Serum concentrations of leptin in pregnant and non-pregnant bitches.

Leptin regulates body weight and several physiological processes including reproduction. We evaluated the circulating levels of leptin in pregnant and non-pregnant bitches as well as their correlation with body weight, food intake and number of foetuses. Nineteen healthy German shepherd bitches were used and divided in two groups (pregnant n = 12 and non-pregnant n = 7). Blood samples were collected every 15 days starting from ovulation (Day 0) throughout pregnancy (pregnant group, P) or throughout luteal phase (non-pregnant group, NP) In pregnant bitches, leptin concentrations increased from the day of ovulation (1.32 ± 0.06 ng/ml) up to day 45 (1.51 ± 0.06 ng/ml; p < .01) and returned to baseline values from day 60 post-ovulation. In non-pregnant bitches, leptin concentrations remained constant throughout the whole observation period (estimated marginal mean ± SE=1.33 ± 0.38 ng/ml). Pairwise comparisons showed significant differences between P and NP at day 45 post-ovulation (p < .05). Multivariable models indicated that, controlling for time and litter size, there was a positive relationship between leptin concentration and BW (p < .05) although Pearson coefficients showed that the correlation between BW and leptin was only significant in NP animals at day 45 (r = 0.76, p < .05). The multivariable approach also suggested that, holding BW and time constant, leptin concentrations tend to increase as the number of puppies increased (p = .06). Our study supports indirectly the contribution of the feto-placental unit to the circulating maternal leptin concentration.

Effect of aglepristone (RU534) administration during follicular phase on progesterone, estradiol-17ß and LH serum concentrations in bitches.

Aglepristone was administered in bitches during the follicular phase to evaluate its effects on progesterone, estradiol-17ß and LH serum concentrations. Ten German Shepherds were divided into two groups (treated n = 5; control n = 5). Treated bitches received 10 mg/kg BW of aglepristone subcutaneously during the early follicular phase, 24 hr after and then 7 days later. The control group was injected, at the same time periods, with saline solution (0.3 ml/kg BW). For the steroid evaluations, blood was collected daily from the onset of proestrus until the first day of cytological dioestrus. For LH base-line serum determination, blood was also collected every 20 min for 2 hr at the onset of proestrus. For LH surge identification, blood was collected daily (every 6 hr) starting from the day of the first administration of aglepristone or saline solution until the first day of dioestrus. All animals ovulated but the treated group presented longer ovulation-dioestrus intervals than the control group (5.2 ± 2.2 days p < .05). Serum concentrations of the evaluated hormones were similar between experimental animals except for serum LH. Indeed, no LH peaks were detected in the treated group while LH surges were clearly observed in the control group (9 ± 1 days after the beginning of proestrus. In particular, the area under the curve for LH was significantly lower in treated than control animals (12 ± 4 ng/ml x Day; p = .01). In conclusion, administrations of aglepristone during the follicular phase of the bitch does not affect the steroid hormone patterns but does prevent the occurrence of a LH surge. This work raises significant questions and opens perspectives concerning the mechanisms of ovulation in bitches.

Protein expression pattern of PAWP in bull spermatozoa is associated with sperm quality and fertility following artificial insemination.

Post-acrosomal WW-domain binding protein (PAWP) is a signaling molecule located in the post-acrosomal sheath (PAS) of mammalian spermatozoa. We hypothesized that the proper integration of PAWP in the sperm PAS is reflective of bull-sperm quality and fertility. Cryopreserved semen samples from 298 sires of acceptable, but varied, fertility used in artificial insemination services were analyzed using immunofluorescence microscopy and flow cytometry for PAWP protein. In normal spermatozoa, PAWP fluorescence formed a regular band around the proximal PAS. Anomalies of PAWP labeling in defective spermatozoa were reflected in flow cytometry by varied intensities of PAWP-induced fluorescence. Distinct sperm phenotypes were also identified, including morphologically normal and some defective spermatozoa with moderate levels of PAWP; grossly defective spermatozoa with low/no PAWP; and defective spermatozoa with high PAWP. Analysis by ImageStream flow cytometry confirmed the prevalence of abnormal sperm phenotypes in the spermatozoa with abnormal PAWP content. Live/dead staining and video recording showed that some abnormal spermatozoa are viable and capable of progressive motility. Conventional flow-cytometric measurements of PAWP correlated significantly with semen quality and fertility parameters that reflect the sires' artificial insemination fertility, including secondary sperm morphology, conception rate, non-return rate, and residual value. A multiplex, flow-cytometric test detecting PAWP, aggresomes (ubiquitinated protein aggregates), and acrosomal integrity (peanut-agglutinin-lectin labeling) had a predictive value for conception rate, as demonstrated by step-wise regression analysis. We conclude that PAWP correlates with semen/fertility parameters used in the cattle artificial insemination industry, making PAWP a potential biomarker of bull fertility.

Expression of CD13 and CD26 on extracellular vesicles in canine seminal plasma: preliminary results.

Canine seminal plasma is a complex fluid containing proteins, peptides, enzymes, hormones as well as extracellular vesicles that are involved in many physiological and pathological processes including reproduction. We examined the expression of the extracellular vesicles surface antigens Aminopeptidase-N (CD13) and Dipeptidyl peptidase IV (CD26) by flow cytometry. For this study, third fraction of the ejaculate, from fertile adult male German Shepherd dogs, was manually collected twice, two days apart. FACS analyses revealed that CD13 and CD26 are co-expressed on the 69.3 ± 3.7% of extracellular vesicles and only a 2.0 ± 0.5% of extracellular vesicles express CD26 alone. On the other hand, 28.6 ± 3.6% of seminal EVs express CD13 alone. Our results agree with the hypothesis that CD26 needs to be co-expressed with other signal-transducing molecules, while CD13, can perform functions independently of the presence or co-expression of CD26. The results obtained in normal fertile dogs could represent physiological expression of these enzymes. Therefore, it would be interesting to carry out further studies to evaluate the expression of CD13 and CD26 on extracellular vesicles as biomarker for prostate pathological condition in dogs.

Invaders: Recognition of Double-Stranded DNA by Using Duplexes Modified with Interstrand Zippers of 2'-O-(Pyren-1-yl)methyl-ribonucleotides.

The invasion has begun: Invaders are shown to recognize DNA hairpins in cell-free assays and chromosomal DNA during non-denaturing fluorescence in situ hybridization (nd-FISH) experiments. As Invaders are devoid of inherent sequence limitations, many previously inaccessible DNA targets could become accessible to exogenous control with important ramifications for karyotyping, in vivo imaging, and gene regulation.

Glycogen storage disease type Ia in canines: a model for human metabolic and genetic liver disease.

A canine model of Glycogen storage disease type Ia (GSDIa) is described. Affected dogs are homozygous for a previously described M121I mutation resulting in a deficiency of glucose-6-phosphatase-α. Metabolic, clinicopathologic, pathologic, and clinical manifestations of GSDIa observed in this model are described and compared to those observed in humans. The canine model shows more complete recapitulation of the clinical manifestations seen in humans including "lactic acidosis", larger size, and longer lifespan compared to other animal models. Use of this model in preclinical trials of gene therapy is described and briefly compared to the murine model. Although the canine model offers a number of advantages for evaluating potential therapies for GSDIa, there are also some significant challenges involved in its use. Despite these challenges, the canine model of GSDIa should continue to provide valuable information about the potential for generating curative therapies for GSDIa as well as other genetic hepatic diseases.

Evaluation of adrenocortical function in Florida manatees (Trichechus manatus latirostris).

The study objectives were to determine the predominant manatee glucocorticoid; validate assays to measure this glucocorticoid and adrenocorticotropic hormone (ACTH); determine diagnostic thresholds to distinguish physiological vs. pathological concentrations; identify differences associated with sex, age class, female reproductive status, capture time, and lactate; and determine the best methods for manatee biologists and clinicians to diagnose stress. Cortisol is the predominant manatee glucocorticoid. IMMULITE 1000 assays for cortisol and ACTH were validated. Precision yielded intra- and inter-assay coefficients of variation for serum cortisol: ≤23.5 and ≤16.7%; and ACTH: ≤6.9 and ≤8.5%. Accuracy resulted in a mean adjusted R(2)≥0.87 for serum cortisol and ≥0.96 for ACTH. Assay analytical sensitivities for cortisol (0.1 µg/dl) and ACTH (10.0 pg/ml) were verified. Methods were highly correlated with another IMMULITE 1000 for serum cortisol (r=0.97) and ACTH (r=0.98). There was no significant variation in cortisol or ACTH with sex or age class and no correlation with female progesterone concentrations. Cortisol concentrations were highest in unhealthy manatees, chronically stressed by disease or injury. ACTH was greatest in healthy free-ranging or short-term rehabilitating individuals, peracutely stressed by capture and handling. Cortisol concentrations ≥1.0 µg/dl were diagnostic of chronic stress; ACTH concentrations ≥87.5 pg/ml were diagnostic of peracute stress. In healthy long-term captive manatees, cortisol (0.4±0.2 µg/dl) and ACTH (47.7±15.9 pg/ml) concentrations were lower than healthy free-ranging, short-term rehabilitated or unhealthy manatees. Capture time was not significantly correlated with cortisol; ACTH correlation was borderline significant. Cortisol and ACTH were positively correlated with lactate.

Preliminary evaluation of deslorelin, a GnRH agonist for contraception of the captive variable flying fox Pteropus hypomelanus.

BACKGROUND: This study was conducted to study the effects of a gonadotropin-releasing hormone (GnRH) agonist, deslorelin, on luteinizing hormone (LH), testosterone (males), semen characteristics and pregnancy in the variable flying fox Pteropus hypomelanus. STUDY DESIGN: Male (n = 3) and female (n = 5) bats received a 4.7-mg implant and were housed with untreated bats (eight females and three males, respectively). Plasma was collected twice monthly and analyzed for hormone concentrations, and semen was collected from untreated and treated males 1 month preimplantation, 3 months postimplantation and 4 months postimplantation. RESULTS: Administration of a GnRH challenge 1 month postimplantation showed an attenuated response in treated (n = 4), but not in untreated (n = 4), male and female bats. Plasma LH was lower in treated versus untreated males (p = .04), but not in females. Testosterone was lower in treated versus untreated males (p < .001). Spermic ejaculates were obtained from treated males, although no untreated females became pregnant during the 8-month study. One treated female became pregnant 6 months after implantation. CONCLUSION: Deslorelin is a useful and reversible contraceptive for P. hypomelanus.

Effect of ghrelin in dry matter intake and energy metabolism in prepartum sheep: a preliminary study.

The objective of this study was to determine the effect of ghrelin on dry matter intake and energy metabolites in ewes during the last 10d of gestation. Ewes were randomly assigned to either a treatment (n=6) or a control group (n=6). One hour after feeding (08:00 and 16:00), ewes in the treated group were given ovine ghrelin (3 microg/kg, i.m.) and the control group received an equivalent volume of saline. Treatments were continued until lambing. Daily blood samples were taken starting at assignment. The principal findings were that: (1) ewes treated with ghrelin had a higher dry matter intake only on the first day of treatment; (2) treated ewes had lower serum concentrations of non-esterified fatty acids and insulin but higher serum concentrations of growth hormone; (3) serum concentrations of beta-hydroxy butyrate and cortisol were not different between treated and non-treated ewes. In conclusion, treatment with ghrelin affected dry matter intake and energy metabolites of pre-partum ewes during the last 10 d of pregnancy.

Evaluation of the urodynamic and hemodynamic effects of orally administered phenylpropanolamine and ephedrine in female dogs.

OBJECTIVE: To compare the urodynamic and hemodynamic effects of different dosages of phenylpropanolamine and ephedrine and determine effective dosages in increasing urethral resistance in female dogs. ANIMALS: 20 sexually intact female Beagles. PROCEDURE: Dogs were allocated into 4 groups and received phenylpropanolamine once, twice, or 3 times daily, or ephedrine twice daily, for 14 days. On days 0, 7, and 14, urethral pressure profiles were performed while dogs were anesthetized with propofol. Variables recorded included maximum urethral pressure, maximum urethral closure pressure, integrated pressure, functional profile length, anatomic profile length, plateau distance, distance before maximum urethral pressure, and maximum meatus pressure. Arterial and central venous pressures were measured before anesthetic induction and 10 and 35 minutes after induction. RESULTS: Administration of phenylpropanolamine once daily or ephedrine twice daily significantly increased maximum urethral pressure and maximum urethral closure pressure. Values for integrated pressure were significantly increased after 14 days of once-daily administration of phenylpropanolamine. Variables did not change significantly from day 7 to day 14. Diastolic and mean arterial blood pressures increased significantly during the treatment periods, and arterial pressure decreased during propofol infusion. CONCLUSIONS AND CLINICAL RELEVANCE: Oral administration of phenylpropanolamine once daily or ephedrine twice daily increased urethral resistance in clinically normal dogs and may be recommended for management of urethral sphincter mechanism incompetence. Treatment efficacy may be assessed after 1 week. Dogs with concurrent cardiovascular disease should be monitored for blood pressure while receiving alpha-adrenergic agents because of the effects on diastolic and mean arterial pressure.

Urodynamic and morphologic changes in the lower portion of the urogenital tract after administration of estriol alone and in combination with phenylpropanolamine in sexually intact and spayed female dogs.

OBJECTIVE: To compare the urodynamic and morphologic effects of the administration of estriol alone and in combination with phenylpropanolamine on the lower portion of the urogenital tract in female dogs. ANIMALS: 3 sexually intact and 3 spayed female Beagles without urinary incontinence. PROCEDURE: Dogs received estriol (2 mg, PO) once daily for 7 days followed by estriol (2 mg, PO) and phenylpropanolamine (1.5 mg/kg, PO) once daily for 7 days. Urethral pressure profilometry, diuresis cystometry, and vaginourethrography were performed before treatment (day 0) and at days 7 and 14. The maximum urethral pressure (MUP) and closure pressure (MUCP), urethral functional and anatomic profile lengths, integrated pressure (IP), plateau, distance before MUP, maximum meatus pressure, threshold pressure, threshold volume, compliance, urethral length, and vaginal length and width were measured. RESULTS: Before treatment, no urodynamic differences were observed between the 2 groups; however, vaginal length and width were significantly shorter in spayed dogs. Compared with day 0 values, estriol treatment significantly increased MUP, MUCP, and IP values at day 7, but at day 14, this effect decreased despite phenylpropanolamine administration. No morphologic changes from baseline were detected after either treatment in any dog. CONCLUSIONS AND CLINICAL RELEVANCE: Data suggest that estriol mainly acts on the urethral sphincter mechanism by increasing urethral resistance in sexually intact and spayed female dogs without urinary incontinence. Administration of estriol and phenylpropanolamine did not increase the urethral resistance more than estriol alone. The urodynamic effects of estriol in female dogs with urinary incontinence remain to be elucidated.

Canine herpesvirus-1 (CHV-1): clinical, serological and virological patterns in breeding colonies.

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with reproductive disorders and neonatal mortality. To advise dog breeders towards an effective management of CHV-1 infected colonies, 27 breeding bitches were studied during one reproductive cycle in field conditions: the effect of cycle stage, kennel size, initial antibody titre, mating and gestation on serologic and viral excretion patterns was evaluated, while the association between reproductive disorders and CHV-1 antibody titres and viral excretion was also analysed. All initially seronegative bitches seroconverted, while 40% of the initially seropositive bitches became seronegative at one or two occasions. No difference in antibody patterns was observed between mated and unmated bitches. Of the mated bitches, 46% experienced infertility, foetal resorption or mummification. No difference in antibody patterns was observed depending on the occurrence of reproductive disorders even if a decrease in antibody titres during early or late-di-oestrus was often present. Significantly higher titres were observed at all cycle stages in large kennels. None of the vaginal and nasal samples or buffy coats tested positive for CHV-1 DNA. The mixed image of clinical and sub-clinical carriage in this study demonstrated CHV-1 has a complex and difficult to predict clinical behavior. Preventive management with vaccination of reproducing bitches in kennels with reproductive disorders should therefore be advised.

Influence of the estrous cycle on urodynamic and morphometric measurements of the lower portion of the urogenital tract in dogs.

OBJECTIVES: To compare the values of the urodynamic parameters of the lower portion of the urinary tract and vaginourethral measurements obtained during the phases of the estrous cycle in dogs and determine possible functional or anatomic modifications of the lower portion of the urinary tract associated with those phases. ANIMALS: 7 adult female Beagles. PROCEDURE: Urethral pressure profilometry, diuresis cystometry, and vaginourethrography were performed in each dog during proestrus; estrus; early, mid, and late diestrus; and early and late anestrus. The maximum urethral pressure (MUP), maximum urethral closure pressure (MUCP), urethral functional and anatomic profile lengths (UFPL and UAPL, respectively), integrated pressure, threshold pressure, threshold volume, compliance, urethral length, and vaginal length and width were measured. RESULTS: For all measurements, significant interindividual variation was detected. Integrated and threshold pressures, APL, and each morphometric value significantly increased from late anestrus to proestrus. Compared with other phases, MUP, MUCP, and integrated pressure values were significantly lower in estrus and early diestrus; UAPL and UFPL values were significantly lower in late diestrus. At each cycle phase in old dogs, MUP, MUCP, threshold pressure, and vaginal length and width were significantly lower (except in proestrus for vaginal measurements) and threshold volume and compliance values were significantly higher, compared with middle-aged dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Urodynamic and morphometric measurements of the lower portion of the urogenital tract are affected by the changes in hormonal balance that occur during the estrous cycle. In sexually intact female dogs, estrous phase determination is important for the interpretation of urodynamic data.

Invader probes: Harnessing the energy of intercalation to facilitate recognition of chromosomal DNA for diagnostic applications.

Development of probes capable of recognizing specific regions of chromosomal DNA has been a long-standing goal for chemical biologists. Current strategies such as PNA, triplex-forming oligonucleotides, and polyamides are subject to target choice limitations and/or necessitate non-physiological conditions, leaving a need for alternative approaches. Toward this end, we have recently introduced double-stranded oligonucleotide probes that are energetically activated for DNA recognition through modification with +1 interstrand zippers of intercalator-functionalized nucleotide monomers. Here, probes with different chemistries and architectures - varying in the position, number, and distance between the intercalator zippers - are studied with respect to hybridization energetics and DNA-targeting properties. Experiments with model DNA targets demonstrate that optimized probes enable efficient (C50 < 1 µM), fast (t50 < 3h), kinetically stable (> 24h), and single nucleotide specific recognition of DNA targets at physiologically relevant ionic strengths. Optimized probes were used in non-denaturing fluorescence in situ hybridization experiments for detection of gender-specific mixed-sequence chromosomal DNA target regions. These probes present themselves as a promising strategy for recognition of chromosomal DNA, which will enable development of new tools for applications in molecular biology, genomic engineering and nanotechnology.

Risk factors and reproductive disorders associated with canine herpesvirus-1 (CHV-1).

Canine herpesvirus-1 (CHV-1) is presumed to be enzootic in the dog population and is associated with fertility disorders and neonatal mortality. In this study we screened for risk factors affecting CHV-1 antibody titers and investigated the association between antibody titers and reproductive disorders. Therefore, serum from 545 dogs used for reproduction was analysed with an ELISA. Using a forward stepwise procedure and retaining significant risk factors (P<0.05), best fitting multifactorial generalized linear model (glm) procedures were built for males and females. The effect of antibody titers on reproductive disorders was analysed with logistic regression analysis. The association between reproductive disorders and seroprevalence was analysed in chi-square analyses using contingency tables. In both sexes, kennel cough and breeding management were found to have an impact on the CHV-1 antibody titer. Also, the influence of kennel cough on the antibody titer was correlated to the hygienic status of the kennel. In females, age, kennel size and cycle stage had an effect on CHV-1 antibody titers. Furthermore, kennel size and hygiene were found to be correlated. In males, mating experience had an impact on CHV-1 antibody titers. An association was observed between serological status and a history of abortion in bitches. In conclusion, this study suggests CHV-1 antibody titers may be affected by many factors, both on an environmental and host level. Therefore, interpretation of the serological status requires precaution. Furthermore, oronasal and venereal transmission seem to play a role in the spreading of infection.

Validation and comparison of the use of diuresis cystometry and retrograde filling cystometry at various infusion rates in female Beagle dogs.

OBJECTIVES: To compare retrograde filling cystometry at infusion rates of 5, 10, and 20 mL/min with diuresis cystometry for determination of an appropriate infusion rate and to confirm the reproducibility of measurements obtained by urethral pressure profilometry (UPP) and cystometry in female Beagles. ANIMALS: Adult female Beagles. PROCEDURE: Successive UPP and cystometry were performed by use of a water perfusion catheter on dogs anesthetized with propofol. Dogs randomly underwent each of the following at 1-week intervals: retrograde filling cystometry at 5, 10, and 20 mL/min, and diuresis cystometry. The maximum urethral pressure and closure pressure, functional and anatomic profile lengths, threshold pressure, threshold volume, and compliance were measured. RESULTS: For each UPP variable, significant differences were found among dogs, but no significant differences were found in intra- or interstudy measurements for individual dogs. For retrograde filling cystometry, threshold pressure was not significantly different between a 5 and 10 mL/min infusion rate. Threshold pressure was significantly higher during retrograde filling cystometry at 20 mL/min, compared with 5 and 10 mL/min, and was associated with bladder wall damages. Threshold pressure was significantly lower during diuresis cystometry, compared with retrograde filling cystometries. Threshold volume and compliance were not significantly different among retrograde filling cystometries but were significantly higher during diuresis cystometry. CONCLUSIONS AND CLINICAL RELEVANCE: Retrograde filling cystometry at 20 mL/min leads to unacceptable sudden increase in threshold bladder pressure. Retrograde filling cystometry at 10 mL/min can be recommended in a clinical setting, shortening the anesthesia time. However, diuresis cystometry approximates physiologic bladder filling most accurately.

Effects of a medetomidine-midazolam-butorphanol combination on renal cortical, intestinal and muscle microvascular blood flow in isoflurane anaesthetized dogs: a laser Doppler study.

OBJECTIVE: To obtain renal cortical, ileal, colonic and skeletal muscle microvascular blood flow measurements in dogs using the laser Doppler technique and to characterize the effects of medetomidine-midazolam-butorphanol combination on these flows. STUDY DESIGN: Prospective randomized experimental study. ANIMALS: Fourteen clinically normal beagles (two groups of seven), aged 1-4 years and weighing 13.2 ± 1.8 kg. METHODS: All dogs were anaesthetized with 1.7% end-tidal isoflurane in oxygen. In the treatment group, after instrumentation and when anaesthesia was considered stable, medetomidine (1 mg m-2 body surface area (BSA)) was administered intramuscularly (time 0). Midazolam (1 mg kg-1) and butorphanol (0.1 mg kg-1) were administered intravenously 20 minutes later. Atipamezole (2.5 mg m-2 BSA) was administered intramuscularly 60 minutes after medetomidine. In the control group, saline (0.5, 2.5 and 0.25 mL) was administered at the corresponding times. Heart rate, systolic, diastolic and mean arterial pressures, body temperature, renal cortical, ileal, colonic and skeletal muscle microvascular blood flows were measured before time 0, and 5, 15, 25, 40, 60, 65, 70 and 90 minutes thereafter. RESULTS: Heart rate, ileal and skeletal muscle blood flows decreased in the treatment group, while no changes were observed in the control group. CONCLUSIONS: Laser Doppler flowmetry allowed the measurement of microvascular blood flow in different organs. The medetomidine-midazolam-butorphanol combination decreases intestinal and skeletal muscle microvascular blood flows, while renal cortical blood flow is maintained. CLINICAL RELEVANCE: Medetomidine-midazolam-butorphanol combination can be used to induce a short duration anaesthesia in dogs, but it will induce cardiovascular depression. This depression appears to be accompanied by a redistribution of blood flow.

Characterization of the altered gene expression profile in early porcine embryos generated from parthenogenesis and somatic cell chromatin transfer.

The in vitro production of early porcine embryos is of particular scientific and economic interest. In general, embryos produced from in vitro Assisted Reproductive Technologies (ART) manipulations, such as somatic cell chromatin transfer (CT) and parthenogenetic activation (PA), are less developmentally competent than in vivo-derived embryos. The mechanisms underlying the deficiencies of embryos generated from PA and CT have not been completely understood. To characterize the altered genes and gene networks in embryos generated from CT and PA, comparative transcriptomic analyses of in vivo (IVV) expanded blastocysts (XB), IVV hatched blastocyst (HB), PA XB, PA HB, and CT HB were performed using a custom microarray platform enriched for genes expressed during early embryonic development. Differential expressions of 1492 and 103 genes were identified in PA and CT HB, respectively, in comparison with IVV HB. The "eIF2 signalling", "mitochondrial dysfunction", "regulation of eIF4 and p70S6K signalling", "protein ubiquitination", and "mTOR signalling" pathways were down-regulated in PA HB. Dysregulation of notch signalling-associated genes were observed in both PA and CT HB. TP53 was predicted to be activated in both PA and CT HB, as 136 and 23 regulation targets of TP53 showed significant differential expression in PA and CT HB, respectively, in comparison with IVV HB. In addition, dysregulations of several critical pluripotency, trophoblast development, and implantation-associated genes (NANOG, GATA2, KRT8, LGMN, and DPP4) were observed in PA HB during the blastocyst hatching process. The critical genes that were observed to be dysregulated in CT and PA embryos could be indicative of underlying developmental deficiencies of embryos produced from these technologies.