Cytological Diestrus Research Articles

Use of a Point-of-Care Progesterone Assay to Predict Onset of Parturition in the Bitch.

An assay of circulating progesterone (P4) is commonly used to estimate progress through late gestation in the bitch. Point-of-care assays provide rapid results, a major advantage over laboratory-based assays. This study aims to compare P4 levels determined by the Catalyst® Progesterone point-of-care assay with those determined by chemiluminescent immunoassay (CLIA) and to identify the expected distribution of Catalyst P4 levels at time intervals 3 days prior to the onset of parturition in pregnant bitches. Twenty-eight pregnant bitches carrying two or more fetuses were admitted to a specialist veterinary reproduction hospital 53 days after the onset of cytological diestrus or, when that date was not known, 57 days after the last mating. Vaginal speculum examinations were performed every 6 h until the onset of cervical dilatation (TCD). Serum samples were collected twice daily (08h00 and 18h00) until TCD. For most samples, fresh serum was assayed for P4 immediately using the Catalyst assay (CatP4), then frozen until assayed by CLIA (IMMULITE 2000; ImmP4). However, for some samples, CatP4 was not analyzed prior to freezing. For these data points (n = 33), CatP4 for fresh serum was estimated from CatP4 assayed on frozen-thawed serum, based on a comparison between CatP4 on fresh vs. frozen-thawed sera. In comparison to ImmP4, CatP4 levels up to and including 7 nmol/L appear to have a constant bias of −1.69 nmol/L (limits of agreement −4.91 to 1.52), while levels >7 nmol/L appear to have a proportional bias of −17.9% (limits of agreement −68.6% to 32.7%). Bootstrapped percentiles of CatP4 results spanned 0.4–9 nmol/L within 12 h of TCD, 0.9–11 nmol/L 12–24 h from TCD, and 2.2–13.5 nmol/L 24–36 h from TCD. A CatP4 >9 nmol/L indicates a bitch that is unlikely to reach TCD within 12 h. Bitches with CatP4s below 3.5 nmol/L are likely to reach TCD within 36 h and bitches with a CatP4 below 2.2 nmol/L are likely to reach TCD within 24 h. In conclusion, the Catalyst Progesterone assay provides rapid assessment of circulating P4 in the bitch, with clinical application in the monitoring of late term pregnant bitches.

Kisspeptin and Sex Steroid Hormone Receptors Are Upregulated at the Maternal‐Fetal Interface of the Preeclamptic‐like BPH/5 Mouse

The main features of preeclampsia (PE), a devastating syndrome that affects 5‐8% of pregnancies worldwide, are spontaneously recapitulated by the Blood Pressure High Subline 5 (BPH/5) mouse model. Comparable to women with early‐onset PE, BPH/5 females develop poor placentation with decreased trophoblast cell invasion, shallower placental discs and reduced placental endothelial remodelling than control C57 mice. Kisspeptins (KP), encoded by Kiss1, have been implicated in the etiology of PE: KP upregulation at the human maternal‐fetal interface is suggested to inhibit trophoblast cell invasion and angiogenesis. However, uterine KP expression pre‐pregnancy in women that later develop PE and the upstream signaling mechanisms leading to uterine/placental KP upregulation remain vastly unexplored. Interestingly, administration of estradiol‐17ß (E2) and/or progesterone (P4) to non‐pregnant ovariectomized CD‐1 mice resulted in uterine Kiss1upregulation. We have previously shown that BPH/5 females present abnormal circulating E2 and P4 throughout the estrous cycle and early pregnancy. Furthermore, KP and KP receptor (Kiss1r) are upregulated at the BPH/5 maternal‐fetal interface during the peak of decidualization, at embryonic day (e) 7.5. Therefore, the goal of this study was to investigate mRNA expression of Kiss1/Kiss1r,and the receptors for Progesterone (PR‐a/b/c)and Estrogen (ER‐αand GPER‐1)in BPH/5 non‐pregnant uterus during the first day of diestrus (NP‐D) and embryonic implantation sites (eIS) at e4.5. We hypothesized that the sex steroid hormone receptors would be upregulated in the uterus/eIS of BPH/5 during NP‐D and e4.5, respectively, potentially contributing with Kiss1/Kiss1rupregulation. Daily vaginal cytology samples were collected from adult BPH/5 and C57 females to determine the stage of the estrous cycle, and uterine samples were collected on the first day of cytologic diestrus (NP‐D, n = 3‐6/strain). Moreover, adult BPH/5 and C57 females were bred, and eIS were collected at e4.5 (n = 7‐9/strain). Gene expression was investigated using quantitative RT‐PCR and comparisons were performed using unpaired T‐tests. Welch’s corrections were performed for unequal variances and the significance level was set at α ≤ 0.05. Kiss1expression was higher in BPH/5 NP‐D uterus (p = 0.04) and e4.5 eIS (p = 0.01) when compared with C57. Contrarily, Kiss1rwas not different between BPH and C57 in NP‐D or e4.5 (p > 0.05). Interestingly, PR‐a/b/cand ER‐αwere also higher in the uterus/eIS of BPH/5 females both during NP‐D (p = 0.05 and 0.02, respectively) and e4.5 (p = 0.003 and 0.02, respectively). The trans‐membrane G‐protein‐coupled estrogen receptor 1 (GPER‐1), largely associated with rapid, non‐genomic cellular responses to estrogens, was also higher in BPH/5 eIS at e4.5 when compared with controls C57 (p < 0.001), but not different between groups in NP‐D (p > 0.05). In conclusion, uterine Kiss1upregulation precedes pregnancy in the BPH/5 mouse model. Furthermore, PR‐a/b/cand ER‐α expression in the NP‐D uterus and early maternal fetal interface parallels the expression levels of Kiss1in the PE‐like BPH/5 mouse.

Aglepristone Administration in Mid-Proestrus Reduces the LH Peak but Does Not Prevent Ovulation in the Bitch.

Simple SummaryThe role of preovulatory progesterone for LH release and ovulation in the bitch is not clear. The aim of the study was to evaluate the influence of administration of aglepristone in mid-proestrus on progesterone concentration, LH release, and occurrence of ovulation in the bitch. Experimental bitches (n = 7) were treated on days 4 and 5 of proestrus with aglepristone (Alizin®, Virbac) at the dose of 10 mg/kg body weight s.c. (i.e., the two treatments were 24 h apart). The progesterone concentration showed a similar pattern in both groups. The LH peak value and area under the curve for LH in bitches treated with aglepristone were significantly lower than those in control bitches. The ovulation occurred in all animals in both groups. The presented study showed that withdrawal of progesterone by administration of aglepristone in the mid-proestrus significantly reduced the preovulatory LH surge, but it had no effect on periovulatory progesterone concentration or the occurrence of ovulation.The aim of the study was to evaluate the influence of administration of aglepristone in mid-proestrus on progesterone concentration, LH release, and occurrence of ovulation in the bitch. Experimental bitches (n = 7) were treated on days 4 and 5 of proestrus with aglepristone at the dose of 10 mg/kg body weight s.c. (i.e., the two treatments were 24 h apart). Control animals (n = 7) received s.c. injections of saline. For progesterone determination, blood was collected daily until the first day of cytological diestrus. For LH determination, blood was collected daily and in the periovulatory phase every 8 h. The progesterone concentration showed a similar pattern in both groups. The LH peak value in bitches treated with aglepristone was significantly lower (p < 0.05) than that in control bitches (4.83 ± 1.20 vs. 13.66 ± 1.21 ng/mL). The area under the curve (AUC) for LH was significantly (p < 0.05) lower in treated than in control animals (6.85 ± 1.21 ng/mL/d vs. 12.25 ± 1.35 ng/mL/d). The ovulation occurred in all animals in both groups. The study showed that administration of aglepristone in the mid-proestrus significantly reduced the preovulatory LH surge, but it had no effect on progesterone concentration and the occurrence of ovulation.

Curtailing parturition observation and performing preparturient cesarean section in bitches

Properly planned elective cesarean section (CS) in bitches is considered safe and justified for some breeds. Therefore, planning a scheduled (fixed date and time) preparturient CS in bitches belonging to a subpopulation where a CS is unavoidable, seems justified. The first day of cytological diestrus (D0) was used to predict the date of parturition. The aims of this study were to (1) compare the mortality of puppies delivered by preparturient CS to those delivered by parturient CS, (2) determine the extent to which a preparturient CS (performed at 08:00 on D57, while the cervix is closed) would shorten gestation, (3) compare the level of progesterone at the time of preparturient CS to that at the time of parturient CS, (4) compare the change in hematocrit before and after CS for parturient- and preparturient CSs. Out of 99 gestations for which D0 was known and a preparturient CS planned, the CS was performed at the scheduled time in 61%, before the scheduled time because cervical dilatation had started in 32% and before the scheduled time in 7% because the bitches had started showing signs of impending parturition, although their cervices were still closed. This study showed high neonatal survival ratios and good Apgar scores after preparturient CS with 99% of puppies delivered by preparturient CS born alive and 98.8% surviving to 2 h. Performing preparturient CSs shortened gestation by four to 52 h. Progesterone level was significantly higher at the time of preparturient CSs than at the time of parturient CSs. Further studies are required to determine whether preparturient CSs on D57 are routinely safe in all breeds, does not affect long term survival of the puppies and to timeously identify bitches in which parturition starts prior to the date and time for the scheduled elective preparturient CS.

Blood periovulatory progesterone quantification using different techniques in the dog

Blood progesterone concentration is used in several procedures related to the reproduction in the bitch, such as ovulation monitoring, estimating time of parturition, or hypo-luteoidism management. Several techniques are available to evaluate blood progesterone concentration, such as the radioimmunoassay (RIA), the chemiluminescent immunoassay (CLIA), and the enzyme-linked immunosorbent assay (ELISA). The aim of this study was to compare the blood progesterone concentration using these three methods during the periovulatory period of 23 bitches. Vaginal cytology was used to classify cytologic estrus (CE) and cytologic diestrus (CD), and blood samples were collected once during proestrus and every other day between CE and CD. The samples were retrospectively classified in the different phases of the estrus based on CD. Pregnancy rate and gestational length were also recorded. A significant increase of the circulating progesterone during the progression of the estrus was recorded, and there were significant differences in the values when using the different methods, with lesser, intermediate, and greatest values with use of the RIA, CLIA, and ELISA, respectively. There was a high correlation (Pearson’s correlation coefficient = 0.978) and substantial strength-of-agreement (Lin’s concordance correlation coefficient = 0.966) between values obtained when using CLIA and RIA, while there was a high correlation (Pearson’s correlation coefficient = 0.955) but poor strength-of-agreement (Lin’s concordance correlation coefficient = 0.866) with use of the ELISA and RIA. The data reported in this study provide evidence that the method used for measuring the blood progesterone concentration during the periovulatory phase of the bitch significantly affected the progesterone values.

Dynamics of Vaginal pH in the Bitch during Proestrus and Estrus

The aim of this study was to investigate the dynamics of the pH of vaginal secretions during proestrus and estrus in bitches and its association with the optimal time for insemination and the values of steroid hormones. Sixteen bitches from different breeds and age were included in this investigation. Vaginal pH of each bitch was measured once daily, starting from the first day of vulval bleeding and continuing until the first day of cytological diestrus. In each bitch vaginal smears were taken to determine the onset of estrus and diestrus and blood samples were taken to determine serum progesterone (ng/ml) and estradiol-17β (pg/ml) for detection of the onset of ovulation and their influence on values and the dynamics of vaginal pH. Vaginal pH values in the bitches at the first day of proestrus were between 7.4 and 8.7. This parameter decreased with the progress of the proestrus and the entering into the estrus. Compared to the rest of the estrus, at the time of ovulation and on the next day, a consistent decrease of mean values (6.61±0.15 and 6.63±0.15 respectively) was observed (р<0,01). Individually at the time of ovulation vaginal pH ranged between 6.5 and 6.8. In the days following ovulation a gradual increase in mean values was detected. At the first day of diestrus vaginal pH values were measured significantly higher than those in estrus (р<0,001) and were between 7.4 and 8.3 (7.74±0.26). In our study we found that the vaginal pH values in the bitch are lowest at the time of ovulation, with individual variations and exceptions.

Power and pulsed Doppler evaluation of ovarian hemodynamic changes during diestrus in pregnant and nonpregnant bitches

The aim of the study was to further characterize the relationship between hemodynamic changes in the ovary and luteal function in pregnant and nonpregnant bitches. Fourteen German Shepherd bitches were monitored three times a week from the first day of cytological diestrus (D1) until parturition or the end of diestrus (progesterone <2 ng/mL) by color Doppler, pulsed wave spectral Doppler, and power Doppler (PD) ultrasonography. By means of PD the total number of color pixels were calculated. The Doppler parameters evaluated were: peak systolic velocity (PSV), end diastolic velocity (EDV), and both resistive and pulsatility indices. Blood samples were collected three times a week throughout the experiment to determine progesterone (P4) concentrations. The length of diestrus in pregnant versus nonpregnant group was significantly shorter (P < 0.01; 57 ± 1 vs. 63 ± 1, respectively). By means of pulsed wave spectral Doppler the waveform showed a typical pattern of a low–resistive vessel characterized by a rapid systolic peak followed by a slow telediastolic decrease with a relatively high end-diastolic velocity. Blood flow parameters did not differ between left and right ovary. In both groups PSV and EDV showed a gradual decrease with the progress of diestrus; however, the values of PSV and EDV were significantly higher (P < 0.05) in the pregnant group versus nonpregnant group from D31 to D61 and from D49 to D58 respectively. Moreover, a significantly decrease (P < 0.05) of PSV and EDV in the pregnant group was observed from D46 to D58 and from D49 to D55, respectively. The resistive and pulsatility indices showed an increase during diestrus and the values were significantly lower (P < 0.05) in the pregnant group from D49 to D61. By means of PD, the pixel number was significantly higher (P < 0.05) in the pregnant versus nonpregnant group from D40 to D61. In particular, a significant decrease (P < 0.05) in the pixel number in the pregnant group was observed from D46 to D61. The comparison of the P4 values with the ovarian pixel number in the pregnant and nonpregnant group showed a direct correlation (r = 0.792, N = 59 and r = 0.774, N = 59, respectively). In particular, the P4 values were higher (P < 0.05) in the pregnant than in the nonpregnant group from D37 to D52. In conclusion, significant physiological differences between pregnant and nonpregnant bitches in terms of P4 and ovarian blood supply are reported. In addition it was possible to define that blood flow pattern during diestrus in pregnant bitches is not always closely related with P4 production.

Pharmacokinetics of eCG and induction of fertile estrus in bitches using eCG followed by hCG

The aim was to design a protocol combining eCG followed by hCG for estrus induction in the bitch. In Experiment 1, three ovariohysterectomized bitches received 10 000 IU of eCG iv, and 15 days later 10 000 IU of eCG im. Blood samples were taken up to 144 h after each injection to measure eCG concentrations. In Experiment 2, 25 healthy, intact late anestrous bitches were assigned to one of five doses of eCG (5, 10, 15, 20, 44, or 50 IU/kg eCG im; [TRT5-TRT50]). Sexual behavior (SB), clinical signs of estrus (CSE) and vaginal cytology (VC) samples were obtained and scored before eCG administration and every other day until onset of estrus, or for 14 days. In Experiment 3, intact late anestrous bitches were assigned to a treatment group (TRT; n = 16) and received eCG (50 IU/kg im) followed by hCG (500 IU im) 7 days later; or to a placebo group (PLA; n = 8) where they received 1 mL saline solution im. All bitches that were induced in estrus were mated or AI with fresh semen. In Experiment 1, maximum observed concentration (Cmax) eCG were similar between im and iv routes (6.1 ± 0.9 vs. 8.6 ± 0.5 IU/mL, P > 0.08), whereas time for maximum observed concentration (Tmax.) was longer for im compared to iv routes (17.5 ± 0.5 vs. 11.6 ± 0.3 h, P < 0.01). The area under the curve (AUC) was similar for im and iv routes (P > 0.48), and eCG was detectable in serum for at least 144 h for both routes. In Experiment 2, 3 days or 3 to 5 days after treatment, all bitches in TRT50 had higher scores compared to TRT5-44 animals (P < 0.01). In TRT50, the mean interval from treatment to estrus was 4.0 ± 0.4 days. In Experiment 3, the mean interval from treatment to estrus was shorter in the TRT group compared to the PLA group (4.1 ± 3.3 vs. 68.5 ± 4.4 days, P < 0.01). The previous interestrus interval was similar for TRT and PLA groups (199.6 ± 7.2 vs. 197.5 ± 10.2 days), but the new interestrus interval was shorter for the TRT compared to the PLA group (164.0 ± 7.2 vs. 212.2 ± 10.2 days; treatment by interval interaction, P < 0.007). Serum P4 concentrations increased on the first day of cytologic diestrus after treatment in bitches in TRT (0.7 ± 0.3 vs. 22.8 ± 4.2 ng/mL; P < 0.01); but did not change in PLA (P > 0.84). Ninety-four percent of animals were bred (15/16; AI, n = 7; natural mating, n = 8), and 80% (12/15) became pregnant. None of the bitches had any side effects from the eCG and hCG therapy. We concluded that 50 IU/kg of eCG combined 7 days later with 500 IU of hCG was effective to induce normal and fertile estrus in bitches at 164 days post estrus, with an 80% pregnancy rate, with no side effects, and with a reduction of 48 days of the interestrus interval.

Comparison Between Vestibular and Subcutaneous Insertion of Deslorelin Implants for Oestrus Induction in Bitches

Investigations using sustained-release deslorelin implants at various insertion sites have shown that this method consistently induces oestrus in anoestus bitches. However, fertility comparisons between implant insertion sites have not been performed. Anestrous beagle bitches received one 2.1 mg deslorelin implant beneath the vestibular mucosa (VM group; n = 6) or in the subcutaneous tissue between the shoulder blades (SubQ group; n = 8). Vestibular implants were removed when serum progesterone concentrations first exceeded 1.5 ng/ml. Vaginal cytologies and blood samples were collected daily and bitches were inseminated during oestrus. Serum progesterone and deslorelin concentrations were measured and pregnancy status was determined using ultrasonography. There were no differences between groups in the intervals between implant administration and the onset of proestrus, the time of the luteinizing hormone surge and the onset of cytologic diestrus. There were also no differences in the number of corpora lutea or foetuses. However, conception rate was significantly lower in the SubQ group. The pregnancy rate did not differ significantly between the VM and SubQ groups [4 out of 6 (66.7%) and 3 out of 8 (37.5%), respectively]. One bitch (16.7%) in the VM group and three bitches (37.5%) in the SubQ group suffered distinct, premature declines in serum progesterone concentrations starting 1-4 weeks after cytologic diestrus. Serum progesterone concentrations did not recover (premature luteal failure), resulting in abortion. Bitches with premature luteal failure in the SubQ group still had serum deslorelin concentrations >100 pg/ml 20 days after implant insertion, suggesting a possible association between prolonged deslorelin release and luteal failure.

Factors affecting gestation duration in the bitch

A retrospective analysis was performed to determine the effects of age, breed, parity, and litter size on the duration of gestation in the bitch. Bitches at two locations were monitored from breeding to whelping. A total of 764 litters whelped from 308 bitches (36 large hounds, 34 Golden Retrievers, 23 German Shepherd Dogs (GSD), and 215 Labrador Retrievers). By breed, the number of whelpings was 152, 72, 58, and 482 for the hounds, Golden Retrievers, German Shepherd Dogs, and Labrador Retrievers, respectively. Whelping was predicted to be 57 d from the first day of cytologic diestrus in the hounds or 65 d from the initial progesterone rise in the other breeds. The average gestation duration (calculated as 8 d prior to Day 1 of cytologic diestrus in hounds or measured from the initial progesterone rise in other breeds) by breed (days ± S.D.) was 66.0 ± 2.8, 64.7 ± 1.5, 63.6 ± 2.1, and 62.9 ± 1.3 for the hounds, Golden Retrievers, German Shepherd Dogs, and Labrador Retrievers, respectively. The relationship of age, breed, parity, and litter size with the difference in gestation duration was evaluated using log linear modeling. Age or parity had no effect on gestation duration. Compared to Labrador Retrievers, the German Shepherd Dogs, Golden Retrievers and hounds were more likely to have a longer gestation duration; three, four and nearly eight times as likely, respectively. Bitches whelping four or fewer pups were significantly more likely to have a longer gestation duration than those whelping five or more pups; the prolongation averaging 1 d.

Failure of luteolysis leads to prolonged gestation in a bitch: A case report

A healthy, 5-yr-old rough collie bitch exhibited prolonged gestation, characterized by intrauterine fetal death and plasma progesterone concentrations (PPC) above 6 nmol/L/d until Day 65 or 66 of cytologic diestrus, which then only dropped following treatment with dinoprost tromethamine. The bitch showed no signs of parturition prior to laparotomy and hysterotomy on Day 63, which revealed 5 corpora lutea (CL) on the left ovary and 1 on the right ovary; 5 full-term, dead conceptuses without obvious autolysis but with 4 of the 5 placentas detached; and a tightly closed cervix. All conceptuses were in the left uterine horn. Treatment with dinoprost tromethamine was initiated 1 d after hysterotomy and was continued for IO d (120 μg/kg twice daily for a total dose of 240 μg kg/d). The discharge of lochia through the cervix and lactation started 3 d after the onset of treatment. Plasma progesterone concentration was 14.4, 16.2 and 1.6 nmol/L/d on the day of hysterotomy and at 1 and 3 d after the onset of prostaglandin therapy, respectively. The reason for the failure of luteolysis to occur is not known.

Luteolytic effects of prostaglandin F2α on day 8 to 19 corpora lutea in the bitch

Luteolysis was induced in 5 experimental Beagle (8 cycles) and 7 client-owned bitches treated with 150 to 200 μg/kg, sc of prostaglandin F2α administered twice daily for 4 d, starting on Days 8 to 19 after the onset of cytological diestrus. Five experimental Beagle bitches had been mated during the estrus preceding treatment, and copulation had been confirmed in 2 7 client-owned bitches presented for termination of unwanted pregnancy. Serum progesterone concentration (mean ± SD) declined from 26.1 ± 66 ng/ml before treatment to 0.3 ± 0.4 ng/ml on the fourth day of treatment One of the 7 client-owned bitches maintained her pregnancy even though serum progesterone concentrations were less than 0.5 ng/ml on the third and fourth day of treatment. Mean (± SEM) interestrous intervals before and following prostaglandin-induced luteolysis were 207.3 ± 12.4 (n = 11 cycles in 6 bitches) and 95.5 ± 20.0 d (n = 6 cycles in the same 6 bitches; P < 0.0001), respectively These results suggest that effective prostaglandin-induced luteolysis can be achieved with administration of 180 μg/kg during the third week of diestrus in pregnant and nonpregnant bitches.

Use of Prostaglandin F2α for Early Pregnancy Termination in the Mismated Bitch

Natural PGF administered at a dose of at least 250 micrograms/kg twice daily subcutaneously for at least 4 days starting no earlier than day 5 of cytologic diestrus induces luteolysis and pregnancy termination in the mated bitch. The resulting shortening of the luteal phase is associated with a shortening of the interestrous interval from 1 to 4 months. Bitches treated with PGF show emesis, diarrhea, and panting within 5 minutes and transient hypothermia which lasts 2 to 3 hours but generally have no further reaction. Bitches with cardiac or respiratory dysfunctions are not considered safe patients for early pregnancy termination with PGF because of the cardiovascular effects of this drug. Bitches treated with this regime early in diestrus resorb their conceptuses; those treated after days 35 to 40 show clinical abortion of viable fetuses.

Luteolysis in early diestrous beagle bitches

To induce luteolysis early in diestrus, 15 mated beagles were treated with 250μg/kg prostaglandin F 2α (PG), administered subcutaneously twice daily for 4 d, from Day 5 to Day 8 after the onset of cytological diestrus. They were divided into three groups of five bitches each: Group 1 received PG only. Group 2 received PG and 20 μg/kg/day bromocryptine orally. Group 3 received PG and 0.1mg/kg/day dexamethasone intramuscularly. Fifteen untreated beagles served as controls. In each of the three treatment groups, four of the five bitches (80%) underwent complete luteolysis and did not whelp. The three bitches which did not undergo luteolysis whelped normally. All control bitches whelped normally. It was concluded that luteolysis is possible in bitches early in diestrus and the inclusion of bromocryptine or dexamethasone did not appear to influence luteolysis.