Live Pups Research Articles

Influence of nonylphenol exposure on basic growth, development, and thyroid tissue structure in F1 male rats

ObjectiveEnvironmental endocrine disruptors (EEDs) with a weak ability to mimic estrogen have been associated with thyroid dysfunction. However, little is known about the effect of nonylphenol (NP), a well-known EED, on thyroid structure. The present study evaluates whether gestational and lactational exposure to NP impacts growth and thyroid structure in F1 male rats.MethodsA total of 60 rats were gavaged with NP (25, 50, and 100 mg/kg), estradiol (E2, 30 μg/kg/day), and corn oil alone (vehicle control) from gestational day 6 to postnatal day (PND) 21. Serum thyroid hormones free triiodothyronine (FT3), free thyroxine (FT4) and thyroid stimulating hormone levels were detected by automated chemiluminescence immunoassay analyzer. The NP level in the thyroid was measured using high-performance liquid chromatography. The ultrastructure of follicular epithelial cells was examined using transmission electron microscopy. Histopathology was conducted using hematoxylin and eosin staining.ResultsOn PND 0, exposure to 50 and 100 mg/kg/day NP led to a significant decrease in the average litter size, litter weight and number of live pups per litter compared to the control group (P < 0.05). Dams exposed to NP during perinatal period demonstrated decreased serum levels of FT3 and FT4 in F1 male rats, when compared to the control group (P < 0.05). The NP level in the control group was 3.39 ± 0.08 ng/mg, while NP levels in the low, middle, and high dose groups ranged from 5.20 to 11.00 ng/mg. Exposure caused a dose-related increase in NP level in the thyroid of male pups (P < 0.01). The thicknesses of the thyroid follicular epithelium were 2.06 ± 0.37 μm in the control group and 3.97 ± 1.61 μm in the high-dose group. The thickness of the thyroid follicular epithelium increased with an increase in treatment dose in a dose-dependent manner (P < 0.05). The sizes of the thyroid follicles were 1,405.53 ± 866.62 μm2 in the control group and 317.49 ± 231.15 μm2 in the high-dose group. With increasing NP dosages, animals showed a decreased size of the thyroid follicle (P < 0.01). Thyroid follicular cells of NP-treated rats showed mildly swollen mitochondria and dilated rough endoplasmic reticulum in the cytoplasm.ConclusionNonylphenol can cross the placental barrier and accumulate in the thyroid of F1 male rats. Gestational and lactational exposure to NP in dams impacted both development and growth of pups and damaged the ultrastructure of their thyroid tissue, which may further negatively influence normal thyroid function.

Effect of Predator Stress on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer.

Predator Stress Can Exert Detrimental Effects on Female Mammals, Leading to Disrupted Reproduction. Although Many Studies Have Addressed the Effects of Predator Stress on Reproductive Output in Rodents, Few Studies Have Focused on the Effect of Visual or Auditory Stress on Pregnant Females. in This Study, We Investigated the Possible Effect of Predator Stress, Either Visual Only or Combined Visual and Auditory (visual+auditory), on the Reproductive Performance of Female Mice After Nonsurgical Embryo Transfer. Reproductive Performance Was Assessed As Pregnancy Rate, Implantation Rate, Gestation Length, Live Pup Rate, and Neonatal Birth Weight. Moreover, Serum Cortisol and Progesterone Levels in Dams Were Measured by Using Electrochemiluminescence Immunoassay. Exposure to Predator (cat) Stress Did Not Lead to a Significant Change in Pregnancy Rates in the Tested Mice. However, the Stressed Mice Showed Significantly Decreased Implantation Rates Compared with the Control Group. Similarly, the Live Pup Rate and Neonatal Birth Weight Were Significantly Lower in the Group Exposed to Preda- Tor Stress Than in the Control Group. Furthermore, Mice Exposed to Visual+auditory Stress Showed a Significant Reduction in Gestation Length Compared with the Control Mice. Our Data Showed That Predator Visual+auditory Stress As Combined Stimuli Significantly Increased Serum Cortisol Level. in Contrast, Progesterone Levels Did Not Significantly Vary Among the Experimental Groups. Taken Together, Our Findings Imply That Predator Stress Adversely Affects the Reproductive Efficiency of Pregnant Mice By Decreasing the Implantation Rate, Live Birth Rate, and Neonatal Birth Weight and by Prolonging Gestation Length.

The GLP‐1 agonist liraglutide lowers blood pressure in a placental ischemic model of preeclampsia

Preeclampsia is a multi‐system disorder of pregnancy characterized by widespread vascular dysfunction. Though the precise etiology of preeclampsia remains largely enigmatic, emerging evidence suggests that an ischemic placenta releases circulating vasoactive factors all of which culminate in endothelial dysfunction. The incretin hormone glucagon‐like peptide 1 (GLP‐1) has been extensively studied for its therapeutic benefit in diabetes mellitus, where it acts to enhance endogenous insulin secretion. In addition to its role in glucose homeostasis, GLP‐1 receptors are present on endothelial cells and activate the endothelial nitric oxide synthase (eNOS) signaling axis. In turn, GLP‐1 receptor agonists have been demonstrated to improve endothelial dysfunction. Therefore, we hypothesized that administration of a GLP‐1 receptor agonist would attenuate endothelial dysfunction and improve blood pressure in our model of preeclampsia.In order to test this hypothesis, timed pregnant CD‐001 rats (an outbred sub‐strain of Sprague‐Dawley rats) underwent surgery on gestation day 15 to place silver clips on the aorta and ovarian‐uterine arterial anastomoses. This procedure restricts blood flow to the fetal‐placental unit by 60%, producing a model of reduced uterine perfusion pressure (RUPP) that mimics several characteristics of human preeclampsia. From gestation day 15 to 20, animals received daily subcutaneous injections of 0.3 mg/kg liraglutide or vehicle. Animals were instrumented with carotid catheters on gestation day 19 and blood pressure determined on day 20 while the animals were conscious and restrained. Daily weight, food intake, and pup outcome were collected.We found that blood pressure was significantly lower in RUPP animals treated with liraglutide (107.8 ±2.5 vs 117.6 ±4.0 mmHg, n = 5 and 8 respectively, p&lt;0.05). Though food intake was diminished in the liraglutide‐treated group immediately following surgery (2.0 ±0.6 vs. 10.8 ±1.6 grams/day on gestation day 15), it rapidly recovered (16.3 ±1.6 vs. 14.5 ±1.8 grams/day on gestation day 19), and there was no significant difference between body weight (259±10 vs 287 ±14 grams on gestation day 20), litter size (8 ±2 vs 10 ±1 live pups), or pup weight (2.07 ±0.05 vs 2.15 ±0.11 grams).This data shows that GLP‐1 receptor agonism effectively lowers blood pressure in a placental ischemic model of preeclampsia. Although the mechanisms contributing to the improved blood pressure remain unclear, these data suggest that the GLP‐1 system represents a new potential therapeutic target in preeclampsia. Importantly, given its role in diabetes, several GLP‐1 receptor agonists are already FDA approved. Future studies will examine vascular mechanisms by which GLP‐1 receptor agonism protects against the development of placental ischemia‐induced hypertension.Support or Funding Information18PRE34050024HL136684P20GM104357This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Widespread neonatal infection with phocid herpesvirus 1 in free-ranging and stranded grey seals Halichoerus grypus

Phocid herpesvirus 1 (PhHV-1) is known to infect grey seals Halichoerus grypus but little is known about its pathogenicity or true prevalence in this species. To investigate the prevalence of and risk factors associated with PHV-1 infection, nasal swabs were collected from grey seal pups and yearlings on the Isle of May, a well-studied grey seal breeding colony, and from stranded grey seal pups submitted to a rehabilitation centre. PhHV-1 nucleic acids were detected in nasal swabs from 58% (52/90) of live free-ranging grey seal pups, 62% (18/29) of live stranded grey seal pups and 28% (5/18) of live free-ranging yearlings, suggesting recrudescence in the latter. Location within the colony, pup body mass and stranding were determined to be risk factors for shedding PhHV-1 in live seal pups with a significantly higher prevalence of PhHV-1 in pups born on the tidal boulder beach when compared to other sites; a significantly positive correlation of PhHV-1 shedding and pup body mass and a higher prevalence in stranded grey seal pups compared to their free-ranging conspecifics. The prevalence of PhHV1 in dead pups on the Isle of May was 56% (27/48) with a positive PhHV-1 PCR status significantly associated with hepatic necrosis (p = 0.01), thymic atrophy (p < 0.001) and buccal ulceration (p = 0.027). Results indicate that PhHV1 was widespread in the pups in the Isle of May grey seal breeding colony.

Identifying risk factors for canine dystocia and stillbirths

Canine dystocia is a common problem that increases the risk of mortality to the dam and stillbirths for the pups. Understanding the risk factors for dystocia and stillbirth can help guide decision making to improve outcomes of whelping management. This study aimed to 1) evaluate risk factors for canine dystocia; and 2) assess risk factors for stillbirth in puppies, by examining all whelpings and puppies born in two breeding populations of guide dogs for a defined period of time. Dystocia risk factors were evaluated using a repeated measures model on 2,489 litters with an overall dystocia rate of 23.8%. Risk factors identified for dystocia were colony, litter size, and age of dam. Small litter size (<5 pups) had the highest dystocia rate of 36.5%, large litter size (>9 pups) had an intermediate dystocia rate of 26.9% and medium litter size had the lowest dystocia rate of 22.1%. Dystocia risk increased as the age of dam increased. Stillbirth risk factors were evaluated for 19,577 pups and the overall stillbirth rate was 5.9%. Risk factors identified for stillbirths were dystocia, litter size, cesarean section performed and breed. Pups from litters that had dystocia were 2.35 times more likely to be stillborn compared with normal litters born without dystocia. Stillbirth risk increased as litter size increased. Pups from litters of 11 or more pups were 1.45 times more likely to be stillborn. Specific information was available for a subset of pups and was used to evaluate the association with stillbirth. Low birth weight was associated with increased stillbirth risk but large pups were also at higher risk of stillbirth compared with average size pups. Stillborn pups had longer mean (102 vs 65 min) and median (47 and 32 min) inter-pup intervals compared with live pups. The positive predictive values for stillbirth were 13.5%, 28.4%, 31.8% and 36.5% when inter-pup interval threshold was set at 1, 2, 3 and 4 h respectively. There is a tradeoff between over diagnosing stillbirth and increased positive predictive value for stillbirth when inter-pup interval threshold is extended but the improvements were modest after 2 h. The last pup born in a litter regardless of litter size was significantly more likely to be stillborn. In conclusion, this paper details data driven parameters to use when making the decision to intervene during whelping that optimize survival for dams and puppies.

The effect of Cynara scolymus (artichoke) on maternal reproductive outcomes and fetal development in rats

Cynara scolymus (C.scolymus) is a plant employed worldwide as an herbal medicine. However, there is a paucity of data related to the evaluation of its toxicity in commercial preparations; thus, the aim of this study was to evaluate the possible teratogenic effect of the dry extract of C.scolymus leaves in Wistar rats. Females were treated, from gestation day (GD) 6 until GD19, with 0.0, 1.0, 2.0 or 4.0 g/kg body weight of C.scolymus extract. At GD20, a cesarean section was performed for evaluation of maternal and fetal parameters. C.scolymus did not induce changes in food consumption, preimplantation or postimplantation losses, placental weight or biochemical profile. An increase in water consumption was observed in pregnant females treated with the higher doses of C.scolymus. Experimental groups showed lower body weight gain during pregnancy and lower gravid uterus weight. Maternal body weight minus the gravid uterus weight did not result in significant differences. Reductions in fetal weight and length were observed in experimental groups. The number of live pups per litter was lower in the highest dose group. No fetal skeletal or visceral malformations were detected. The results showed that the consumption of artichoke during pregnancy clearly has a negative impact on fetuses.

Evaluation of the effects of Uncaria rhynchophylla alkaloid extract on LPS-induced preeclampsia symptoms and inflammation in a pregnant rat model.

Excessive pro-inflammatory cytokines result in adverse pregnancy outcomes, including preeclampsia-like phenotypes, and fetal growth restriction. Anti-inflammation might be an effective therapy. The aim of this research was to investigate whether Uncaria rhynchophylla alkaloid extract (URE), a highly safe anti-inflammation constituent of the herb, can inhibit inflammation and improve clinical characteristics of preeclampsia in a lipopolysaccharide (LPS)-induced preeclampsia rat model. The rat model was established by daily administration of LPS (1 μg/kg body weight per day) from gestational day (GD) 14 to 19. Different doses of URE (35, 70, and 140 mg/kg body weight per day) were administered from GD 14 to GD 19. The effects of URE on proteinuria, maternal hypertension, pregnancy outcomes, as well as pro-inflammatory cytokines levels in serum and placenta were measured. High-dose URE (HURE) treatment decreased LPS-induced mean 24-h proteinuria and systolic blood pressure, and increased fetal weight, placental weight, and the number of live pups (P<0.05). Moreover, increased serum and placental levels of interleukin (IL)-6, IL-1β, tumor necrosis factor-α, and interferon-γ in the LPS-treated group were obviously inhibited after HURE administration (P<0.01). URE improved preeclampsia symptoms and mitigated inflammatory responses in the LPS-induced preeclampsia rat model, which suggests that the anti-inflammation effect of URE might be an alternative therapy for preeclampsia.

A way for in vitro/ex vivo egg production in mammals.

Eggs are female germ cells that are required for producing offspring through sexual reproduction. In mammals, eggs are produced in the ovary and ovulated into the oviduct. It is well known that over 99% of eggs are degenerated without ovulation, so that many studies have attempted in vitro folliculogenesis to produce many eggs in different species for a few decades. Although many methods have been developed, a success of in vitro egg production with the resultant live birth of offspring has been limited, especially in livestock animals. More recently, we have succeeded in producing live pups derived from in vitro/ex vivo egg production in mice. This review aims to introduce our recent findings with a brief history of in vitro/ex vivo culture systems for follicles and ovaries.

101: Maternal microbiome diminishes fetal infection and demise in a murine model of congenital zika syndrome

Maternal ZIKV infection periconception results in CZS in a significant minority of cases. Our recent data demonstrated that ZIKA infection also results in early pregnancy loss in several non-human primate models (PMID 29967348, 29717225). However, estimates of rates of ZIKV vertical transmission and associated pregnancy loss vary in human populations, and are likely dependent on host, viral and other factors. Commensal bacterial microbes, for example, may be important mediators of immunity and viral infection. Here we hypothesized that host bacteria would mediate ZIKV vertical transmission and associated pregnancy loss. Timed-pregnant Swiss-Webster mice, that were either conventional or bred and housed in the absence of bacteria (gnotobiotic germ-free; Fig. A), were inoculated with 1x104PFU of a contemporaneous first passage ZIKV or a mock injection (M) on embryonic days 4 through 7. A group was also treated with anti-IFNI1 antibody + ZIKV as a positive control. Caesarean deliveries of pups and placentae were performed at day 18.5. Animals were assessed for viral levels, as well as fetal demise and growth restriction. Mothers were asymptomatic for ZIKV infection based on daily monitoring. Overall, germ-free (GF) mice had higher rates of vertical transmission as measured by fetal growth restriction (1.0 g vs 1.2 g, GF vs conventional p<0.01), and a significantly higher rate intrauterine demise on day 18.5 (OR 8.7 c2p=0.01) (Fig. B). Among ZIKA infected gestations, placentae were positive in 82% of demised fetus’ compared to 52% of live pups (p=0.04) (Fig. C). 11 days after infection, viral RNA was recovered from maternal spleen and uterus, and was highest by an order of magnitude in fetal placental tissue overall (7.9x103, 1.9x103vs 6.1x104, respectively, p<0.001). Of particular interest, ZIKV levels among GF mice when compared with conventional were higher in spleen (2.1 x 104vs 8.7 x103 RNA copies/g; p=0.04) and uterus (1.0 x104 vs 6.1 x 102 RNA copies/g, p=0.04) (Fig D). Overall, 25% of conventional dams were infected compared to 78% of GF (c2, p=0.03). We tested the novel hypothesis that maternal antiviral immunity may be stimulated or augmented by the presence of commensal bacteria in pregnancy. We have shown for the first time that germ-free dams and their pups are more susceptible to ZIKV infection and CZS, suggesting the mitigating potential of commensal bacteria in congenital viral infections.

POSSIBILITIES OF LECTIN HELIX POMATIA AGGLUTININ (HPA) USE IN RABBITS INSEMINATION

The aim of this work was to analyse the effect of incubation HPA (lectin Helix pomatia agglutinin) with ID (insemination dose) on reproduction parameters (conception ratio, number of live and dead born of pups and birth weight of pups) of rabbits. The experiments were carried out under conditions of 2 rabbit breeds (production breed P and experimental breed E). Fresh ID, in sperm concentrations 25 mil. / 0.5 ml ID, were divided into control ID (without HPA) and experimental ID (with HPA). HPA was added to the experimental ID in concentrations 1µg HPA / 1x106 spermatozoa (30 - 60 minutes of incubation before AI – artificial insemination). Conception ratios obtained from 2 different breeds (P, E) showed minor improvement ( + 2.67 % P; + 7.36 % E) between the monitored groups of females, however without statistically significant differences (P χ2 = 0.03; E χ2 = 0.11). Number of live born pups in litter obtained from breeds P and E showed minor improvement (+ 0.37 pcs P (p&gt;0.05); + 0.95 pcs E (p≤0.05)) compared to the control groups. The weight of live pups among the monitored groups was comparable. It can be concluded from the present study that incubation of ID with lectin HPA had been positively affected by the observed reproductive parameters. The statistically significant difference was found only in the number of live-born pups in the litter. The price of lectin is too high for breeding in common breeding practice.

Tocotrienol-rich fraction supplementation prevents foetal loss in females mated with corticosterone-treated male Sprague-Dawley rats

This study examined whether tocotrienol supplementation to corticosterone-treated male rats could prevent foetal loss in females upon their mating. Epididymides of adult male Sprague-Dawley (SD) rats with proven fertility were surgically separated at the testis-caput junction. Twenty-four hours post-surgery, these animals received for 7days either: tocopherol-stripped corn oil (Control), corticosterone 25mg/kg s.c. (CORT), CORT 25mg/kg s.c. and tocotrienol-rich fraction (TRF) 100mg/kg orally (CORT+TRF) or TRF 100mg/kg orally (TRF). On day 8, males were cohabited with proestrus females. A spermatozoa-positive vaginal smear indicated pregnancy. Males were euthanised for analysis of testosterone and antioxidant activities. Reproductive organs were weighed. On day 8 of pregnancy, females were laparotomised to count the number of implantation sites. Pregnancy was continued until term. Number of pups delivered and their weights were determined. Data were analysed using ANOVA. Malondialdehyde levels were significantly lower in CORT+TRF group compared with CORT group. Enzymatic antioxidant activities, testosterone level and reproductive organ weights were significantly higher in CORT+TRF group compared with CORT group. Number of implantation sites and live pups delivered, and their birth weights from females mated with CORT+TRF males were significantly higher compared to CORT group. Therefore, TRF prevents foetal loss in females mated with CORT+TRF-treated males.

Understanding meta-population trends of the Australian fur seal, with insights for adaptive monitoring.

Effective ecosystem-based management requires estimates of abundance and population trends of species of interest. Trend analyses are often limited due to sparse or short-term abundance estimates for populations that can be logistically difficult to monitor over time. Therefore it is critical to assess regularly the quality of the metrics in long-term monitoring programs. For a monitoring program to provide meaningful data and remain relevant, it needs to incorporate technological improvements and the changing requirements of stakeholders, while maintaining the integrity of the data. In this paper we critically examine the monitoring program for the Australian fur seal (AFS) Arctocephalus pusillus doriferus as an example of an ad-hoc monitoring program that was co-ordinated across multiple stakeholders as a range-wide census of live pups in the Austral summers of 2002, 2007 and 2013. This 5-yearly census, combined with historic counts at individual sites, successfully tracked increasing population trends as signs of population recovery up to 2007. The 2013 census identified the first reduction in AFS pup numbers (14,248 live pups, -4.2% change per annum since 2007), however we have limited information to understand this change. We analyse the trends at breeding colonies and perform a power analysis to critically examine the reliability of those trends. We then assess the gaps in the monitoring program and discuss how we may transition this surveillance style program to an adaptive monitoring program than can evolve over time and achieve its goals. The census results are used for ecosystem-based modelling for fisheries management and emergency response planning. The ultimate goal for this program is to obtain the data we need with minimal cost, effort and impact on the fur seals. In conclusion we identify the importance of power analyses for interpreting trends, the value of regularly assessing long-term monitoring programs and proper design so that adaptive monitoring principles can be applied.

Oocyte retrieval after heterotopic transplantation of ovarian tissue cryopreserved by closed vitrification protocol.

A device for closed vitrification was designed to reduce the risk of contamination and investigated on its efficacy for ovarian function recovery after cryopreservation and heterotopic transplantation. Ovarian tissues from green fluorescence protein transgenic mice (10 GFP mice) were vitrified using the device, and warmed ovarian tissues were transplanted into the ovarian bursa region in wild-type female mice (6 mice). Fresh ovarian tissues were similarly transplanted as a control. After recovery of the estrous cycle, mice were mated with male mice. Ovarian tissues from six cynomolgus monkeys were vitrified and warmed with the device for autologous, heterotopic transplantation. Fresh tissue transplantation was not performed for the control. Ovarian function was examined by recovery of the hormonal cycle. Histological examination was conducted. The number of live pups per recipient mouse was not significantly different after transplantation of fresh or vitrified-warmed ovarian tissue, although the pregnancy rate was reduced with vitrified tissues. The hormonal cycle was restored in 5/6 monkeys after heterotopic transplantation of vitrified-warmed ovarian tissue. Follicles were harvested at eight sites in the omentum and 13 sites in the mesosalpinx. In vitro maturation (IVM)/IVF produced embryo but did not develop. Resumption of the hormonal cycles, follicle development, and oocyte retrieval from vitrified-warmed ovarian tissue transplants may indicate that the use of vitrification for ovarian tissue in a closed system has a potential of clinical application without the risk of contaminations. More detailed analyses of the effects of vitrification on ovarian tissue, such as gene expression patterns in oocytes and granulosa cells, may be needed for establishing a standard procedure for cryopreservation of ovarian tissues in human.

Influence of high ambient temperature on reproductive performance and physiology of rabbit does in a commercial rabbitry in Greece

The reproductive performance and physiological traits of intensively raised does were evaluated under farm conditions in Greece, during the hot (average farm temperature 19.5°C) and cold period (average farm temperature 12.4°C). The size of litter at birth (6.94 vs. 7.84 alive pups, P&lt;0.001), the size of litter at weaning (5.95 vs. 7.06, P&lt;0.001), the pre-weaning mortality rate (16.94% vs. 9.60%, P&lt;0.001, the number of born dead (0.39 vs. 0.19, P&lt;0.001) and the number of stillborn (0,05 vs 0,02 P&lt;0.01) were significantly affected during the hot period, while litter weight and individual weight at birth did not seem to differ between hot and cold period (461 g vs. 466 g (P&lt;0.05) and 68 vs. 65 g (P&lt;0.05), respectively). Rectal temperature of does and respiration rate were higher in the hot period (39.09°C vs. 38.93°C (NS) and 128.08 respirations/min vs. 115.37 respirations/min; P&lt;0.001). The effects of parity order and animals were also studied revealing that parity order influenced significantly total born, born alive, stillborn and weaned rabbits, litter weight and individual weight at birth, as well as the does' respiration rate. The interaction between parity and period was not significant for any of the recorded parameters. In conclusion, high ambient temperature impaired does' reproductive and physiological traits under those conditions, similar to most natural environmental conditions in Greece.

Effect of mouse ovarian tissue cryopreservation by vitrification with Rapid-i closed system.

Currently, open systems are mainly used for cryopreservation of ovarian tissue, oocytes, and embryos, but there is a potential risk of contamination. This study was performed to assess ovarian tissue cryopreservation by a closed vitrification system (Rapid-i vitrification system™), which is already used clinically for oocyte/embryo cryopreservation. Ovaries of C57BL/6J mice were frozen and thawed by using the Rapid-i vitrification system™ (Rapid-i) followed by implantation into recipient mice. Hematoxylin-eosin staining was performed for histological examination of the frozen-thawed ovaries to assess follicle grade. Fertility after implantation of the ovaries was assessed from the live birth rate and the number of live pups. There was no significant difference in grade 1 primary follicles between fresh ovaries (control group, 94.2 ± 2.9%) and frozen-thawed ovaries (Rapid-i group, 87.1 ± 1.8%). However, there was a significant decrease in grade 1 early and late secondary follicles in the Rapid-i group compared with the control group. The live-birth rate was significantly lower in the Rapid-i group compared with the control group (29.2 vs. 83.3%, p < 0.05). On the other hand, there was no significant difference in the average number of live pups between the control group and the Rapid-i group (3 ± 0.4 vs. 2.7 ± 0.3). The Rapid-i seems to be effective for cryopreservation of mouse ovarian tissue. Under appropriate conditions, the Rapid-i could be employed for ovarian tissue cryopreservation and preservation of fertility in humans.

Studies of wolf x coyote hybridization via artificial insemination.

Following the production of western gray wolf (Canis lupus) x western coyote (Canis latrans) hybrids via artificial insemination (AI), the present article documents that the hybrids survived in captivity for at least 4 years and successfully bred with each other. It further reports that backcrossing one of the hybrids to a male gray wolf by AI also resulted in the birth of live pups that have survived for at least 10 months. All male hybrids (F1 and F2) produced sperm by about 10 months of age, and sperm quality of the F1 males fell within the fertile range for domestic dogs, but sperm motility and morphology, in particular, were low in F2 males at 10 months but improved in samples taken at 22 months of age. These studies are relevant to a long-standing controversy about the identity of the red wolf (Canis rufus), the existence of a proposed new species (Canis lycaon) of gray wolf, and to the role of hybridization in mammalian evolution.

Risk Assessment for Toxicity Experiments with Discrete and Continuous Outcomes: A Bayesian Nonparametric Approach

We present a Bayesian nonparametric modeling approach to inference and risk assessment for developmental toxicity studies. The primary objective of these studies is to determine the relationship between the level of exposure to a toxic chemical and the probability of a physiological or biochemical response. We consider a general data setting involving clustered categorical responses on the number of prenatal deaths, the number of live pups, and the number of live malformed pups from each laboratory animal, as well as continuous outcomes (e.g., body weight) on each of the live pups. We utilize mixture modeling to provide flexibility in the functional form of both the multivariate response distribution and the various dose–response curves of interest. The nonparametric model is built from a structured mixture kernel and a dose-dependent Dirichlet process prior for the mixing distribution. The modeling framework enables general inference for the implied dose–response relationships and for dose-dependent correlations between the different endpoints, features which provide practical advances relative to traditional parametric models for developmental toxicology. We use data from a toxicity experiment that investigated the toxic effects of an organic solvent (diethylene glycol dimethyl ether) to demonstrate the range of inferences obtained from the nonparametric mixture model, including comparison with a parametric hierarchical model. Supplementary materials accompanying this paper appear on-line.

Live, full-term mouse pups from oocytes grown and matured in vitro with serum substitutes

For in vitro growth and maturation of mouse oocytes (IVG-IVM), serum is added to media up to and including the stage of oocyte maturation; this subsequently supports oocytes through fertilization and early embryo development. However, problems may occur with sera, such as batch differences and issues of biosafety. The purpose of the present study was to determine the capacity for fertilization and pre- and post-implantation development of oocytes that underwent IVG-IVM with a serum substitute. Oocyte-granulosa cell complexes from preantral follicles were cultured in medium with either fetal bovine serum (FBS), Serum Substitute Supplement™ (SSS), or Knockout™ Serum Replacement (KSR) for 10days, and were then allowed to mature for 17 h. Subsequently, more than 90% of oocytes underwent germinal vesicle breakdown (GVBD) and more than 70% reached metaphase II, with no significant difference between the groups. A lower fertilization rate, presumably due to zona hardening, was found in the serum substitute groups. Nevertheless, more than 50% of the inseminated oocytes were fertilized and 35%–45% of them underwent first cleavage and developed to the blastocyst stage. Following embryo transfer, one and four live offspring were produced from the SSS and KSR groups, respectively. The present study demonstrated that murine IVG-IVM oocytes cultured in media with a serum substitute, achieved fertilization in vitro, pre- and post-implantation development, and the delivery of live pups, although the efficiency of the process is reduced compared to FBS supplementation.

CRISPR/Cas9-Mediated Mutation of αA-Crystallin Gene Induces Congenital Cataracts in Rabbits.

The present study aimed to investigate the role of the αA-crystallin gene in inducing congenital cataracts in rabbits and to construct a novel animal model for characterization and pathologic analysis of congenital cataracts for future research. We generated αA-crystallin gene knockout rabbits with congenital cataracts by coinjection of Cas9 mRNA and sgRNA into zygotes. Cataract phenotypes were investigated in a repeated study of 19 F0-generation and 11 F1-generation rabbits with αA-crystallin gene mutations. Heritability was analyzed by PCR, sequencing, slim lamp, hematoxylin eosin staining, immunohistochemistry, and Western blot. We found αA-crystallin gene mutations in all 19 F0-generation pups (100%) with indel mutations in the αA-crystallin gene ranging from 3 to 52 bp. Off-target assay revealed that none of the potential off-target sites exhibited mutations, demonstrating that off-target mutagenesis was not induced by cytoplasmic microinjection of in vitro-transcribed Cas9 mRNA. Slim lamp assay revealed that 15 of 19 live pups (78.9%) exhibited typical phenotypes, including congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. Histologic hematoxylin and eosin staining showed that αA-crystallin gene knockout rabbits exhibited smaller lenses. Production of the αA-crystallin protein was determined to be dramatically reduced in αA-crystallin gene knockout rabbits. We induced αA-crystallin gene mutations and phenotypes in F1-generation rabbits. Our data suggest that CRISPR/Cas9-mediated mutation of the αA-crystallin gene in rabbits recapitulates phenotypes of congenital cataracts, microphthalmia, obscurity, and early atrophy of the lens, and failed differentiation of lens fibers. These findings suggest the possibility of a new animal model of congenital cataracts, which should be used to further investigate the association between mutations in αA-crystallin gene and congenital cataracts in humans.

Composition of the gut microbiota influences infection severity and pregnancy outcome in Plasmodium chabaudi-infected pregnant mice

Abstract Placental malaria, a severe clinical manifestation of Plasmodium falciparum infection, is a major cause of pregnancy loss, neonatal mortality, and severe maternal illness. We have developed a novel mouse model for pregnancy maintenance during maternal malaria infection utilizing outbred Swiss Webster mice. When infected with Plasmodium chabaudi in early gestation, several inbred mouse strains will abort their pregnancies at midgestation. However, outbred Swiss Webster mice infected with P. chabaudi in early gestation carry their pregnancies to term, allowing us to explore the immunological balance between parasite clearance and pregnancy success. The composition of the gut microbiota may alter this balance. As described by Villarino et al., C57BL/6 mice sourced from different vendors display microbiota-dependent differences in Plasmodium infection severity resulting in a susceptible or resistant phenotype. Similarly, pregnant Swiss Webster mice with susceptible gut microbes develop higher parasite burdens than mice with resistant gut microbes, although both cohorts produce live pups at term. Despite the lower parasite burdens observed in resistant mice, these dams tend to produce fewer viable pups per litter, suggesting a trade-off between accelerated parasite clearance and pregnancy success. We hypothesize that the difference in litter size between susceptible and resistant dams is a result of the elevated levels of pro-inflammatory cytokines observed in resistant dams. The levels of systemic TNF and IFN-γ, which have previously been associated with the clearance of blood-stage malaria infection and pregnancy loss in P. chabaudi-infected pregnant C57BL/6 mice, are expected to be higher in resistant mice.