Cues play a critical role in estrous cycle-dependent enhancement of cocaine reinforcement.

While preclinical work has aimed to outline the neural mechanisms of drug addiction, it has overwhelmingly focused on male subjects. There has been a push in recent years to incorporate females into existing addiction models; however, males and females often have different behavioral strategies, making it important to not only include females, but to develop models that assess the factors that comprise female drug addiction. Traditional self-administration models often include light or tone cues that serve as discriminative stimuli and/or consequent stimuli, making it nearly impossible to disentangle the effects of cue learning, the cues themselves, and acute effects of psychostimulant drugs. To disentangle the interaction between drug-associated cues and the consummatory and appetitive responding driven by cocaine, we have developed a new behavioral procedure that combines Pavlovian-instrumental transfer with behavioral economic analysis. This task can be completed within a single session, allowing for studies looking at estrous cycle stage-dependent effects in intact cycling females, something that has been difficult in the past. In this study, we found no differences in self-administration across the estrous cycle in the absence of cues; however, when cues were introduced, the cues that acquired value during estrus-but not during diestrus or in males-increased motivation. Cues paired during estrus also increased c-fos expression to a greater extent in striatal regions, an effect that may underlie the observed increases in seeking induced by these cues, even weeks later. Together, these data suggest that fundamental differences in the motivational properties of psychostimulant drugs between males and females are complex and are driven primarily by the interaction between drug-associated stimuli and drug effects.

Safety assessment of propylparaben in juvenile rats.

There are conflicting literature reports that parabens, useful antimicrobial additives in pharmaceuticals, may have estrogenic activity. We conducted a comprehensive study to determine whether propylparaben (PP) administration to juvenile rats is associated with adverse effects on reproductive development and function. PP was administered orally once daily to groups of Crl:CD(SD) rats at doses of 0 (vehicle), 10, 100, or 1,000 mg/kg on Postnatal Days (PNDs) 4-90. In-life observations, clinical pathology, reproductive organ weights and histopathology, landmarks of sexual maturation, estrous cyclicity and functional reproductive competence were assessed. A conventional uterotrophic assay was conducted separately using the same doses. Systemic exposures to PP and 3 metabolites were evaluated on PND 7, 21 and 83. These studies demonstrated that PP was well tolerated when administered from PND 4-90 at all doses (AUC[0-T] on PND 83 = 69.9 ng•h/mL). Para-hydroxybenzoic acid, a non-estrogenic compound, was the predominant metabolite contributing to 95% of the total exposure at 1,000 mg/kg/day on PND 7. There was no evidence of estrogenic activity at any dose, and no effects on reproductive organs or function. The No-Observed-Adverse-Effect-Level (NOAEL) was 1,000 mg/kg/day.

Doxorubicin-induced female reproductive toxicity: an assessment of ovarian follicular apoptosis, cyclicity and reproductive tissue histology in Wistar rats.

Doxorubicin is a widely used chemotherapeutic agent for various cancers, particularly for the female breast cancer patients. Although the rate of young female cancer patients is increasing every year, conversely the lack of knowledge of adverse effects of doxorubicin on female reproductive system insisted us to assess the toxic effects of doxorubicin on the female reproductive tissue histoarchitecture, cyclicity, and mammary glands in Wistar rats. The rats were divided into two groups depending on the treatment period, i.e., 24 h and 28 d and further subdivided into three subgroups and administered with doxorubicin at 3 mg/kg bw (subgroup I), 6 mg/kg bw (subgroup II), and equal volume of normal saline (subgroup III) intraperitoneally once during the whole treatment period. We observed a significantly altered estrous cycle with a prolonged diestrous and short proestrous in higher dose group and dose-dependent significant changes in the uteri and mammary gland histoarchitecture in 28 days treated rats as compared to control. Moreover, the micronuclei and chromosomal aberration frequency were increased significantly in both treatment groups. A significant increase in follicular atresia in ovaries of the 28 days treated rats was observed. The immunohistochemical analysis of ovarian tissues showed an increased p53 and caspase 3 expression and apoptosis in primordial follicles of treated rats. The results suggest that though doxorubicin is a potential chemotherapeutic drug for many tumors, but the risk of adverse effects on the female reproductive system is there even at low doses.

Synchronized ovulation for first insemination improves reproductive performance and reduces cost per pregnancy in dairy heifers.

The objectives were to evaluate the effects of synchronizing estrus and ovulation to implement a timed artificial insemination (AI) at first insemination on reproductive performance and cost per pregnancy in dairy heifers. Six hundred eleven Holsteins heifers at approximately 400 d of age from 3 farms were enrolled in the study. Six days before moving to the breeding pens, heifers were allocated randomly to AI after detected estrus from study d 0 to 84 (CON, n=306), or to timed AI for first AI followed by detected estrus for the remainder of the 84-d study (TAI, n=305). Heifers receiving TAI were enrolled in the 5-d timed AI protocol on study d -6 (d -6, GnRH and a progesterone insert; d -1, PGF2α and insert removal; d 0, PGF2α; d 2, GnRH + AI), and they were allowed to be bred the day before scheduled timed AI if detected in estrus. Starting on study d 0, estrus was detected daily. Heifers in estrus were inseminated on the same morning as detected estrus. Control heifers not inseminated by study d 7 received PGF2α and this treatment was repeated every 2 wk until AI. The study lasted 84 d to allow a period of breeding equivalent to four 21-d estrous cycles. A herd budget accounting for inputs for both treatments was created to determine the cost per pregnancy. Sensitivity analysis compared economic differences between the 2 treatments under different input scenarios when detection of estrus after the first AI varied from 50 to 80%. Interval to first AI was 8 d shorter for TAI than for CON. Pregnancy at first AI did not differ between treatments (CON=58.3 vs. TAI=62.8%). In contrast, TAI increased pregnancy per AI (P/AI) compared with CON in heifers inseminated with sex-sorted semen (CON=31.6 vs. TAI=54.8%). The 21-d cycle insemination rate was greater for TAI (91.4%) than for CON (82.4%), even when evaluated after the first 21 d in the study (CON=68.2 vs. TAI=77.1%). The increased insemination rate improved the 21-d cycle pregnancy rate from 47.9% in CON to 57.2% in TAI heifers. In fact, the hazard of pregnancy was 60% greater for TAI than CON. The increased pregnancy rate in TAI reduced the median days to pregnancy by 12 d (CON=2.0 vs. TAI=14.0) and increased the proportion of pregnant heifers by 6.3 percentage points by study d 84 (CON=85.2 vs. TAI=91.5%). The cost per pregnancy was $17.00 less for TAI than CON. The sensitivity analysis indicated that TAI was economically more advantageous to produce a pregnancy compared with CON. Only when insemination rate after the first 21 d of breeding was very high and P/AI was relatively low did the cost per pregnancy become similar for the 2 treatments. Collectively, inseminating all heifers within 2 d of breeding with the 5-d timed AI protocol maintains P/AI, improves pregnancy rate, and reduces cost per pregnancy compared with insemination after detected estrus.

Assessment of estrogenic potential of di-n-butyl phthalate and butyl benzyl phthalate in vivo.

Phthalate compounds are widely used industrial chemicals; when incorporated into polyvinyl chloride, they are not covalently bound and released into the surrounding media. Some of them have estrogenic potential in vitro but data on in vivo studies are scanty. For the 3-day uterotrophic assay, di-n-butyl phthalate (DBP;10 and 100 mg/kg), butyl benzyl phthalate (BBP; 20 and 200 mg/kg), and diethylstilbestrol (DES, 40 µg/kg, positive control) were administered orally to immature female rats for three consecutive days from postnatal day (PND) 21. For the 20-day pubertal onset assay, DBP (10 and 20 mg/kg), BBP (20 and 200 mg/kg), and DES (6 µg/kg) were administered orally from PND 21 daily for 20 days. In the uterotrophic assay, in groups treated with higher dose of DBP and BBP, the uterine wet weight significantly decreased in the higher dose, and there were minor variations in the ovary wet weight, while the wet weight of these organs increased significantly in DES-treated group. In the 20-day pubertal assay, the weight of uterus and ovary declined significantly and changes in vaginal weight were nonsignificant in DBP- and BBP-treated groups. However, in DES-treated group nonsignificant elevation in vagina weight was observed. All the DES-treated animals showed the vaginal opening (VO) on day 26.17 ± 0.16. However, VO was not observed in any of the animals in control, vehicle control, BBP-, and DBP-treated groups up to PND 42, except in one animal each in vehicle control and DBP (100 mg/kg)-treated groups. The data indicated that both DBP and BBP were unable to induce elevation in the uterine and ovarian weight. While DES treatment can accelerate the growth of uterus and ovary and alter the onset of puberty and estrous cyclicity in prepubertal rats. These suggest that these compounds may not have estrogenic potential in vivo.

Evaluation of three synchrony programs for pasture-based dairy heifers.

The objective was to evaluate the efficacy and economic benefits of three synchrony programs in 1137 heifers from 10 pasture-based dairy herds. Heifers were randomly assigned to one of three treatments within each herd on Day -13 (Day 0 = start of the breeding program). They were treated with: (1) PGF(2α) on Days -13 and -2, with AI after detection of estrus between Days 0 and 3 (Double PG); (2) GnRH, PGF(2α), and GnRH on Days -9, -2, and 0, respectively, with placement of an intravaginal progesterone (P4)-releasing device between Days -9 and -2, and set time AI on Day 1 (GPG + P4); or (3) same as the GPG + P4 group but with the set time AI on Day 0 (Cosynch + P4). Plasma P4 concentrations were determined on Days -20 and -13 to determine pubertal status. The Cosynch + P4 treatment had a higher (P < 0.05) conception rate to AI (57% vs. 47% vs. 48% for Cosynch + P4, GPG + P4, and Double PG, respectively), 21-day in-calf rate (76% vs. 72% vs. 63% for Cosynch + P4, GPG + P4, and Double PG), and a shorter median interval from the start of the breeding program to conception (0, 14, and 19 days for Cosynch + P4, GPG + P4, and Double PG). Heifers that had reached puberty before breeding, compared with those that had not, had higher (P < 0.05) in-calf rates to AI (53% vs. 47%) at 21 days (74% vs. 64%) and at 42 days (91% vs. 84%). Pubertal status was associated with herd, breed, age, and body condition score at the start of mating (P < 0.05). A partial budget model demonstrated that, compared with the Double PG program, there was an economic benefit from the Cosynch + P4 (mean, NZ$25.73; 95% confidence interval, 2.99-50.69), but not the GPG + P4 program (mean, NZ$-0.65; 95% confidence interval, -21.87 to 21.58). We concluded that the Cosynch + P4 program resulted in the highest fertility and economic benefit of the three programs evaluated, and that reproductive response was affected by pubertal status.

Scientific and regulatory policy committee (SRPC) paper: assessment of circulating hormones in nonclinical toxicity studies III. female reproductive hormones.

Hormonally mediated effects on the female reproductive system may manifest as pathologic changes of endocrine-responsive organs and altered reproductive function. Identification of these effects requires proper assessment, which may include investigative studies to profile female reproductive hormones. Here, we briefly describe normal hormonal patterns across the estrous or menstrual cycle and provide general guidance on measuring female reproductive hormones and characterizing hormonal disturbances in nonclinical toxicity studies. Although species used in standard toxicity studies share basic features of reproductive endocrinology, there are important species differences that affect both study design and interpretation of results. Diagnosing female reproductive hormone disturbances can be complicated by many factors, including estrous/menstrual cyclicity, diurnal variation, and age- and stress-related factors. Thus, female reproductive hormonal measurements should not generally be included in first-tier toxicity studies of standard design with groups of unsynchronized intact female animals. Rather, appropriately designed and statistically powered investigative studies are recommended in order to properly identify ovarian and/or pituitary hormone changes and bridge these effects to mechanistic evaluations and safety assessments. This article is intended to provide general considerations and approaches for these types of targeted studies.

Non-invasive assessment of the reproductive cycle in free-ranging female African elephants (Loxodonta africana) treated with a gonadotropin-releasing hormone (GnRH) vaccine for inducing anoestrus.

BACKGROUND: In southern Africa, various options to manage elephant populations are being considered. Immunocontraception is considered to be the most ethically acceptable and logistically feasible method for control of smaller and confined populations. In this regard, the use of gonadotropin-releasing hormone (GnRH) vaccine has not been investigated in female elephants, although it has been reported to be safe and effective in several domestic and wildlife species. The aims of this study were to monitor the oestrous cycles of free-ranging African elephant cows using faecal progestagen metabolites and to evaluate the efficacy of a GnRH vaccine to induce anoestrus in treated cows. METHODS: Between May 2009-June 2010, luteal activity of 12 elephant cows was monitored non-invasively using an enzyme immunoassay detecting faecal 5alpha-reduced pregnanes (faecal progestagen metabolites, FPM) on a private game reserve in South Africa. No bulls of breeding age were present on the reserve prior to and for the duration of the study. After a 3-month control period, 8 randomly-selected females were treated twice with 600 micrograms of GnRH vaccine (Improvac®, Pfizer Animal Health, Sandton, South Africa) 5-7 weeks apart. Four of these females had been treated previously with the porcine zona pellucida (pZP) vaccine for four years (2004-2007). RESULTS: All 12 monitored females (8 treated and 4 controls) showed signs of luteal activity as evidenced by FPM concentrations exceeding individual baseline values more than once. A total of 16 oestrous cycles could be identified in 8 cows with four of these within the 13 to 17 weeks range previously reported for captive African elephants. According to the FPM concentrations the GnRH vaccine was unable to induce anoestrus in the treated cows. Overall FPM levels in samples collected during the wet season (mean 4.03 micrograms/gram dry faeces) were significantly higher (P<0.002) than the dry season (mean 2.59 micrograms/gram dry faeces). CONCLUSIONS: The GnRH vaccination protocol failed to induce anoestrus in the treated female elephants. These results indicate that irregular oestrous cycles occur amongst free-ranging elephants and are not restricted to elephants in captivity. The relationship between ecological conditions and endocrine activity were confirmed. Free-ranging female elephants were observed to not cycle continuously throughout the year in the absence of adult bulls.

Reproductive toxicity assessment of sunitinib, a multitargeted receptor tyrosine kinase inhibitor, in male and female rats.

BACKGROUND: Sunitinib (SUTENT, Pfizer Inc., New York, NY) is a multitargeted inhibitor of selected receptor tyrosine kinases, which produces an antiproliferative and antiangiogenic effect by blocking pathways fundamental to tumor growth and survival. We investigated the effects of sunitinib on male and female fertility and early embryonic development in the rat. METHODS: In the female fertility and early embryonic development phase, untreated males were paired with treated females dosed at 0 (control), 0.5, 1.5, and 5 mg/kg/day from 14 days premating, through mating, to gestation day 7. In the male fertility phase, the same males were then treated 58 days at doses of 0 (control), 1, 3, and 10 mg/kg/day, mated with untreated females, with continued daily dosing for a total of 74 days. RESULTS: There was no systemic toxicity- or treatment-related effects on fertility in female rats. Females exposed at 5 mg/kg/day had an increase in the number of early resorptions with associated decrease in viable embryos. In the males, body weight and food consumption were decreased at 10 mg/kg/day compared to the controls. Male reproductive capacity, as assessed by copulation, fertility, and conception indices, was not impacted at any dose level. Sperm morphology, concentration, and motility were also unaffected by treatment. CONCLUSIONS: There were no effects on male reproduction. An increase in corpora lutea and an increase in early resorptions with associated reduction in viable embryos was noted in the females dosed 5 mg/kg/day. Sunitinib at doses up to 1.5 and 10 mg/kg/day had no effects on female and male reproduction, respectively.

Comparison of diagnostic approaches, and a cost-benefit analysis of different diagnostic approaches and treatments of anoestrous dairy cows.

AIM: To compare diagnostic techniques, and to assess the economic effects of diagnosing and treating dairy cows not detected in oestrus before the planned start of mating (PSM). METHODS: Cows from 12 herds were defined as anoestrus at Day -9 (where Day 0=PSM) based on presence of tail paint that had been applied at Day -35. The presence of a corpus luteum (CL) was diagnosed by palpation or ultrasonography on Day -9, or by determining the concentration of progesterone (P4) in milk at Days -16 and -9. Cows with concentrations of P4 in milk >1 ng/ml at one or both times were defined as CL+. Cows were randomly assigned to be treated with (a) nothing (Control; n=558); (b) gonadotrophin-releasing hormone (GnRH) on Day -9, prostaglandin F(2alpha) (PGF(2alpha)) on Day -2, and GnRH on Day 0, with set-time artificial insemination (AI) 16-20 h after the second GnRH treatment (Ovsynch; n=553); or (c) as for (b) but with placement of an intravaginal P4-releasing device on Day -9, and removal on Day -2 (Ovsynch+P4; n=551). Cows detected in oestrus between Day -2 and the second GnRH treatment did not receive the second GnRH treatment. Pregnancy diagnosis took place on three occasions, and the date of conception estimated, from which the PSM-to-conception interval was calculated. Agreement between the three diagnostic techniques for CL status was evaluated using Kappa analyses, and sensitivities and specificities were calculated using a Bayesian Monte Carlo approach that does not assume a gold standard. Partial budgets and decision trees were constructed to assess the cost effectiveness of diagnosis and treatment. RESULTS: The level of agreement was higher between ultrasonography and concentration of P4 in milk (0.64) than for palpation and ultrasonography or concentration of P4 in milk (0.50 and 0.49, respectively). The Ovsynch+P4 treatment had a higher net benefit than Ovsynch relative to no treatment (NZ$80.40 and NZ$47.50/cow treated, respectively) in the absence of diagnosis of CL status. Following diagnosis, the Ovsynch+P4 treatment remained the most cost-effective option for both CL+ and CL- cows. It was concluded that the Ovsynch+P4 treatment without any diagnostic procedure was the most cost-effective option. CLINICAL RELEVANCE: Treatment of anoestrous cows was more cost-effective than no treatment, with Ovsynch+P4 more cost-effective than Ovsynch in cows with or without a CL. Differentiation of anoestrous cows into CL+ and CL- groups for treatment was not cost-effective.

Safety assessment of dietary diacylglycerol oil: a two-generation reproductive toxicity study in rats.

Diacylglycerol (DAG) oil is a novel edible oil with similar taste and usability characteristics as conventional edible oils. Recent studies suggest that DAG oil may be helpful in the prevention and management of obesity. The objective of the present two-generation study was to evaluate potential adverse effects of DAG oil on reproductive processes. DAG oil was administered via gavage to rats (30/sex/group) for at least 70 days prior to mating, at dose levels of 0, 1.25, 2.5 or 5.0 ml/kg/day (0, 1160, 2320 and 4630 mg/kg/day). An additional group received a triacylglycerol (TAG) oil with a similar fatty acid composition to DAG oil. The rats were treated throughout the mating, gestation and lactation periods. Administration of DAG or TAG oil did not reveal any toxicologically significant effects on reproductive performance (mating, fertility and copulation/conception indices). DAG oil did not affect mean gestation lengths, the process of parturition, spermatogenic parameters, organ weights, histopathologic findings, mean numbers of pups born, implantation sites and unaccounted sites. F1 and F2 pup viability, live litter sizes, body weights, mean age of attainment of balanopreputial separation and vaginal patency were similar to those in the control group. Based on the results of this study, a dose level of 5.0 ml/kg (4630 mg/kg/day) was considered as the no-observed-adverse-effect level for reproductive and systemic toxicity, and neonatal toxicity.

Histological assessment of ovaries and uterus of rats subjected to nandrolone decanoate treatment.

This study aimed to analyze the effects of nandrolone decanoate on the ovaries and uterus of adult females rats. This drug was administered intraperitoneally, at one, two and three doses of 3 mg nandrolone decanoate/kg of body weight, respectively, in the first, second and third week of treatment. The females of the control group received a physiological solution. The rats treated with nandrolone decanoate showed estral acyclicity and there was destruction of follicular units and an absence of corpus luteum in the ovaries. In the uterus, the drug promoted morphological alterations, characterized by vacuolated epithelium and endometrial stroma fibrosis. Ovary, uterus and pituitary weights were not affected by the steroid treatment. Nandrolone decanoate affects the sexual cycle and promotes histological alterations in the ovaries and uterus of adult female rats.

Reproductive toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in female rats.

BACKGROUND: Lasofoxifene is a nonsteroidal selective estrogen receptor modulator (SERM). With high affinity to the alpha and beta human estrogen receptors and greater potency than other SERMs, lasofoxifene is potentially a superior treatment for postmenopausal osteoporosis. In light of the known effects of estrogen-modulating compounds on female reproductive indices, two studies were conducted to evaluate the effects of lasofoxifene on female rat cyclicity, reproduction, and parturition. METHODS: One study evaluated effects of lasofoxifene on estrous cyclicity, and the second study assessed effects on implantation and parturition. In the cyclicity study, lasofoxifene was administered to female rats at doses of 0.1, 0.3, and 1.0 mg/kg/day for 14 consecutive days. After treatment, there was a 3-week reversibility phase followed by a mating phase. In the implantation study, lasofoxifene was administered to pregnant female rats at doses of 0.01, 0.03, and 0.1 mg/kg/day for 7 consecutive days (gestation day [GD] 0-6). Some animals were euthanized on GD 21, and the remainder of the group was allowed to deliver the F1 generation. Several developmental indices were evaluated in the F1 pups through post-natal day (PND) 21. RESULTS: In the cyclicity study, all lasofoxifene-treated females were anestrous by Study Day 7 (1.0 mg/kg) or 9 (0.3 and 0.1 mg/kg). The reversibility phase resulted in restoration of normal estrous cycles by the end of 1 (0.1 mg/kg) or 2 weeks (0.3 and 1.0 mg/kg). During the mating phase, no adverse effects occurred in pregnancy success or reproductive parameters. In the implantation study, all doses of lasofoxifene increased pre- and post-implantation losses, increased gestation length, and reduced litter size. None of the developmental parameters measured on the F1 generation was adversely affected. CONCLUSION: Lasofoxifene reversibly altered the estrous cycle and inhibited implantation, consistent with what would be expected from a member of the SERM class.