer. Ejaculates were collected throughout the year; however, peaks in average sperm production were observed during the summer for all species. In summary, this study has identi®ed several species differences in male testicular traits among ocelots, margays and tigrinas. Results of longitudinal reproductive assessments suggest males of each species are capable of breeding throughout the year.

Research paper thumbnail of Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis)

Reproduction, Fertility and Development, 2014

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon ... more The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March–June and September–November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March–June than September–November) or an interaction between year and season (...

Research paper thumbnail of Natural Versus Chorionic Gonadotropin-lnduced Ovarian Responses in the Clouded Leopard (Neofelis nebulosa) Assessed by Fecal Steroid Analysis

The clouded leopard (Neofelis nebulosa) is an endangered species difficult to propagate in captiv... more The clouded leopard (Neofelis nebulosa) is an endangered species difficult to propagate in captivity because of sexual incompatibility between paired individuals. Fecal estradiol (E2) and progesterone (P 4) metabolites were quantified in 14 females to noninvasively monitor seasonal reproductive patterns and compare ovarian responses to natural mating vs. exogenous gonadotropins. Increased E excretion was associated with behavioral estrus or eCG treatment, whereas elevated P,4 metabolites were observed during natural and hCG-induced pregnant and nonpregnant luteal phases. On the basis of fecal E, profiles, duration of the estrous cycle was 24 2 days, with estrus lasting 6 1 days. Mean gestation length was 89 2 days, whereas duration of the nonpregnant luteal phase was 47 2 days. Females experienced a seasonal anestrus during the late summer and early fall. One female demonstrated a lactational anestrus after the birth of three cubs. On nine occasions, clouded leopards ovulated sponta...

Research paper thumbnail of Enzyme-immunoassay for the measurement of luteinizing hormone in the serum of African elephants (Loxodonta africana)

Zoo Biology, 2002

Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well ... more Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well characterized, and routine monitoring of these hormones is now viewed as a valuable tool for making informed decisions about the reproductive management of elephants in captivity. Currently, LH monitoring in elephants is done with radio-immunoassays (RIAs); unfortunately, the use of radioactive materials in RIAs limits their application to institutions with laboratory facilities equipped for the storage and disposal of radioactive waste. Enzyme-immunoassays (EIAs) offer an inexpensive and more zoo-friendly alternative to RIA. This work reports on an EIA capable of quantifying circulating LH in African elephants. The EIA employs a biotin label and microtiter plates coated with goat anti-mouse gamma globulin. LH surges in African elephants (n=3) increased fivefold over baseline concentrations (1.0070.1 ng/ml vs. 0.270.1 ng/ml) and occurred 19.370.2 days apart. Ovulatory LH surges were associated with an increase in serum progestogens from 4.870.4 ng/ml to 11.770.4 ng/ml. The ability to quantify reproductive hormones in elephants via EIA is an important step in the process of making endocrine monitoring more accessible to zoos housing these species. Zoo Biol 21:403-408, 2002.

Research paper thumbnail of The reproductive endocrinology and behavior of Vancouver Island marmot (Marmota vancouverensis)

Zoo Biology, 2012

The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America&amp;amp;amp... more The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America&amp;amp;amp;amp;amp;amp;#39;s most endangered species with fewer than 150 individuals remaining in the wild. A captive breeding program was established across four facilities in Canada as an insurance population and source of animals for reintroduction to the wild. The purpose of this study was to gather information about the basic reproductive biology and behavior of this species, which is essential to improve captive breeding programs. Regular fecal samples were obtained from adult female (n = 14) and male (n = 10) marmots, 2 years of age and older, over 1-3 breeding seasons (2-3 months duration posthibernation) for steroid hormone analysis. Enzyme immunoassays were validated for quantifying fecal testosterone metabolite concentrations for males, and fecal estrogen and progesterone metabolite concentrations for females. Results indicated that fecal progesterone metabolite concentrations can be used to monitor ovulation and pregnancy. Behavioral monitoring through infrared video surveillance was conducted in four breeding pairs over a 2-year period (n = 7 behavioral profiles). Breeding behaviors correlated strongly with changes in reproductive endocrine profiles. A high frequency of play behavior or &amp;amp;amp;amp;amp;amp;quot;wrestling&amp;amp;amp;amp;amp;amp;quot; was observed in conjunction with breeding activity before an elevation in progesterone metabolite concentrations. Impending parturition was associated with increased aggression and exclusion of the male from the maternal nestbox as well as an increase in nesting activity. Observational data combined with hormonal analysis suggest that female VIMs are induced ovulators and that multiple breeding attempts may be required for ovulation and conception. Gestation appears to be approximately 34 days from peak breeding activity (32 days from estimated ovulation). Fecal testosterone concentrations suggest that testicular activity is seasonal with the reproductive activity occurring immediately posthibernation. Monitoring breeding behavior is a useful means of indicating estrus, conception and pregnancy, which can also be supported by the hormonal analysis of daily fecal samples of individual animals.

Research paper thumbnail of A versatile enzyme immunoassay for the determination of progestogens in feces and serum

Zoo Biology, 2001

The ability of zoos to monitor the reproductive status of their animals can vastly improve the ef... more The ability of zoos to monitor the reproductive status of their animals can vastly improve the effectiveness of husbandry/management practices, and noninvasive methods such as fecal steroid analysis are the easiest to apply in a zoo setting. Furthermore, enzyme immunoassay (EIA) is preferred to radioimmunoassay (RIA) as the method of quantifying hormones because EIAs do not involve the use, storage, and disposal of radioactive materials. However, progesterone is excreted in the feces as predominantly unconjugated metabolites (progestogens) and, until recently, antibodies able to cross-react with a variety of progestogens were used primarily in RIAs. An EIA using a broad-spectrum progestogen antibody is described and applied to serum and/or fecal samples from female African elephants, black rhinoceros, white rhinoceros, okapi, and hippopotami. The clear progestogen profiles generated in these species suggest that the described EIA would be as versatile as the RIA using the same antibody and could be a practical and economical alternative to RIAs for monitoring gonadal function via progestogen analysis in zoo species.

Research paper thumbnail of Fecal androgen metabolite analysis for noninvasive monitoring of testicular steroidogenic activity in felids

Zoo Biology, 1996

Limited data are available on long-term, seasonal changes in testicular ste- roidogenic activity ... more Limited data are available on long-term, seasonal changes in testicular ste- roidogenic activity in nondomestic felids, primarily because of the difficulties associated with longitudinal blood sampling (eg, handling, restraint, ...

Research paper thumbnail of Reproductive endocrine responses to photoperiod and exogenous gonadotropins in the pallas' cat (Otocolobus manul)

Zoo Biology, 2002

Fecal samples were collected for 14-26 months from three male and six female Pallas' cats (Otocol... more Fecal samples were collected for 14-26 months from three male and six female Pallas' cats (Otocolobus manul) to examine gonadal steroidogenic activity in response to changes in photoperiod and treatment with exogenous gonadotropins. Females exhibited a seasonal anestrus from May-December, excreting consistently low concentrations of fecal estrogens (overall mean, 50.278.5 ng/g). During the breeding season (January-April), baseline fecal estrogen concentrations were higher, averaging 128.4718.9 ng/g, with peak concentrations ranging from 455.8-909.6 ng/g. Interpeak intervals in estrogen excretion ranged between 7 and 21 days, with an average estrous cycle length of 14.371.7 days. Two females became pregnant after natural mating, with overall luteal progestogen concentrations averaging B40 mg/g throughout gestation. Fecal estrogens increased in mid-gestation, peaking just before birth. Induction of follicular development with eCG (100-300 IU, i.m.) resulted in an increase in fecal estrogens (peak range, 263.1-1198.1 ng/g), followed by a postovulatory increase in fecal progestogens (overall mean, 41.1711.9 mg/g) after hCG (75-150 IU, i.m.). Despite apparently normal ovarian responses, none of the females conceived after artificial insemination (AI). The gonadotropin-induced nonpregnant luteal phase lasted 49.875.3 days (range, 30-60 days), whereas gestation lasted B70 days. In the male Pallas' cat, fecal androgens were elevated from November-April

Research paper thumbnail of Successful artificial insemination of an Asian elephant at the National Zoological Park

Zoo Biology, 2004

For decades, attempts to breed elephants using artificial insemination (AI) have failed despite c... more For decades, attempts to breed elephants using artificial insemination (AI) have failed despite considerable efforts and the use of various approaches. However, recent advances in equipment technology and endocrine-monitoring techniques have resulted in 12 elephants conceiving by AI within a 4-year period (1998-2002). The successful AI technique employs a unique endoscope-guided catheter and transrectal ultrasound to deliver semen into the anterior vagina or cervix, and uses the ''double LH surge'' (i.e., identifying the anovulatory LH (anLH) surge that predictably occurs 3 weeks before the ovulatory LH (ovLH) surge to time insemination. This study describes the 6-year collaboration between the National Zoological Park (NZP) and the Institute for Zoo Biology and Wildlife Research (IZW), Berlin, Germany, that led to the refinement of this AI technique and subsequent production of an Asian elephant calf. The NZP female was the first elephant to be inseminated using the new AI approach, and was the fifth to conceive. A total of six AI trials were conducted beginning in 1995, and

Research paper thumbnail of Comparison of fecal storage methods for steroid analysis in black rhinoceroses (Diceros bicornis)

Zoo Biology, 2004

Many field studies and conservation programs for wildlife species include noninvasive endocrine m... more Many field studies and conservation programs for wildlife species include noninvasive endocrine monitoring of gonadal function. Freezing fecal samples immediately after collection until further analysis is often not a viable option for researchers in remote areas. Phase 1 of this study was designed to compare different methods of preserving fecal samples over several time periods (30, 90, or 180 days) in order to determine which method provided the most accurate and reliable technique for measuring fecal progestagens. Fecal samples were collected from two female black rhinoceroses (Diceros bicornis) housed at Disney's Animal Kingdom, Lake Buena Vista, FL. We compared three storage methods: 1) storing fecal samples without processing or preservatives (untreated), 2) storing an aliquot of fecal sample in 80% methanol (MeOH), and 3) drying the fecal sample in a solar box cooker prior to storage. Control samples (day 0) were collected and extracted, and then stored at À201C until they were analyzed. Phase 2 of the study was designed to examine the effects of long-term storage (up to 180 days) on fecal progestagen profiles that reflect reproductive activity (pregnancy and estrous cycles). In samples obtained from a pregnant female and stored for 30 days, there were no significant differences in fecal progestagen concentrations between the three treatment conditions. However, the mean concentrations of progestagens (7 SE) in untreated samples increased significantly from 8.3 7 0.3 mg/g wet weight feces at day 0 to 17.7 7 5.1 mg/g feces at day 90, and 17.8 7 4.7 mg/g feces at day 180. Samples that were collected from a pregnant female and stored in 80% MeOH or dried in the solar box correlated with controls (r ¼ 0.86 and 0.87, respectively; Po0.05) at day 180. In contrast, samples that were stored without preservatives for 180 days did not correlate with controls (r ¼ 0.35, P40.05). Progestagen concentrations from samples of the estrous cycling female showed similar results. In conclusion, fecal samples dried in a solar box cooker or stored in 80% MeOH maintained absolute and relative progestagen concentrations for at least 180 days when they were stored outdoors and exposed to the climatic conditions of central Florida. Both methods can have significant applications for

Research paper thumbnail of Non-invasive monitoring of ovarian function in several felid species by measurement of fecal estradiol-17β and progestins

Zoo Biology, 1995

An extraction and assay procedure to measure fecal estradiol-17p and progestin concentrations in ... more An extraction and assay procedure to measure fecal estradiol-17p and progestin concentrations in several cat species was developed and validated for use for noninvasive monitoring of ovarian function. Fecal samples were collected over a range of 3-20 months from female tigers (three), lions (three), snow leopards (three), cheetahs (two), caracals (two), and domestic cats (five). Samples were extracted with 90% methanol, lipids removed with petroleum ether, and the estradiol and progestins in the methanol measured by radioimmunoassay (RIA). High Performance Liquid Chromatography (HPLC) fractionation and subsequent RIA of the fractions indicated that the estradiol-17p antiserum cross-reacted primarily with estradiol-17P in the feces of lions and tigers and was assumed to be specific for estradiol-17P in the feces of other species as well. However, there were several immunoreactive compounds, presumably progesterone metabolites, excreted in the feces which varied both quantitatively and qualitatively among species. The behavior of tigers, lions, cheetahs, and caracals was visually monitored during the collection period and frequency of sexual behaviors was positively correlated with increases in fecal estradiol in all species observed. The mean fecal estradiol-17P peaks were as follows: tigers, 128.0 t 13.1; lions, 186.0 5 14.8; snow leopards, 136.7 k 15.9; cheetahs, 140.9 k 9.0; caracals, 24.5 ? 4.0; and domestic cats 158.9 2 19.3 ng/gm. Fecal progestin concentrations rose significantly (P < 0.001) only after breeding or during pregnancy and were as follows: tigers, 5.6 t 0.6; lions, 1.9 t 0.1; cheetahs, 8.4 5 1.1; and caracals, 2.4 5 0.4 pg/gm. Fecal progestins were elevated for one-half to twothirds of the gestation length during presumed pseudopregnancy but remained elevated throughout successful pregnancies. These results suggest that ovarian function can be monitored noninvasively in the family Felidae by the measurement of fecal estradiol-17P and progestin concentrations 0 1995 Wiley-Liss, Inc.

Research paper thumbnail of Behavioral indices of estrus in a group of captive African elephants (Loxodonta africana)

Zoo Biology, 2005

This study investigated behavioral signals of estrus by systematically monitoring the interaction... more This study investigated behavioral signals of estrus by systematically monitoring the interactions of one male with four female African elephants housed in a naturalistic outdoor enclosure at Disney's Animal Kingdom over a period of 11 months. We measured changes in five spatial behaviors and 22 tactile-contact behaviors, as well as changes in serum progestagen and LH concentrations, across three ovarian cycles for each female. Two females did not cycle during the study. Three different phases of the ovarian cycle were identified: mid luteal, anovulatory follicular, ovulatory follicular. The male followed more and carried out more genital inspections, flehmen, and trunk-to-mouth behaviors toward cycling females during their ovulatory phase. Genital inspections by the male peaked above baseline levels on the day of an LH surge, and up to 9 days before, in both cycling females and, thus, might be a useful behavioral index of estrus. The male also carried out more genital inspections, flehmen, and trunk touches to the back leg toward ovulatory cycling than noncycling females. Overall, our results indicated that: 1) a single subadult African elephant male could discriminate two females in the ovulatory phase of their cycle (i.e., during the 3 weeks preceding ovulation) from the mid luteal phase; 2) the male also discriminated two cycling females in the ovulatory and anovulatory follicular phases from two noncycling females; 3) two females in the

Research paper thumbnail of Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport

Theriogenology, 2000

Fecal steroid analysis was used to investigate relationships between endocrine parameters and emb... more Fecal steroid analysis was used to investigate relationships between endocrine parameters and embryo characteristics in domestic cats subjected to chorionic gonadotropin stimulation and artificial insemination (AI). In Study 1, normal endocrine patterns were assessed in 12 cycling domestic queens. Fecal estradiol (E) patterns established an anovulatory cycle length of 18.3 + 0.4 d with estrus lasting 6.3 f 0.3 d. Eight females (67%) exhibited at least one spontaneous ovulation based on sustained increases in fecal progestagens (P). In Study 2, queens were mated during natural estrus (NE, n = 5) or subjected to exogenous i.m. gonadotropin stimulation, 100 IU eCG followed by 75 IU hCG 80 h later, (GS, n = 5). Compared with NE queens, fecal E concentrations were higher (P < 0.05) and remained elevated longer after ovulation induction with hCG. In Study 3, gonadotropin-stimulated queens (n = 7) were artificially inseminated and ovariohysterectomized 160 h after hCG. Ancillary follicles and/or corpora lutea were observed in 5 of 6 (83%) ovulating queens. Both fecal E and number of unovulated follicles observed at ovariohysterectomy were negatively correlated with the percentage of embryos recovered from the uterus (r =-0.91 and r =-0.87, respectively; P < 0.05). In summary, exogenous gonadotropin administration causes an abnormal endocrine environment in domestic cats, likely due to ancillary follicle development. The sustained elevations in estradiol appear to impair oviductal transport of embryos, possibly leading to the reduced fertility typically observed in cats subjected to gonadotropin stimulation and AI.

Research paper thumbnail of Influence of oral melatonin on natural and gonadotropin-induced ovarian function in the domestic cat

Theriogenology, 2004

Ovarian hyperstimulation after exogenous gonadotropin stimulation is believed to be a cause of po... more Ovarian hyperstimulation after exogenous gonadotropin stimulation is believed to be a cause of poor success after artificial insemination (AI) in felids. The objectives of this study were to assess the effect of oral melatonin on endogenous ovarian activity in the domestic cat and subsequent eCG/hCG-induced ovarian activity. Serum melatonin concentrations peaked $1 h after a single oral dose of 30 mg melatonin and remained elevated above endogenous daytime concentrations for >8 h. The calculated circulating half-life (mean AE S:E:M:) of oral melatonin was 45:4 AE 3:5 min, and the elimination rate constant (k 10) was 55:2 AE 4:2 min À1. Oral melatonin (30 mg per day) administered 3 h before lights-off effectively and reversibly suppressed estrous elevations in fecal estrogens after 25 days of treatment. There was a progressive decrease in baseline estrogen concentrations from inter-estrous concentrations after 25 days of treatment to below inter-estrous concentrations after 35 days of treatment. Oral melatonin treatment (30 mg per day for 30 days) prior to eCG/hCG administration only marginally reduced ancillary follicle development and had no significant effect on the quantity or quality of embryos produced by AI. Thus, oral melatonin effectively inhibited endogenous ovarian activity and had no adverse impact on embryo quality after AI in the domestic cat; however, this treatment was only marginally effective in minimizing eCG/hCG-induced ovarian hyperstimulation.

Research paper thumbnail of Reproductive activity in captive female cheetahs (Acinonyx jubatus) assessed by faecal steroids

Reproduction, 1996

Faecal oestradiol and progestogen metabolite excretion was monitored in adult, female cheetahs (A... more Faecal oestradiol and progestogen metabolite excretion was monitored in adult, female cheetahs (Acinonyx jubatus) (n = 26) for 1\p=n-\24months. Increased faecal oestradiol excretion was associated with mating or equine chorionic gonadotrophin (eCG) administration for artificial insemination, whereas increased progestogen metabolites were observed during natural and human chorionic gonadotrophin (hCG)-induced pregnant and nonpregnant luteal phases. On the basis of oestradiol excretory patterns, duration of the oestrous cycle (mean \ m=+-\ SEM) was 13.6 \ m=+-\ 1.2 days with high oestradiol concentrations lasting for 4.1 \ m=+-\ 0.8 days. In non-gonadotrophin-treated cheetahs, 75% showed evidence of oestrous cyclicity; however, none evaluated for 1 year or longer were continuously cyclic. Rather, cyclicity was interrupted by periods of anoestrus, often exceeding several months in duration. These inactive ovarian periods were unrelated to season and were not synchronous among females. Mean duration of gestation (breeding to parturition) was 94.2 \ m=+-\ 0.5 days, whereas duration of faecal progestogen metabolite excretion during the nonpregnant luteal phase was 51.2 \ m=+-\ 3.5 days. On the basis of progestogen metabolite evaluations, spontaneous ovulation (non-mating induced) occurred only once in two females (2 of 184 oestrous cycles; 1.1%). Peak eCG-stimulated, preovulatory oestradiol concentrations were similar to those associated with natural oestrus, whereas progestogen metabolite profiles after hCG resembled those during pregnant and nonpregnant luteal phases after natural mating. In summary, results confirm that the cheetah is polyoestrus and ovulation is almost always induced. However, new evidence suggests that many females inexplicably experience periods of anoestrus unrelated to season, while 25% of the cheetahs examined expressed no ovarian activity during the study period.

Research paper thumbnail of The use of low-frequency vocalizations in African elephant(Loxodonta africana) reproductive strategies

Hormones and Behavior, 2003

Fertility-advertisement calls in females are predicted to occur in nonmonogamous species where ma... more Fertility-advertisement calls in females are predicted to occur in nonmonogamous species where males and females are widely separated in space. In African elephants, low-frequency vocalizations have thus been suggested as a reproductive strategy used by fertile females to attract mates. This study examined the use of low-frequency vocalizations with respect to different phases of the estrous cycle in African elephants by simultaneously monitoring vocalizations, behavior, and hormonal profiles. Subjects were one male and six female African elephants housed at Disney's Animal Kingdom. No acoustically distinct vocalizations were restricted to the ovulatory follicular phase. However, overall rate of low-frequency vocalization as well as the rate of one acoustically distinct vocalization changed over the estrous cycle, with highest rates of calling related to the first period of follicular growth, or anovulatory follicular phase. Elevated rates of vocalization thus were not restricted to behavioral estrus and occurred much earlier in the estrous cycle than in most species that produce fertility-advertisement calls. Both herd composition and elephant identity also affected rates of vocalization. Vocalizations therefore may not be reliable signals of actual fertility. However, the increase in vocalizations in advance of estrus may attract males to the herd prior to ovulation, facilitating both male-male competition and female choice. Once present in the herd, males may then switch strategies to use more reliable chemical and visual cues to detect ovulating females.

Research paper thumbnail of Endocrine patterns associated with reproduction in the Nile hippopotamus (Hippopotamus amphibius) as assessed by fecal progestagen analysis

General and Comparative Endocrinology, 2002

The hippopotamus (Hippopotamus amphibius) is a popular zoo animal and an integral part of wetland... more The hippopotamus (Hippopotamus amphibius) is a popular zoo animal and an integral part of wetland ecosystems in its native continent of Africa. However, information about the reproductive physiology of the hippopotamus is limited compared to the other megaherbivores of Africa such as the elephant and the rhinoceros. This study is the first to report on the endocrine patterns associated with ovulation and pregnancy in the Nile hippopotamus. Fecal samples were collected 3 times per week from female hippopotami (N = 9) housed in captivity in Florida. Progesterone metabolites were extracted from the feces with 80% MeOH and quantified using a progestagen EIA. Fecal progestagen analysis indicated hippopotami had a cycle length of 35.3 +/- 1.3 days and ovulated throughout the year. Fecal progestagen concentrations during pregnancy (N = 11 pregnancies) were higher on average (833.5 +/- 115.2 ng/g; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than those observed during non-pregnant luteal phases (309.5 +/- 20.9 ng/g) and remained elevated throughout gestation. Fecal progestagen analysis revealed the onset of puberty in three females between 3 and 4 years of age and all conceived by their fourth cycle. Lactational suppression of ovulation during nursing was usually, but not always, observed and lasted for 33.9 +/- 1.8 weeks. The observed calving interval in females with lactational anovulation was 17 months. The majority of births (63.6%) occurring during the summer rainy season and the inconsistency of lactational anovulation suggested the possibility of environmental influences on reproduction.

Research paper thumbnail of Natural and gonadotropin-induced ovarian activity in tigers (Panthera tigris) assessed by fecal steroid analyses

General and Comparative Endocrinology, 2006

Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated... more Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated with natural ovarian activity with those after chorionic gonadotropin ovulation induction and artificial insemination (AI). Baseline fecal estradiol concentrations were 65.77 +/- 3.61 ng/g with estrual peaks of 167.39 +/- 9.92 ng/g and an anovulatory cycle length of 17.96 +/- 0.70 days. Peak fecal estradiol was higher when females were housed with a male for breeding (262.30 +/- 41.43 vs. 165.30 +/- 3.67 ng/g; P &amp;amp;lt; 0.05). The majority of animals showed some seasonal differences in fecal estradiol however, patterns were inconsistent. Fecal progestagens increased only after breeding confirming tigers are primarily induced ovulators. The non-pregnant luteal phase was 34.50 +/- 1.85 days in duration. In pregnant tigers, fecal progestagens remained elevated for 108 days until parturition and the diagnosis of pregnancy was possible based on the elevated fecal progestagens after 35 days of gestation. Tigers were administered equine chorionic gonadotropin (eCG) to stimulate follicular growth and human chorionic gonadotropin (hCG) to induce ovulation prior to AI [200 IU eCG/100 IU hCG (n = 5); 400 IU eCG/200 IU hCG (n = 2); 500 IU eCG/100 IU hCG (n = 2); 1000 IU eCG/750 IU hCG (n = 11); 1000 IU eCG/1000 IU hCG (n = 4)]. None of the tigers subjected to AI became pregnant (n = 9). Fecal endocrine patterns in gonadotropin-stimulated tigers were considerably different from those observed in naturally bred tigers. In particular, fecal estradiol concentrations were higher than those observed during natural estrus and remained elevated for longer periods of time in tigers administered the higher doses of gonadotropins typically used in conjunction with AI in this species. These abnormal endocrine patterns may help explain the poor success rate of AI in this species.

Research paper thumbnail of Hormone Secretion in the Asian Elephant (Elephas maximus): Characterizationof Ovulatory and Anovulatory Luteinizing Hormone Surges

Biology of Reproduction, 1999

In the elephant, two distinct LH surges occur 3 wl< apart during the nonluteal phase of the estro... more In the elephant, two distinct LH surges occur 3 wl< apart during the nonluteal phase of the estrous cycle, but only the second surge (ovLH) induces ovulation. The function of the first, anovulatory surge (anLH) is unknown, nor is it clear what regulates the timing of these two surges. To further study this observation in the Asian elephant, serum concentrations of LH, FSH, progesterone, inhibin, estradiol, and prolactin were quantified throughout the estrous cycle to establish temporal hormonal relationships. To examine long-term dynamics of hormone secretion, analyses were conducted in weekly blood samples collected from 3 Asian elephants for up to 3 yr. To determine whether differences existed in secretory patterns between the anLH and ovLH surges, daily blood samples were analyzed from 21 nonluteal-phase periods from 7 Asian elephants. During the nonluteal phase, serum LH was elevated for 1-2 days during anLH and ovLH surges with no differences in peak concentration between the two surges. The anLH surge occurred 19.9 ± 1.2 days after the end of the luteal phase and was followed by the ovLH surge 20.8 ± 0.5 days later. Serum FSH concentrations were highest at the beginning of the nonluteal phase and gradually declined to nadir concentrations within 4 days of the ovLH surge. FSH remained low until after the ovLH surge and then increased during the luteal phase. Serum inhibin concentrations were negatively correlated with FSH during the nonluteal phase (r = •0.53). Concentrations of estradiol and prolactin fluctuated throughout the estrous cycle with no discernible patterns evident. In sum, there were no clear differences in associated hormone secretory patterns between the anLH and ovLH surge. However, elevated FSH at the beginning of the nonluteal phase may be important for follicle recruitment, with the first anLH surge acting to complete the follicle selection process before ovulation.

Research paper thumbnail of A two-stage method to approach weaning stress in horses using a physical barrier to prevent nursing

Applied Animal Behaviour Science

Research paper thumbnail of Seasonal analysis of semen characteristics, serum testosterone and fecal androgens in the ocelot (Leopardus pardalis), margay (L. wiedii) and tigrina (L. tigrinus)

Theriogenology, 2002

P < 0:05) in the summer. Ejaculates were collected throughout the year; however, peaks in average... more P < 0:05) in the summer. Ejaculates were collected throughout the year; however, peaks in average sperm production were observed during the summer for all species. In summary, this study has identi®ed several species differences in male testicular traits among ocelots, margays and tigrinas. Results of longitudinal reproductive assessments suggest males of each species are capable of breeding throughout the year.

Research paper thumbnail of Seasonal variation in urinary and salivary reproductive hormone levels in Amazonian manatees (Trichechus inunguis)

Reproduction, Fertility and Development, 2014

The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon ... more The Amazonian manatee (Trichechus inunguis) is a threatened aquatic mammal endemic to the Amazon basin. The aim of this study was to evaluate the urinary and salivary reproductive hormone levels of captive Amazonian manatees collected during two seasons of the year. Salivary samples from four males and urinary and salivary samples from three females were collected during two seasons (March–June and September–November) over two consecutive years. Salivary testosterone in males was measured by radioimmunoassay and reproductive hormones in females (salivary progesterone and oestradiol and urinary progestogens, oestrogens and luteinising hormone) were measured by enzyme immunoassay. The data were analysed in a 2×2 factorial design, where the factors were year and season. There was no effect of year or season for salivary testosterone. All female hormones showed a seasonal effect (higher hormone levels during March–June than September–November) or an interaction between year and season (...

Research paper thumbnail of Natural Versus Chorionic Gonadotropin-lnduced Ovarian Responses in the Clouded Leopard (Neofelis nebulosa) Assessed by Fecal Steroid Analysis

The clouded leopard (Neofelis nebulosa) is an endangered species difficult to propagate in captiv... more The clouded leopard (Neofelis nebulosa) is an endangered species difficult to propagate in captivity because of sexual incompatibility between paired individuals. Fecal estradiol (E2) and progesterone (P 4) metabolites were quantified in 14 females to noninvasively monitor seasonal reproductive patterns and compare ovarian responses to natural mating vs. exogenous gonadotropins. Increased E excretion was associated with behavioral estrus or eCG treatment, whereas elevated P,4 metabolites were observed during natural and hCG-induced pregnant and nonpregnant luteal phases. On the basis of fecal E, profiles, duration of the estrous cycle was 24 2 days, with estrus lasting 6 1 days. Mean gestation length was 89 2 days, whereas duration of the nonpregnant luteal phase was 47 2 days. Females experienced a seasonal anestrus during the late summer and early fall. One female demonstrated a lactational anestrus after the birth of three cubs. On nine occasions, clouded leopards ovulated sponta...

Research paper thumbnail of Enzyme-immunoassay for the measurement of luteinizing hormone in the serum of African elephants (Loxodonta africana)

Zoo Biology, 2002

Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well ... more Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well characterized, and routine monitoring of these hormones is now viewed as a valuable tool for making informed decisions about the reproductive management of elephants in captivity. Currently, LH monitoring in elephants is done with radio-immunoassays (RIAs); unfortunately, the use of radioactive materials in RIAs limits their application to institutions with laboratory facilities equipped for the storage and disposal of radioactive waste. Enzyme-immunoassays (EIAs) offer an inexpensive and more zoo-friendly alternative to RIA. This work reports on an EIA capable of quantifying circulating LH in African elephants. The EIA employs a biotin label and microtiter plates coated with goat anti-mouse gamma globulin. LH surges in African elephants (n=3) increased fivefold over baseline concentrations (1.0070.1 ng/ml vs. 0.270.1 ng/ml) and occurred 19.370.2 days apart. Ovulatory LH surges were associated with an increase in serum progestogens from 4.870.4 ng/ml to 11.770.4 ng/ml. The ability to quantify reproductive hormones in elephants via EIA is an important step in the process of making endocrine monitoring more accessible to zoos housing these species. Zoo Biol 21:403-408, 2002.

Research paper thumbnail of The reproductive endocrinology and behavior of Vancouver Island marmot (Marmota vancouverensis)

Zoo Biology, 2012

The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America&amp;amp;amp... more The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America&amp;amp;amp;amp;amp;amp;#39;s most endangered species with fewer than 150 individuals remaining in the wild. A captive breeding program was established across four facilities in Canada as an insurance population and source of animals for reintroduction to the wild. The purpose of this study was to gather information about the basic reproductive biology and behavior of this species, which is essential to improve captive breeding programs. Regular fecal samples were obtained from adult female (n = 14) and male (n = 10) marmots, 2 years of age and older, over 1-3 breeding seasons (2-3 months duration posthibernation) for steroid hormone analysis. Enzyme immunoassays were validated for quantifying fecal testosterone metabolite concentrations for males, and fecal estrogen and progesterone metabolite concentrations for females. Results indicated that fecal progesterone metabolite concentrations can be used to monitor ovulation and pregnancy. Behavioral monitoring through infrared video surveillance was conducted in four breeding pairs over a 2-year period (n = 7 behavioral profiles). Breeding behaviors correlated strongly with changes in reproductive endocrine profiles. A high frequency of play behavior or &amp;amp;amp;amp;amp;amp;quot;wrestling&amp;amp;amp;amp;amp;amp;quot; was observed in conjunction with breeding activity before an elevation in progesterone metabolite concentrations. Impending parturition was associated with increased aggression and exclusion of the male from the maternal nestbox as well as an increase in nesting activity. Observational data combined with hormonal analysis suggest that female VIMs are induced ovulators and that multiple breeding attempts may be required for ovulation and conception. Gestation appears to be approximately 34 days from peak breeding activity (32 days from estimated ovulation). Fecal testosterone concentrations suggest that testicular activity is seasonal with the reproductive activity occurring immediately posthibernation. Monitoring breeding behavior is a useful means of indicating estrus, conception and pregnancy, which can also be supported by the hormonal analysis of daily fecal samples of individual animals.

Research paper thumbnail of A versatile enzyme immunoassay for the determination of progestogens in feces and serum

Zoo Biology, 2001

The ability of zoos to monitor the reproductive status of their animals can vastly improve the ef... more The ability of zoos to monitor the reproductive status of their animals can vastly improve the effectiveness of husbandry/management practices, and noninvasive methods such as fecal steroid analysis are the easiest to apply in a zoo setting. Furthermore, enzyme immunoassay (EIA) is preferred to radioimmunoassay (RIA) as the method of quantifying hormones because EIAs do not involve the use, storage, and disposal of radioactive materials. However, progesterone is excreted in the feces as predominantly unconjugated metabolites (progestogens) and, until recently, antibodies able to cross-react with a variety of progestogens were used primarily in RIAs. An EIA using a broad-spectrum progestogen antibody is described and applied to serum and/or fecal samples from female African elephants, black rhinoceros, white rhinoceros, okapi, and hippopotami. The clear progestogen profiles generated in these species suggest that the described EIA would be as versatile as the RIA using the same antibody and could be a practical and economical alternative to RIAs for monitoring gonadal function via progestogen analysis in zoo species.

Research paper thumbnail of Fecal androgen metabolite analysis for noninvasive monitoring of testicular steroidogenic activity in felids

Zoo Biology, 1996

Limited data are available on long-term, seasonal changes in testicular ste- roidogenic activity ... more Limited data are available on long-term, seasonal changes in testicular ste- roidogenic activity in nondomestic felids, primarily because of the difficulties associated with longitudinal blood sampling (eg, handling, restraint, ...

Research paper thumbnail of Reproductive endocrine responses to photoperiod and exogenous gonadotropins in the pallas' cat (Otocolobus manul)

Zoo Biology, 2002

Fecal samples were collected for 14-26 months from three male and six female Pallas' cats (Otocol... more Fecal samples were collected for 14-26 months from three male and six female Pallas' cats (Otocolobus manul) to examine gonadal steroidogenic activity in response to changes in photoperiod and treatment with exogenous gonadotropins. Females exhibited a seasonal anestrus from May-December, excreting consistently low concentrations of fecal estrogens (overall mean, 50.278.5 ng/g). During the breeding season (January-April), baseline fecal estrogen concentrations were higher, averaging 128.4718.9 ng/g, with peak concentrations ranging from 455.8-909.6 ng/g. Interpeak intervals in estrogen excretion ranged between 7 and 21 days, with an average estrous cycle length of 14.371.7 days. Two females became pregnant after natural mating, with overall luteal progestogen concentrations averaging B40 mg/g throughout gestation. Fecal estrogens increased in mid-gestation, peaking just before birth. Induction of follicular development with eCG (100-300 IU, i.m.) resulted in an increase in fecal estrogens (peak range, 263.1-1198.1 ng/g), followed by a postovulatory increase in fecal progestogens (overall mean, 41.1711.9 mg/g) after hCG (75-150 IU, i.m.). Despite apparently normal ovarian responses, none of the females conceived after artificial insemination (AI). The gonadotropin-induced nonpregnant luteal phase lasted 49.875.3 days (range, 30-60 days), whereas gestation lasted B70 days. In the male Pallas' cat, fecal androgens were elevated from November-April

Research paper thumbnail of Successful artificial insemination of an Asian elephant at the National Zoological Park

Zoo Biology, 2004

For decades, attempts to breed elephants using artificial insemination (AI) have failed despite c... more For decades, attempts to breed elephants using artificial insemination (AI) have failed despite considerable efforts and the use of various approaches. However, recent advances in equipment technology and endocrine-monitoring techniques have resulted in 12 elephants conceiving by AI within a 4-year period (1998-2002). The successful AI technique employs a unique endoscope-guided catheter and transrectal ultrasound to deliver semen into the anterior vagina or cervix, and uses the ''double LH surge'' (i.e., identifying the anovulatory LH (anLH) surge that predictably occurs 3 weeks before the ovulatory LH (ovLH) surge to time insemination. This study describes the 6-year collaboration between the National Zoological Park (NZP) and the Institute for Zoo Biology and Wildlife Research (IZW), Berlin, Germany, that led to the refinement of this AI technique and subsequent production of an Asian elephant calf. The NZP female was the first elephant to be inseminated using the new AI approach, and was the fifth to conceive. A total of six AI trials were conducted beginning in 1995, and

Research paper thumbnail of Comparison of fecal storage methods for steroid analysis in black rhinoceroses (Diceros bicornis)

Zoo Biology, 2004

Many field studies and conservation programs for wildlife species include noninvasive endocrine m... more Many field studies and conservation programs for wildlife species include noninvasive endocrine monitoring of gonadal function. Freezing fecal samples immediately after collection until further analysis is often not a viable option for researchers in remote areas. Phase 1 of this study was designed to compare different methods of preserving fecal samples over several time periods (30, 90, or 180 days) in order to determine which method provided the most accurate and reliable technique for measuring fecal progestagens. Fecal samples were collected from two female black rhinoceroses (Diceros bicornis) housed at Disney's Animal Kingdom, Lake Buena Vista, FL. We compared three storage methods: 1) storing fecal samples without processing or preservatives (untreated), 2) storing an aliquot of fecal sample in 80% methanol (MeOH), and 3) drying the fecal sample in a solar box cooker prior to storage. Control samples (day 0) were collected and extracted, and then stored at À201C until they were analyzed. Phase 2 of the study was designed to examine the effects of long-term storage (up to 180 days) on fecal progestagen profiles that reflect reproductive activity (pregnancy and estrous cycles). In samples obtained from a pregnant female and stored for 30 days, there were no significant differences in fecal progestagen concentrations between the three treatment conditions. However, the mean concentrations of progestagens (7 SE) in untreated samples increased significantly from 8.3 7 0.3 mg/g wet weight feces at day 0 to 17.7 7 5.1 mg/g feces at day 90, and 17.8 7 4.7 mg/g feces at day 180. Samples that were collected from a pregnant female and stored in 80% MeOH or dried in the solar box correlated with controls (r ¼ 0.86 and 0.87, respectively; Po0.05) at day 180. In contrast, samples that were stored without preservatives for 180 days did not correlate with controls (r ¼ 0.35, P40.05). Progestagen concentrations from samples of the estrous cycling female showed similar results. In conclusion, fecal samples dried in a solar box cooker or stored in 80% MeOH maintained absolute and relative progestagen concentrations for at least 180 days when they were stored outdoors and exposed to the climatic conditions of central Florida. Both methods can have significant applications for

Research paper thumbnail of Non-invasive monitoring of ovarian function in several felid species by measurement of fecal estradiol-17β and progestins

Zoo Biology, 1995

An extraction and assay procedure to measure fecal estradiol-17p and progestin concentrations in ... more An extraction and assay procedure to measure fecal estradiol-17p and progestin concentrations in several cat species was developed and validated for use for noninvasive monitoring of ovarian function. Fecal samples were collected over a range of 3-20 months from female tigers (three), lions (three), snow leopards (three), cheetahs (two), caracals (two), and domestic cats (five). Samples were extracted with 90% methanol, lipids removed with petroleum ether, and the estradiol and progestins in the methanol measured by radioimmunoassay (RIA). High Performance Liquid Chromatography (HPLC) fractionation and subsequent RIA of the fractions indicated that the estradiol-17p antiserum cross-reacted primarily with estradiol-17P in the feces of lions and tigers and was assumed to be specific for estradiol-17P in the feces of other species as well. However, there were several immunoreactive compounds, presumably progesterone metabolites, excreted in the feces which varied both quantitatively and qualitatively among species. The behavior of tigers, lions, cheetahs, and caracals was visually monitored during the collection period and frequency of sexual behaviors was positively correlated with increases in fecal estradiol in all species observed. The mean fecal estradiol-17P peaks were as follows: tigers, 128.0 t 13.1; lions, 186.0 5 14.8; snow leopards, 136.7 k 15.9; cheetahs, 140.9 k 9.0; caracals, 24.5 ? 4.0; and domestic cats 158.9 2 19.3 ng/gm. Fecal progestin concentrations rose significantly (P < 0.001) only after breeding or during pregnancy and were as follows: tigers, 5.6 t 0.6; lions, 1.9 t 0.1; cheetahs, 8.4 5 1.1; and caracals, 2.4 5 0.4 pg/gm. Fecal progestins were elevated for one-half to twothirds of the gestation length during presumed pseudopregnancy but remained elevated throughout successful pregnancies. These results suggest that ovarian function can be monitored noninvasively in the family Felidae by the measurement of fecal estradiol-17P and progestin concentrations 0 1995 Wiley-Liss, Inc.

Research paper thumbnail of Behavioral indices of estrus in a group of captive African elephants (Loxodonta africana)

Zoo Biology, 2005

This study investigated behavioral signals of estrus by systematically monitoring the interaction... more This study investigated behavioral signals of estrus by systematically monitoring the interactions of one male with four female African elephants housed in a naturalistic outdoor enclosure at Disney's Animal Kingdom over a period of 11 months. We measured changes in five spatial behaviors and 22 tactile-contact behaviors, as well as changes in serum progestagen and LH concentrations, across three ovarian cycles for each female. Two females did not cycle during the study. Three different phases of the ovarian cycle were identified: mid luteal, anovulatory follicular, ovulatory follicular. The male followed more and carried out more genital inspections, flehmen, and trunk-to-mouth behaviors toward cycling females during their ovulatory phase. Genital inspections by the male peaked above baseline levels on the day of an LH surge, and up to 9 days before, in both cycling females and, thus, might be a useful behavioral index of estrus. The male also carried out more genital inspections, flehmen, and trunk touches to the back leg toward ovulatory cycling than noncycling females. Overall, our results indicated that: 1) a single subadult African elephant male could discriminate two females in the ovulatory phase of their cycle (i.e., during the 3 weeks preceding ovulation) from the mid luteal phase; 2) the male also discriminated two cycling females in the ovulatory and anovulatory follicular phases from two noncycling females; 3) two females in the

Research paper thumbnail of Chorionic gonadotropin administration in domestic cats causes an abnormal endocrine environment that disrupts oviductal embryo transport

Theriogenology, 2000

Fecal steroid analysis was used to investigate relationships between endocrine parameters and emb... more Fecal steroid analysis was used to investigate relationships between endocrine parameters and embryo characteristics in domestic cats subjected to chorionic gonadotropin stimulation and artificial insemination (AI). In Study 1, normal endocrine patterns were assessed in 12 cycling domestic queens. Fecal estradiol (E) patterns established an anovulatory cycle length of 18.3 + 0.4 d with estrus lasting 6.3 f 0.3 d. Eight females (67%) exhibited at least one spontaneous ovulation based on sustained increases in fecal progestagens (P). In Study 2, queens were mated during natural estrus (NE, n = 5) or subjected to exogenous i.m. gonadotropin stimulation, 100 IU eCG followed by 75 IU hCG 80 h later, (GS, n = 5). Compared with NE queens, fecal E concentrations were higher (P < 0.05) and remained elevated longer after ovulation induction with hCG. In Study 3, gonadotropin-stimulated queens (n = 7) were artificially inseminated and ovariohysterectomized 160 h after hCG. Ancillary follicles and/or corpora lutea were observed in 5 of 6 (83%) ovulating queens. Both fecal E and number of unovulated follicles observed at ovariohysterectomy were negatively correlated with the percentage of embryos recovered from the uterus (r =-0.91 and r =-0.87, respectively; P < 0.05). In summary, exogenous gonadotropin administration causes an abnormal endocrine environment in domestic cats, likely due to ancillary follicle development. The sustained elevations in estradiol appear to impair oviductal transport of embryos, possibly leading to the reduced fertility typically observed in cats subjected to gonadotropin stimulation and AI.

Research paper thumbnail of Influence of oral melatonin on natural and gonadotropin-induced ovarian function in the domestic cat

Theriogenology, 2004

Ovarian hyperstimulation after exogenous gonadotropin stimulation is believed to be a cause of po... more Ovarian hyperstimulation after exogenous gonadotropin stimulation is believed to be a cause of poor success after artificial insemination (AI) in felids. The objectives of this study were to assess the effect of oral melatonin on endogenous ovarian activity in the domestic cat and subsequent eCG/hCG-induced ovarian activity. Serum melatonin concentrations peaked $1 h after a single oral dose of 30 mg melatonin and remained elevated above endogenous daytime concentrations for >8 h. The calculated circulating half-life (mean AE S:E:M:) of oral melatonin was 45:4 AE 3:5 min, and the elimination rate constant (k 10) was 55:2 AE 4:2 min À1. Oral melatonin (30 mg per day) administered 3 h before lights-off effectively and reversibly suppressed estrous elevations in fecal estrogens after 25 days of treatment. There was a progressive decrease in baseline estrogen concentrations from inter-estrous concentrations after 25 days of treatment to below inter-estrous concentrations after 35 days of treatment. Oral melatonin treatment (30 mg per day for 30 days) prior to eCG/hCG administration only marginally reduced ancillary follicle development and had no significant effect on the quantity or quality of embryos produced by AI. Thus, oral melatonin effectively inhibited endogenous ovarian activity and had no adverse impact on embryo quality after AI in the domestic cat; however, this treatment was only marginally effective in minimizing eCG/hCG-induced ovarian hyperstimulation.

Research paper thumbnail of Reproductive activity in captive female cheetahs (Acinonyx jubatus) assessed by faecal steroids

Reproduction, 1996

Faecal oestradiol and progestogen metabolite excretion was monitored in adult, female cheetahs (A... more Faecal oestradiol and progestogen metabolite excretion was monitored in adult, female cheetahs (Acinonyx jubatus) (n = 26) for 1\p=n-\24months. Increased faecal oestradiol excretion was associated with mating or equine chorionic gonadotrophin (eCG) administration for artificial insemination, whereas increased progestogen metabolites were observed during natural and human chorionic gonadotrophin (hCG)-induced pregnant and nonpregnant luteal phases. On the basis of oestradiol excretory patterns, duration of the oestrous cycle (mean \ m=+-\ SEM) was 13.6 \ m=+-\ 1.2 days with high oestradiol concentrations lasting for 4.1 \ m=+-\ 0.8 days. In non-gonadotrophin-treated cheetahs, 75% showed evidence of oestrous cyclicity; however, none evaluated for 1 year or longer were continuously cyclic. Rather, cyclicity was interrupted by periods of anoestrus, often exceeding several months in duration. These inactive ovarian periods were unrelated to season and were not synchronous among females. Mean duration of gestation (breeding to parturition) was 94.2 \ m=+-\ 0.5 days, whereas duration of faecal progestogen metabolite excretion during the nonpregnant luteal phase was 51.2 \ m=+-\ 3.5 days. On the basis of progestogen metabolite evaluations, spontaneous ovulation (non-mating induced) occurred only once in two females (2 of 184 oestrous cycles; 1.1%). Peak eCG-stimulated, preovulatory oestradiol concentrations were similar to those associated with natural oestrus, whereas progestogen metabolite profiles after hCG resembled those during pregnant and nonpregnant luteal phases after natural mating. In summary, results confirm that the cheetah is polyoestrus and ovulation is almost always induced. However, new evidence suggests that many females inexplicably experience periods of anoestrus unrelated to season, while 25% of the cheetahs examined expressed no ovarian activity during the study period.

Research paper thumbnail of The use of low-frequency vocalizations in African elephant(Loxodonta africana) reproductive strategies

Hormones and Behavior, 2003

Fertility-advertisement calls in females are predicted to occur in nonmonogamous species where ma... more Fertility-advertisement calls in females are predicted to occur in nonmonogamous species where males and females are widely separated in space. In African elephants, low-frequency vocalizations have thus been suggested as a reproductive strategy used by fertile females to attract mates. This study examined the use of low-frequency vocalizations with respect to different phases of the estrous cycle in African elephants by simultaneously monitoring vocalizations, behavior, and hormonal profiles. Subjects were one male and six female African elephants housed at Disney's Animal Kingdom. No acoustically distinct vocalizations were restricted to the ovulatory follicular phase. However, overall rate of low-frequency vocalization as well as the rate of one acoustically distinct vocalization changed over the estrous cycle, with highest rates of calling related to the first period of follicular growth, or anovulatory follicular phase. Elevated rates of vocalization thus were not restricted to behavioral estrus and occurred much earlier in the estrous cycle than in most species that produce fertility-advertisement calls. Both herd composition and elephant identity also affected rates of vocalization. Vocalizations therefore may not be reliable signals of actual fertility. However, the increase in vocalizations in advance of estrus may attract males to the herd prior to ovulation, facilitating both male-male competition and female choice. Once present in the herd, males may then switch strategies to use more reliable chemical and visual cues to detect ovulating females.

Research paper thumbnail of Endocrine patterns associated with reproduction in the Nile hippopotamus (Hippopotamus amphibius) as assessed by fecal progestagen analysis

General and Comparative Endocrinology, 2002

The hippopotamus (Hippopotamus amphibius) is a popular zoo animal and an integral part of wetland... more The hippopotamus (Hippopotamus amphibius) is a popular zoo animal and an integral part of wetland ecosystems in its native continent of Africa. However, information about the reproductive physiology of the hippopotamus is limited compared to the other megaherbivores of Africa such as the elephant and the rhinoceros. This study is the first to report on the endocrine patterns associated with ovulation and pregnancy in the Nile hippopotamus. Fecal samples were collected 3 times per week from female hippopotami (N = 9) housed in captivity in Florida. Progesterone metabolites were extracted from the feces with 80% MeOH and quantified using a progestagen EIA. Fecal progestagen analysis indicated hippopotami had a cycle length of 35.3 +/- 1.3 days and ovulated throughout the year. Fecal progestagen concentrations during pregnancy (N = 11 pregnancies) were higher on average (833.5 +/- 115.2 ng/g; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05) than those observed during non-pregnant luteal phases (309.5 +/- 20.9 ng/g) and remained elevated throughout gestation. Fecal progestagen analysis revealed the onset of puberty in three females between 3 and 4 years of age and all conceived by their fourth cycle. Lactational suppression of ovulation during nursing was usually, but not always, observed and lasted for 33.9 +/- 1.8 weeks. The observed calving interval in females with lactational anovulation was 17 months. The majority of births (63.6%) occurring during the summer rainy season and the inconsistency of lactational anovulation suggested the possibility of environmental influences on reproduction.

Research paper thumbnail of Natural and gonadotropin-induced ovarian activity in tigers (Panthera tigris) assessed by fecal steroid analyses

General and Comparative Endocrinology, 2006

Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated... more Fecal samples were collected from female tigers (n = 17) to compare endocrine patterns associated with natural ovarian activity with those after chorionic gonadotropin ovulation induction and artificial insemination (AI). Baseline fecal estradiol concentrations were 65.77 +/- 3.61 ng/g with estrual peaks of 167.39 +/- 9.92 ng/g and an anovulatory cycle length of 17.96 +/- 0.70 days. Peak fecal estradiol was higher when females were housed with a male for breeding (262.30 +/- 41.43 vs. 165.30 +/- 3.67 ng/g; P &amp;amp;lt; 0.05). The majority of animals showed some seasonal differences in fecal estradiol however, patterns were inconsistent. Fecal progestagens increased only after breeding confirming tigers are primarily induced ovulators. The non-pregnant luteal phase was 34.50 +/- 1.85 days in duration. In pregnant tigers, fecal progestagens remained elevated for 108 days until parturition and the diagnosis of pregnancy was possible based on the elevated fecal progestagens after 35 days of gestation. Tigers were administered equine chorionic gonadotropin (eCG) to stimulate follicular growth and human chorionic gonadotropin (hCG) to induce ovulation prior to AI [200 IU eCG/100 IU hCG (n = 5); 400 IU eCG/200 IU hCG (n = 2); 500 IU eCG/100 IU hCG (n = 2); 1000 IU eCG/750 IU hCG (n = 11); 1000 IU eCG/1000 IU hCG (n = 4)]. None of the tigers subjected to AI became pregnant (n = 9). Fecal endocrine patterns in gonadotropin-stimulated tigers were considerably different from those observed in naturally bred tigers. In particular, fecal estradiol concentrations were higher than those observed during natural estrus and remained elevated for longer periods of time in tigers administered the higher doses of gonadotropins typically used in conjunction with AI in this species. These abnormal endocrine patterns may help explain the poor success rate of AI in this species.

Research paper thumbnail of Hormone Secretion in the Asian Elephant (Elephas maximus): Characterizationof Ovulatory and Anovulatory Luteinizing Hormone Surges

Biology of Reproduction, 1999

In the elephant, two distinct LH surges occur 3 wl< apart during the nonluteal phase of the estro... more In the elephant, two distinct LH surges occur 3 wl< apart during the nonluteal phase of the estrous cycle, but only the second surge (ovLH) induces ovulation. The function of the first, anovulatory surge (anLH) is unknown, nor is it clear what regulates the timing of these two surges. To further study this observation in the Asian elephant, serum concentrations of LH, FSH, progesterone, inhibin, estradiol, and prolactin were quantified throughout the estrous cycle to establish temporal hormonal relationships. To examine long-term dynamics of hormone secretion, analyses were conducted in weekly blood samples collected from 3 Asian elephants for up to 3 yr. To determine whether differences existed in secretory patterns between the anLH and ovLH surges, daily blood samples were analyzed from 21 nonluteal-phase periods from 7 Asian elephants. During the nonluteal phase, serum LH was elevated for 1-2 days during anLH and ovLH surges with no differences in peak concentration between the two surges. The anLH surge occurred 19.9 ± 1.2 days after the end of the luteal phase and was followed by the ovLH surge 20.8 ± 0.5 days later. Serum FSH concentrations were highest at the beginning of the nonluteal phase and gradually declined to nadir concentrations within 4 days of the ovLH surge. FSH remained low until after the ovLH surge and then increased during the luteal phase. Serum inhibin concentrations were negatively correlated with FSH during the nonluteal phase (r = •0.53). Concentrations of estradiol and prolactin fluctuated throughout the estrous cycle with no discernible patterns evident. In sum, there were no clear differences in associated hormone secretory patterns between the anLH and ovLH surge. However, elevated FSH at the beginning of the nonluteal phase may be important for follicle recruitment, with the first anLH surge acting to complete the follicle selection process before ovulation.