Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation.

BACKGROUND: Elevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates. METHODS: The senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation. RESULTS: The high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG. CONCLUSIONS: FSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis ("FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients. Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Maternal age and gonadotrophin elevation cooperatively decrease viable ovulated oocytes and increase ootoxicity, chromosome-, and spindle-misalignments: '2-Hit' and 'FSH-OoToxicity' mechanisms as new reproductive aging hypotheses.

While there is consensus that advanced maternal age (AMA) reduces oocyte yield and quality, the notion that high FSH reduces oocyte quality and causes aneuploidy remains controversial, perhaps due to difficulties controlling the confounding variables of age and FSH levels. Here, contributions of age and gonadotrophin elevation were separately controlled using a mouse model of human female reproductive aging. Ovulated oocytes were collected from young and midlife mice after 0-, 2.6-, or 17-day treatment with the FSH analog equine chorionic gonadotrophin (eCG), to model both exogenous FSH elevation within a single treatment cycle (as in controlled ovarian stimulation (COS)), and chronic endogenous FSH elevation during multiple cycles (as in diminished ovarian reserve). After 17-day eCG, fewer total oocytes/mouse are ovulated in midlife than young mice, and a precipitous decline in viable oocytes/mouse is observed in midlife but not young mice throughout eCG treatment. eCG is potently ootoxic to ovulatory oocytes and strongly induces chromosome- and spindle-misalignments within 2.6 days of eCG in midlife, but only after 17 days in young mice. These data indicate that AMA increases susceptibility to multiple adverse effects of elevated FSH activity in ovulated oocytes, including declines in total and viable oocytes/mouse, and induction of ootoxicity and aneuploidy. Two hypotheses are proposed for underlying causes of infertility in women. The FSH OOToxicity Hypothesis ('FOOT Hypothesis') posits that high FSH is ootoxic to ovulatory oocytes and that FSH ootoxicity is a root cause of low pregnancy success rates in naturally cycling women with high FSH and IUI patients undergoing COS. The '2-Hit Hypothesis' posits that AMA increases susceptibility to FSH-induced ootoxicity and aneuploidy.

Effects of transrectal palpation with the fetal membrane slip technique for early pregnancy diagnosis on the proportion and type of associated pregnancy loss in dairy cattle.

OBJECTIVE: To assess the effect of transrectal palpation (TRP) performed with the fetal membrane slip (FMS) technique for early pregnancy diagnosis on the proportion and type of associated pregnancy losses (PLs) in dairy cattle. ANIMALS: 580 healthy pregnant cattle. PROCEDURES: Data for artificially inseminated females with 1 or 2 viable embryos detected by transrectal ultrasonography (TRUS) at approximately 30 days of gestation were retrospectively assessed. Cattle were assigned to 1 of 2 groups on the basis of whether they did or did not undergo TRP once between 34 and 41 days of gestation (palpation and control group, respectively). At approximately 45 and 60 days of gestation, all cattle were reevaluated by TRUS; PL was categorized as type I (FMS detectable by TRP and TRUS-confirmed evidence of embryo or fetus degeneration and a functional corpus luteum) or type II (FMS undetectable by TRP and no TRUS-confirmed evidence of an embryo or fetus or of a functional corpus luteum). RESULTS: Of the 580 healthy pregnant cattle, 271 underwent TRP and 309 did not. In the palpation and control groups, PL occurred in 40 (14.8%) and 47 (15.2%) cattle, respectively. Among the palpation group's PLs, 17 (43%) were type I and 23 (58%) were type II. Among the control group's PLs, 27 (57%) were type I and 20 (43%) were type II. The prevalance and type of PL did not differ between groups. CONCLUSIONS AND CLINICAL RELEVANCE: TRP with the FMS technique for early pregnancy diagnosis did not increase the prevalence of PL in dairy cattle or alert the proportion of type I versus type II PL.

A rapid and efficient method for the collection of highly developmental murine immature oocytes using cilostazol, a phosphodiesterase 3A inhibitor.

AIMS: The present study aims to define maturation, yield, health, and ease of collection of murine immature oocytes recovered using the conventional method or from mice treated with cilostazol. MAIN METHODS: The conventional method included the superovulation of mice and the recovery of germinal vesicle (GV) or metaphase (MI) oocytes from preovulatory follicles. The cilostazol method included the oral treatment of superovulated mice with 7.5 mg cilostazol once or twice to result in the ovulation of MI or GV oocytes, respectively. KEY FINDINGS: The cilostazol method resulted in >95% of GV or MI oocytes with a diameter range of 60-90 µm or 50.1-70 µm in comparison to <60.0% of GV or MI oocytes resulting from the conventional method, respectively (P < 0.0001). The cilostazol method resulted in GV oocytes having higher levels of co-occurrence of peripheral cortical granules (CG) and chromatin configuration of surrounded nucleolus and MI oocytes having higher levels of co-occurrence of normally organized spindles/chromosomes and peripheral CG with free domains than did the conventional method (P < 0.001). The cilostazol method was more time and labor efficient and resulted in higher oocyte yields of normal morphology than did the conventional method (P < 0.01). SIGNIFICANCE: The presented method provides not only oocytes with uniform size and synchronized developmental maturation but also a technique of oocyte collection that is efficient and resourceful. It is possible that not all immature oocytes resulting from the conventional method are from preovulatory follicles nor have been developed adequately and consequently ovulated as opposed to the presented method.

Improvements in oocyte competence in superovulated mice following treatment with cilostazol: Ovulation of immature oocytes with high developmental rates.

Exogenous administration of superovulatory hormones negatively affects oocyte competence in mammals. Phosphodiesterase 3A inhibitors were found to improve competence of oocytes matured in vitro in several species, including humans. This study was therefore designed to define oocyte maturation synchronization and competence, in vivo, using superovulated mice treated with cilostazol, a selective phosphodiesterase 3A inhibitor. Swiss Webster mice were superovulated and treated orally with 7.5mg cilostazol once or twice to result in ovulation of immature oocytes at the metaphase I (MI) or germinal vesicle (GV) stage, respectively. Control immature oocytes were recovered from preovulatory follicles of superovulated mice not treated with cilostazol. Treated GV oocytes had significantly higher rates of synchronized and advanced chromatin configuration and cortical granule distribution than did control GV oocytes. Treated GV oocytes had a moderate increase in cAMP levels and consequently higher rates of meiotic maturation, IVF, and blastocyst formation than did control GV oocytes (P<0.0001). Treated MI oocytes had higher rates of normal spindles and chromosomes aligned at the metaphase plate than did control MI oocytes (P<0.003). Treated mice ovulating MI oocytes produced litter sizes larger than those observed in control mice ovulating mature oocytes (P<0.002). This study reveals that synchronization of oocyte maturation in superovulated mice improves oocyte development and competence. The capability of cilostazol, a clinically approved medication, to improve mouse oocyte competence suggests the potential benefit of including this compound in ovarian hyperstimulation programs to improve in vitro fertilization outcomes in infertile women.

Cilostazol blocks pregnancy in naturally cycling swine: An animal model.

AIMS: Cilostazol (CLZ) is an FDA approved therapeutic that is indicated for patients with intermittent claudication disease. CLZ is a selective inhibitor for phosphodiesterase 3A (PDE3A); an enzyme that controls oocyte maturation in many mammals including humans. Recently, CLZ has been reported to block pregnancy and oocyte maturation in mice. The objective of the present work was to evaluate the potential non-steroidal contraceptive capacity of CLZ using a more advanced translational model for humans. MAIN METHODS: Three groups of naturally cycling sows were treated orally with 0, 100, or 200mg CLZ, twice a day (bid), for 6days before estrus and continued for three days after estrus. Each sow was mated by one of two proven fertile boars on alternate days during estrus. KEY FINDINGS: CLZ dose of 100mg, bid, completely blocked pregnancy in sows when compared to control sows (P<0.01). However, the 200mg dose of CLZ, bid, failed to significantly block pregnancy in pigs. No significant differences were observed in heart rates of treated and control animals. Re-mating of the previously treated sows exhibited normal pregnancies and litter sizes. SIGNIFICANCE: This study shows that CLZ is capable of producing a reversible non-steroidal contraceptive effect without adverse effects on the heart rate in pigs. The observed contraceptive effect of CLZ was at doses similar to those indicated to humans. This FDA approved agent, for treatment of patients with intermittent claudication, may have an additional therapeutic effect as a non-steroidal contraceptive agent. Cilostazol merits further evaluation in women and might be useful for controlling the population of homeless animals.

Improvement in in vitro fertilization outcome following in vivo synchronization of oocyte maturation in mice.

Synchronization of oocyte maturation in vitro has been shown to produce higher in vitro fertilization (IVF) rates than those observed in oocytes matured in vitro without synchronization. However, the increased IVF rates never exceeded those observed in oocytes matured in vivo without synchronization. This study was therefore designed to define the effect of in vivo synchronization of oocyte maturation on IVF rates. Mice were superovulated and orally treated with 7.5 mg cilostazol (CLZ), a phosphodiesterase 3A (PDE3A) inhibitor, to induce ovulation of immature oocytes at different stages depending on frequency and time of administration of CLZ. Mice treated with CLZ ovulated germinal vesicle (GV) or metaphase I (MI) oocytes that underwent maturation in vitro or in vivo (i.e. in the oviduct) followed by IVF. Superovulated control mice ovulated mature oocytes that underwent IVF directly upon collection. Ovulated MI oocytes matured in vitro or in vivo had similar maturation rates but significantly higher IVF rates, 2-4 cell embryos, than those observed in control oocytes. Ovulated GV oocytes matured in vitro showed similar maturation rates but significantly higher IVF rates than those observed in control oocytes. However, ovulated GV oocytes matured in vivo had significantly lower IVF rates than those noted in control oocytes. It is concluded that CLZ is able to synchronize oocyte maturation and improve IVF rates in superovulated mice. CLZ may be capable of showing similar effects in humans, especially since temporal arrest of human oocyte maturation with other PDE3A inhibitors in vitro was found to improve oocyte competence level. The capability of a clinically approved PDE3A inhibitor to improve oocyte fertilization rates in mice at doses extrapolated from human therapeutic doses suggests the potential scenario of the inclusion of CLZ in superovulation programs. This may improve IVF outcomes in infertile patients.

Shortened estrous cycle length, increased FSH levels, FSH variance, oocyte spindle aberrations, and early declining fertility in aging senescence-accelerated mouse prone-8 (SAMP8) mice: concomitant characteristics of human midlife female reproductive aging.

Women experience a series of specific transitions in their reproductive function with age. Shortening of the menstrual cycle begins in the mid to late 30s and is regarded as the first sign of reproductive aging. Other early changes include elevation and increased variance of serum FSH levels, increased incidences of oocyte spindle aberrations and aneuploidy, and declining fertility. The goal of this study was to investigate whether the mouse strain senescence-accelerated mouse-prone-8 (SAMP8) is a suitable model for the study of these midlife reproductive aging characteristics. Midlife SAMP8 mice aged 6.5-7.85 months (midlife SAMP8) exhibited shortened estrous cycles compared with SAMP8 mice aged 2-3 months (young SAMP8, P = .0040). Midlife SAMP8 mice had high FSH levels compared with young SAMP8 mice, and mice with a single day of high FSH exhibited statistically elevated FSH throughout the cycle, ranging from 1.8- to 3.6-fold elevation on the days of proestrus, estrus, metestrus, and diestrus (P < .05). Midlife SAMP8 mice displayed more variance in FSH than young SAMP8 mice (P = .01). Midlife SAMP8 ovulated fewer oocytes (P = .0155). SAMP8 oocytes stained with fluorescently labeled antitubulin antibodies and scored in fluorescence microscopy exhibited increased incidence of meiotic spindle aberrations with age, from 2/126 (1.59%) in young SAMP8 to 38/139 (27.3%) in midlife SAMP8 (17.2-fold increase, P < .0001). Finally, SAMP8 exhibited declining fertility from 8.9 pups/litter in young SAMP8 to 3.5 pups/litter in midlife SAMP8 mice (P < .0001). The age at which these changes occur is younger than for most mouse strains, and their simultaneous occurrence within a single strain has not been described previously. We propose that SAMP8 mice are a model of midlife human female reproductive aging.

Cilostazol administered to female mice induces ovulation of immature oocytes: a contraceptive animal model.

AIMS: Both Cilostamide and Org 9935 are phosphodiesterase 3A (PDE3A) inhibitors that were evaluated in rodents and monkeys for their non-steroidal contraceptive properties. Although both compounds inhibited oocyte maturation, an adverse effect on heart rate was observed. Cilostazol (CLZ, Pletal) is a safe PDE3A inhibitor that was recently reported to block pregnancy in naturally cycling mice. In this study, the dose, frequency, time of administration, and reversibility effects of CLZ on oocyte maturation were defined using superovulated mice. MAIN METHODS: Superovulated mice were gavaged once or twice with 0, 7.5, or 15 mg CLZ at various times around the ovulatory stimulus. Ovulated oocytes were then evaluated for maturational stages. KEY FINDINGS: CLZ resulted in mice ovulating significant numbers of immature oocytes when administered anytime between 9h before the ovulatory stimulus and 2 h after the stimulus. This inhibitory effect was greater when CLZ dose was increased, administered twice or closer to the time of the ovulatory stimulus. Conversely, ovulated immature oocytes resumed maturation in oviducts when CLZ dose was reduced, administered once and away from the time of the stimulus. SIGNIFICANCE: Controlling CLZ dose, frequency, and time of administration produced mice ovulating immature oocytes at different stages, which may be an advantage over the conventional collection of immature oocytes from ovaries. More importantly, the capability of a clinically approved PDE3A inhibitor, with a reasonable margin of safety, to arrest oocyte maturation over a wide range of administration times and at doses extrapolated from human therapeutic doses demonstrates the contraceptive capacity of CLZ.

In vitro effects of cilostazol, a phosphodiesterase 3A inhibitor, on mouse oocyte maturation and morphology.

Inhibition of phosphodiesterase 3A (PDE3A) in oocytes has been reported to arrest oocyte maturation and to increase intra-oocyte cyclic adenosine monophosphate levels. Although many PDE3A inhibitors have been found to arrest oocyte maturation in different species, including humans, the most commonly prescribed PDE3A inhibitor named cilostazol (CLZ) has not yet been fully evaluated in reproduction. The present study was designed to investigate the potential inhibitory effects of CLZ on oocyte maturation and morphology in vitro. Antral oocytes were recovered from hyperstimulated mice and allocated to 10 different CLZ concentrations (0.00-67.66 µmol/L). Oocytes were then assessed after 24 and 48 h of incubation for maturation and morphology. Some of the evaluated CLZ concentrations (1.06-4.23 µmol/L) were made similar to those observed in human clinical trials. CLZ arrested oocyte maturation at the germinal vesicle (GV) stage at concentrations as low as 1.06 µmol/L (P < 0.0001). A selective degenerative impact of CLZ targeting arrested oocytes at the GV stage was observed during 24 h of incubation (r = -0.781, P < 0.0001). This was not the case with non-arrested oocytes (r = -0.082, P = 0.64). Such degenerative impact was dose-dependent (P < 0.0001), suggesting a role for cyclic adenosine monophosphate in this degenerative process. The degenerated oocytes were of distorted oolema or fragmented cytoplasm. Based on the experiments, it is concluded that CLZ can inhibit oocyte maturation in vitro, at concentrations similar to those observed in humans taking CLZ, and under such conditions the prolonged maintenance of oocytes at the GV stage is harmful.

Cilostazol blocks pregnancy in naturally cycling mice.

BACKGROUND: Administration of a phosphodiesterase three enzyme inhibitor (PDE3-I) in rodents and primates results in ovulation of immature oocytes. Concerns regarding inhibition of PDE3 enzymes that are expressed in heart and blood vessels discouraged further development of PDE3-Is as nonsteroidal contraceptives. Cilostazol (CLZ) is a PDE3A-I that is approved for medical indications in humans and has an additional effect of adenosine uptake inhibition that is believed to counterbalance the undesirable outcomes resulting from PDE inhibition. STUDY DESIGN: Cycling mature female mice were treated with 7.5 or 15 mg CLZ, dimethyl sulfoxide or water beginning on the day of proestrus. Animals were placed with fertility-proven males after 3 days of treatment. Treatments were continued until 1 day after detection of a vaginal plug, and then females were monitored up to 30 days postbreeding to assess the effects of the compounds on pregnancy. Each of the treated female with CLZ was then remated with the same male and again monitored up to 30 days. RESULTS: None of the CLZ-treated mice produced offspring, whereas all of the control animals maintained pregnancy and delivered normal pups (p<.0001). Remating of the previously CLZ-treated females exhibited normal pregnancies and gave birth to live offspring that were not different from the controls. CONCLUSION: CLZ is a potential nonsteroidal contraceptive agent that merits further evaluation in other mammals.

Effects of early pregnancy diagnosis by palpation per rectum on pregnancy loss in dairy cattle.

OBJECTIVE: To determine the effect of palpation per rectum (PPR) by use of 1 or 2 fetal membrane slips (FMSs) for pregnancy diagnosis during early gestation on pregnancy loss in dairy cattle. DESIGN: Controlled, randomized block design. ANIMALS: 928 healthy pregnant cattle. PROCEDURES: All cattle were determined to be pregnant by use of transrectal ultrasonography at approximately day 31 after estrus and randomly allocated into 2 groups (control group [n = 476 cows] and palpation group [452]). The control group was not subjected to pregnancy diagnosis via PPR. The palpation group was subdivided into 2 groups (PPR FMS 1 [n = 230 cows] and PPR FMS 2 [222]), which involved PPR and pregnancy diagnosis via 1 or 2 FMSs, respectively, during the same examination, which was performed by 1 veterinarian between days 34 and 43 after estrus. All cattle were reevaluated by use of transrectal ultrasonography on days 45 and 60 to determine viability of the embryo and fetus, respectively. RESULTS: Overall pregnancy loss between days 31 and 60 was 14.1%. Pregnancy loss for the control, PPR FMS 1, and PPR FMS 2 groups from days 31 to 60 was 14.5%, 12.6%, and 14.9%, respectively. Embryonic pregnancy loss for the control, PPR FMS 1, and PPR FMS 2 groups was 12.4%, 9.1%, and 9.5%, respectively. Fetal pregnancy loss for the same groups was 2.4%, 3.8%, and 5.9%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Pregnancy diagnosis via 1 or 2 FMSs performed during PPR in early gestation did not increase pregnancy loss in dairy cattle.

Delivery of stem cells to porcine arterial wall with echogenic liposomes conjugated to antibodies against CD34 and intercellular adhesion molecule-1.

In atherosclerosis, the loss of vascular stem cells via apoptosis impairs the capacity of the vascular wall to repair or regenerate the tissue damaged by atherogenic factors. Recruitment of exogenous stem cells to the plaque tissue may repopulate vascular cells and help repair the arterial tissue. Ultrasound-enhanced liposomal targeting may provide a feasible method for stem cell delivery into atheroma. Bifunctional echogenic immunoliposomes (BF-ELIP) were generated by covalently coupling two antibodies to liposomes; the first one specific for CD34 antigens on the surface of stem cells and the second directed against the intercellular adhesion molecule-1 (ICAM-1) antigens on the inflammatory endothelium covering atheroma. CD34+ stem cells from adult bone marrow were incubated on the ICAM-1-expressing endothelium of the aorta of swine fed high cholesterol diets, which was preloaded with BF-ELIP. Significantly increased stem cell adherence and penetration were detected in particular in the aortic segments treated with 1 MHz low-amplitude continuous wave ultrasound. Fluorescence and scanning electron microscopy confirmed the presence of BF-ELIP-bound CD34+ cells in the intimal compartment of the atheromatous arterial wall. Ultrasound treatment increased the number of endothelial cell progenitors migrating into the intima. Thus, under ultrasound enhancement, BF-ELIP bound CD34+ stem cells selectively bind to the ICAM-1 expressing endothelium of atherosclerotic lesions.

Molecular characterization of the Rocky Mountain elk (Cervus elaphus nelsoni) PRNP putative promoter.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting deer (Odocoileus spp.), moose (Alces alces), and Rocky Mountain elk (Cervus elaphus nelsoni). Leucine homozygosity at elk PRNP codon 132 has been associated with reduced CWD susceptibility. However, naturally acquired CWD has been detected in elk possessing the 132 Leu/Leu genotype. Recent human and bovine studies indicate that PRNP regulatory polymorphisms may also influence TSE occurrence. Therefore, we generated sequences for the elk PRNP putative promoter (2.2 kb), exon 1 (predicted; 54 bp), intron 1 (predicted; 193 bp), and exon 3 (771 bp). Promoter prediction analysis using CpGProD yielded a single elk PRNP promoter that was homologous to regions of known promoter activity in cow and sheep. Molecular interrogation of the elk PRNP putative promoter revealed 32 diallelic single-nucleotide polymorphisms (SNPs). No variation was detected within the predicted exon 1 or intron 1 sequences. Evaluation of elk PRNP exon 3 revealed 3 SNPs (63Y, 312R, 394W-->Met/Leu). Bayesian haplotype reconstruction resulted in 3 elk PRNP haplotypes, with complete linkage disequilibrium observed between all PRNP putative promoter SNPs and codon 132. The results of this study provide the initial genomic foundation for future comparative and haplotype-based elk PRNP studies.

Cloning of GJA1 (connexin43) and its expression in canine ovarian follicles throughout the estrous cycle.

GJA1 (also known as connexin43 or Cx43) is the most abundant gap junction protein in mammalian tissues including the ovary. Here, it facilitates intercellular communication among granulosa cells and growing oocytes, thereby connecting the developing gamete to the hormonal axis as well as to the essential network of supporting granulosa cells. To date, the pattern of follicular GJA1 expression has not yet been defined for canines, a species with unique reproductive physiology including delays in follicle development, ovulation, oocyte maturation and fertilization. Here, we report the complete mRNA sequence for canine GJA1 and identify not only increases (P<0.05) in GJA1 mRNA expression in follicles at the secondary stage and larger, but also differences in expression levels between estrous cycle stages in both secondary and antral stage follicles. Expression of GJA1 mRNA in secondary follicles during proestrus was higher than in anestrus or estrus (P<0.01), and at diestrus (P<0.10). Antral follicles obtained during estrus expressed lower levels of GJA1 mRNA than any other cycle stage (P<0.01). GJA1 mRNA expression in primary and large antral follicles was similar across the estrous cycle. Despite the extensive length of the canine estrous cycle as compared with that of other mammals, the GJA1 mRNA and protein expression profiles are not significantly different from those reported for other species and suggests that mechanisms regulating GJA1 transcription are not likely to contribute to the extended delays in follicle and oocyte development in the dog.

Early pregnancy diagnosis by palpation per rectum: influence on embryo/fetal viability in dairy cattle.

The objective was to estimate the effect of palpation per rectum (for early pregnancy diagnosis) on embryo/fetal viability in dairy cattle. A controlled, randomized block-design experiment with two blocks, one by category, and the other by number of embryos, was conducted. Five-hundred-and-twenty pregnant dairy cows and heifers with a viable embryo detected by transrectal ultrasonography (TRUS) between days 29 and 32 after AI were included. The pregnant females were randomly allocated into two nearly equal groups: palpation per rectum (PAL group; n=258) and no palpation per rectum (NPAL group; n=262). The PAL group was submitted to palpation per rectum (PPR) using the fetal membrane slip (FMS) technique once between days 34 and 41 of pregnancy. The fetal membrane slip consisted of compressing the pregnant uterine horn and allowing the chorioallantoic membrane to slip between the fingers. Both groups were submitted to two additional TRUS at days 45 and 60 of pregnancy, to monitor the potential immediate and delayed deleterious effects of PPR on embryo and fetal viability, respectively. A diagnosis of embryo/fetal death was made when there was no embryo/fetal heart beat or the absence of positive signs of pregnancy in an animal previously diagnosed pregnant, or the presence of signs of embryo/fetal degeneration. The overall rate of embryo/fetal death was 14.0% (73/520). Embryonic death (10%; 52/520) was higher than fetal death (4.5%; 21/468; P<0.001). Embryo/fetal mortality was higher in cows (16.4%; 59/360) than in heifers (8.8%; 14/160; P<0.025) and in cattle with twin (25.5%; 12/47) versus singleton pregnancies (12.9%; 61/473; P<0.025), but was not different (P>0.05) between PAL (14.7%; 38/258) and NPAL (13.4%; 35/262). In conclusion, PPR between days 34 and 41 of pregnancy using the fetal membrane slip technique did not affect embryo/fetal viability.

Early pregnancy diagnosis by transrectal ultrasonography in dairy cattle.

The objective of the present study was to determine differences in time of detection of pregnancy between heifers and cows and the interval after insemination at which the maximum sensitivity and negative predictive value of transrectal ultrasonography were obtained. One-thousand-four-hundred transrectal ultrasonographies (TRUS-1; 1,079 in cows and 321 in heifers) were performed using a 5-MHz linear-array transducer. The cattle were randomly assigned to have TRUS performed once between days 24 and 30 (estrus=day 0) in cows or between days 21 and 27 in heifers. Every TRUS diagnosis was subsequently compared with a second TRUS diagnosis (TRUS-2), performed 3-8 days later, after day 30 (range 31-38) for cows and after day 27 (range 28-35) for heifers. The sensitivity and specificity between cows and heifers for the common days of TRUS (from 24 to 27) were compared. In cows, sensitivity increased gradually from 74.5% at day 24 to 100% at day 29 (P<0.01). Specificity increased from days 24-25 and reached a plateau of 96.6% on day 26 (P<0.01). In heifers, sensitivity increased from 50% at day 21 to 100% at day 26 (P<0.01). Specificity increased from 87.5% at day 21 and remained steady at 94% starting on day 23 (P>0.05). The sensitivity for cows and heifers was 89.2 and 96.8%, respectively (P<0.05) and the specificity was 93.0 and 93.4% (P>0.05). In this study, heifers were diagnosed pregnant earlier than cows, and the maximum sensitivity and negative predictive value were obtained 3 days earlier in heifers than cows (days 26 and 29, respectively).