Single, transcervical insemination using frozen-thawed semen in the Greyhound: a case series study

Transcervical insemination (TCI) has generated recent interest as an assisted reproductive technique in the dog. A case series study was performed to determine if TCI using frozen-thawed semen was a viable technique to offer in a general veterinary practice setting. Over a period exceeding 28 months, 137 Greyhound bitches were presented for assisted breeding. A single, timed insemination using a rigid cystoscope to aid in transcervical deposition of a frozen-thawed semen dose was given within 72 h after the behaviorally estrual bitch had a > 4 ng/mL serum progesterone concentration and estrus-categorized vaginal cytology. Litter size, pregnancy and whelping rate were collected; their association to semen center and stud dog were quantified. Of the 137 bitches, 117 were bred for one cycle and 20 were bred for two or more cycles, giving a total of 161 single, timed inseminations. Pregnancy rate was 89.4%, with 141 (87.5%) whelping. Litter size was 6.9+/-2.7 (mean+/-S.D.) pups. Semen center (P=0.84) and stud (P=0.79) had no effect on pregnancy. These results were quite favorable when compared to prior TCI studies, and are possibly due to the use of a single breed (i.e., Greyhound) with good fertility. This study supported the application of TCI, in Greyhounds, as a successful and viable service to offer in private practice. Additionally, these results have value in their use for benchmarking future breed-specific and TCI research. Serendipitously, the apparent fecundity results obtained in this observational study suggests a possible greater appreciation be given to breed composition and choice in assisted reproductive technique studies.     

The induction of parturition in the bitch using sodium cloprostenol

The objectives of this studies were to determine a continuous low-dose treatment regimen for the administration of sodium cloprostenol to the bitch that did not cause polydipsia, and whether this treatment would induce normal and timed parturition in bitches during late pregnancy. Non-pregnant greyhound bitches (n=18) received sodium cloprostenol subcutaneously, via a miniosmotic pump, at dose rates of 0.875 to 4.5 microg/kg/24 h, for 7 days (Days 0 to 7). Daily water intake was measured from Day -2 to Day 9. Polydipsia was observed in bitches treated with the higher dose rates but not in bitches treated with the lowest dose rate of 0.875 microg/kg/24 h. In the second experiment, pregnant greyhound bitches received sodium cloprostenol at dose rates of 1 (n=4), 2 (n=1) and 3 microg/kg/24 h (n=1), on Day 57 of pregnancy. Polydipsia was observed in bitches treated at the higher dose rates of 2 and 3 microg/kg/24 h, but not in the bitches treated at the lower dose rate of 1 microg/kg/24 h. These treatments resulted in the successful induction of parturition. Parturition was associated with a decrease in plasma progesterone concentrations, a reduction in body temperature, and an increase in plasma concentrations of 13,14-dihydro-15-keto prostaglandin F2alpha. The first puppy was born 37.7 +/- 2.9 h after the start of treatment (range 28 to 46 h). The duration of whelping was approximately 15.7 +/- 2.2 h (range 10 to 24 h). The litter size was 9.2 +/- 0.8 pups (range 6 to 12 pups), and the puppy survival rate was 6.0 +/- 0.8 per litter (range 4 to 9 pups). This study demonstrated that the administration of sodium cloprostenol in continuous low dose for 24 h is an effective treatment for the induction of parturition in bitches during late pregnancy. This treatment resulted in the birth of healthy pups, with minimal or no side effects to the bitch.     

Artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach

From 1994 to 2003, a total of 526 bitches of 99 different breeds were artificially inseminated in 685 estrus cycles with domestic (n = 353) or imported (n = 332) frozen-thawed semen from 368 males. The overall whelping rate was 73.1% and mean (+/- S.E.M.) litter size 5.7 +/- 0.1 pups. The whelping rate was higher after intrauterine insemination (75.0%; n = 665) than after intravaginal insemination (10.0%, n = 20; P < 0.05). Insemination at the optimal time resulted in a higher whelping rate (78%, n = 559; P < 0.01) and larger litter size (5.8 +/- 0.2; P < 0.05) than inseminations performed late or too late (55.7% and 4.5 +/- 0.5, n = 61). Two inseminations (n = 384) yielded a higher whelping rate (P < 0.05) and mean litter size (P < 0.01) than one insemination (n = 241), 78.1% and 6.0 +/- 0.2 and 70.5% and 5.1 +/- 0.2, respectively. For inseminations performed at the optimal time, however, the whelping rate was not significantly different for bitches inseminated twice (79.3%, n = 358) versus once (76.8%, n = 168), but the litter size was larger (6.0 +/- 0.2 and 5.3 +/- 0.3). Semen classified as of poor quality (progressive motility < 50% or percentage abnormal sperm > 20%) resulted in a lower whelping rate (P < 0.01) than semen classified as of good quality (progressive motility > or = 50% and percentage abnormal sperm < or = 20%), 61 and 77%, respectively. Small breeds (n = 50) had a smaller litter size (3.9 +/- 0.3; P < 0.01) than larger breeds (medium [5.7 +/- 0.3, n = 94], large [5.9 +/- 0.2, n = 295] or giant breeds [6.1 +/- 0.5, n = 62] [P < 0.01]). Bitches older than 6 years had a lower whelping rate (68.2%) than younger ones (77.0%; P < 0.05). The duration of pregnancy was longer (P < 0.01) for bitches with a litter size of < 3 pups (61.7 +/- 0. 4 days, n = 30) than for bitches with larger litters (60.5 +/- 0.1 days, n = 177). These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs. The results with frozen semen inseminations were optimised by inseminating bitches < or = 6 years old 2 and 3 days after ovulation with semen of good quality from males < or = 8 years old.     

Termination of pregnancy and induction of premature luteolysis by the antiprogestagen, mifepristone, in dogs

Five pregnant beagle bitches were treated with 2.5 mg mifepristone/kg body weight, twice a day, for 4.5 days starting at Day 32 of gestation. Results of fetal ultrasonography and assay of serum progesterone concentrations every 2-4 days were compared to those in 5 control bitches. Mifepristone resulted in a premature (P less than 0.01) termination of pregnancy (36 +/- 1 vs 65 +/- 1 days), without side effects. The antiprogestagen also caused progesterone to decline to less than 1 ng/ml by Day 40-45 after the preovulatory LH peak (vs 64-67 days in controls) and reduced (P less than 0.05) mean concentrations on Days 34-50 (2.2 +/- 0.5 vs 6.3 +/- 0.3 ng/ml). The results suggest that antiprogestagen therapy is a safe means to terminate unwanted pregnancy in dogs, and that luteal function in pregnant bitches is dependent on luteotrophic support that is blocked by antiprogestagen treatment, directly or indirectly, due to termination of pregnancy.     

The use of ally-trenbolone as a progestational agent to maintain pregnancy in ovariectomized bitches

The administration of the synthetic progestogen, allytrenbolone, at a dose of 0.088 mg/kg/d per os successfully maintained pregnancy in 3 of 3 bitches ovariectomized at 34 to 42 d of gestation and in 1 of 3 ovariectomized on Day 8 or 9 of gestation. However, a dose of 0.044 mg/kg/d per os maintained pregnancy in only 2 of 6 bitches ovariectomized in mid-gestation. Two bitches that had ovariectomies performed on Day 9 of gestation and were supplemented with ally-trenbolone at a dose of 0.088 mg/kg/d per os did not establish a pregnancy that was detectable by mid-gestation. Although inhibited the first 2 d post partum in some bitches, lactation increased sufficiently to successfully maintain pups.     

Effect of cyproterone acetate,levonorgestrel and progesterone on adrenal glands and reproductive organs in the beagle bitch

Summary The effects of short-term (8 weeks) treatment with different doses of cyproterone acetate (CPA), levonorgestrel (LN) and progesterone (PRO) on the adrenal gland, ovary, uterus and vagina were studied in cycle-synchronised beagle bitches (first anoestrus). The same organs from non-treated primiparous beagle bitches at the 6th and 9th weeks of pregnancy were also included. In the animals treated with the highest doses of CPA (4.0 mg/kg/day orally) and PRO (42.5mg/kg/day subcutaneously), as well as in pregnant bitches (9th week of pregnancy), a decrease in adrenal weight and cortex width and also an apparent loss of cells in the zona fasciculata and zona reticularis were observed. A marked increase in ovarian weight was recorded only in pregnant bitches (6th week of pregnancy). This was reflected by the presence of multiple well-developed corpora lutea. The ovaries of virgin control and progestagen-treated bitches revealed ovarian atrophy. Progestagen treatment caused marked stimulation of the uterus, resulting in dose-related oedematous and hyperplastic changes. Comparable findings were also observed during pregnancy. The vaginal epithelium of the progestagen-treated and pregnant bitches showed marked mucification as compared with control bitches. These structural responses indicate that progestagen treatment stimulates a pseudopregnancy-like condition in the adrenal glands, uterus and vagina of the beagle bitch.Abbreviations for Hormones cited in this Paper ACTH Adrenocorticotropin - CRH Corticotropin Releasing Hormone - FSH Follicle Stimulating Hormone Beta Subunit - LH Luteinizing Hormone Beta Subunit - MSH Melanocyte Stimulating Hormone The author is grateful to Dr. Christel Schöbel and Mrs. P. Kurth for carrying out the experimental work on the animals, to Mrs. B. Schilk and Mrs. U. Tüshaus-Bußmann for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Decrease of body temperature after aglepristone treatment in bitches

Body temperature responses and the timing of abortions were evaluated in pregnant bitches with the anti-progestin aglepristone. Fifteen purebred and crossbred, 25-45 days pregnant, were included in this study and seven untreated bitches at the same stage of pregnancy served as controls. Treated bitches were administered two applications of aglepristone (10 mg/kg SC) 24 h apart for pregnancy termination. Pregnancy termination was confirmed by ultrasonographic assessment. Body temperature was rectally measured three times a day for 6 days beginning 24 h before treatment or pregnancy diagnosis in the treated and control bitches, respectively. Additionally, serum progesterone concentrations were assessed at time points during the study in the treated bitches. Pregnancy was terminated in 14 treated bitches in a mean+/-S.E.M. of 4.3+/-0.7 days after treatment. Control bitches remained pregnant. In the treated bitches, but not in the controls, body temperature significantly decreased 24 h after the beginning of the treatments (P < 0.01) and then gradually returned to pre-treatment values. Correlation between the day of mean minimum body temperature and the day of pregnancy termination was low (0.07; > 0.05). Progesterone did not show significant change throughout the study. Body temperature does not seem to be a suitable variable to clinically monitor the aborting effect of aglepristone. Decrease of body temperature after aglepristone treatment could represent further evidence of its hypothalamic effects.     

Ovulation induction in anestrous bitches by pulsatile administration of gonadotropin-releasing hormone

Preliminary studies in anestrous Beagle bitches demonstrated that a single injection of gonadotropin-releasing hormone (150 micrograms) produced a rapid, physiological rise in serum estradiol lasting 1-3 days while progesterone remained below 1 ng/ml, whereas serial injections of FSH rapidly produced greater elevations in estradiol and a rapid rise in progesterone over 2 ng/ml. Consequently, attempts to induce fertile ovulation by means of pulsatile intravenous administration of GnRH (1 pulse/1.5 hours for 6-12 days; 0.04-0.43 micrograms/kg body weight/pulse) were conducted in eight anestrous bitches. Willingness to mate, serum progesterone levels and results of mating were monitored. In six of the eight bitches, vulval and vaginal signs of proestrus occurred by Day 2-4 after initiation of treatment (Day 0); but, two bitches showed negligible responses. In five of the six bitches in which proestrus was induced, behavioral (n = 4) and vaginal (n = 5) correlates of early estrus occurred by Day 5-7 of treatment and breedings occurred over a period of 4-12 days. Following onset of estrus, four of the five bitches had increases in serum progesterone levels between Days 14 and 18 after initiation of treatment (and 4-11 days after cessation of treatment); three of them became pregnant and whelped normal litters (ranging from 9 to 11 pups). The fifth bitch did not have elevated progesterone during the induced estrus, and upon return to estrus one month later was successfully bred and whelped a normal litter of 10 pups.(ABSTRACT TRUNCATED AT 250 WORDS)     

Induction of fertile estrus in bitches using a sustained-release formulation of a GnRH agonist (leuprolide acetate)

A single subcutaneous injection of a sustained-release formulation of a potent GnRH agonist, leuprolide acetate (LA; [D-Leu6, Pro9NEt]-GnRH), was evaluated as a method of inducing fertile estrus in 12 mature anestrous and 6 prepubertal beagle bitches. The bitches were treated with microencapsulated LA (100 micrograms/kg, s.c.) at 120 or 150 d post partum, or at 1 yr of age, followed by a GnRH-analogue (fertirelin; [Pro9NEt]-GnRH, 3 micrograms/kg, i.m.) on the first day of induced estrus. Signs of estrus were seen within 10.3 +/- 0.9 d after LA administration in all bitches. The interestrous interval in 120- and 150-d post-partum bitches was shortened (P < 0.05) to 191 +/- 3 and 222 +/- 3 d, respectively, compared with 264 +/- 11 d in control bitches. All LA treated dogs demonstrated behavioral estrus and mated. Three of 6 (50%) at 120 d post partum, 6 of 6 (100%) at 150 d post partum and 5 of 6 (83%) of prepubertal (1-yr old) bitches then became pregnant and produced a mean litter size of 4.1 +/- 0.8 pups. A normal circulating estrogen and progesterone response pattern was observed in mature anestrous bitches. A prepubertal bitch that failed to become pregnant had a similar estrogen response pattern but an insufficient progesterone profile. The results suggest that microencapsulated LA can be useful in inducing fertile estrus in the domestic dogs.     

Termination of pregnancy in dogs by oral administration of dexamethasone

Dexamethasone was administered orally for 7.5 or 10 d to each of 20 pregnant bitches beginning at an estimated 28 to 51 d of gestation, using 1 of 2 dose regimens. Five bitches were given dexamethasone 3 times a day for 10 d, with the highest dose of 0.2 mg/kg for 5 d and then at progressively decreasing doses of 0.16-0.02 mg/kg for 5 d. The 15 remaining bitches were given dexamethasone 2 times a day for 7.5 d, increasing from 0.1 to 0.2 mg/kg over the first 3 administrations, then remaining at 0.2 mg/kg on Days 2 to 5, and decreasing from 0.16 to 0.02 mg/kg over the last 5 administrations. The side effects, including mild polydipsia and polyuria, disappeared when treatment was discontinued. Depending on the stage of pregnancy, uterine contents were either resorbed or aborted, or both. Pregnancy was terminated within 2 to 16 d after the start of treatment in all treated bitches, at 2 to 5 d of treatment in 2 of 3 bitches treated at 40 to 51 d of pregnancy, and at 0 to 4 d after the end of treatment in most of the 17 bitches treated at 28 to 35 days of pregnancy. Oral administration of dexamethasone appears to be a potentially useful pharmacologic treatment for the termination of unwanted pregnancy in the bitch.     

Fertile estrus induced in bitches by bromocryptine, a dopamine agonist: a clinical trial

The dopamine agonist bromocryptine, probably through amplifying gonadotroph (mainly FSH) secretion, was found to be suitable for provoking fertile estrus during the anestrous phase in bitches without functional cycles and/or ovarian activity. We studied estrus induction in 48 bitches after treatment with semisynthetic ergot alkaloid bromocryptine. For habituation a fractional dose of 0.3 mg/bitch was administered for three days followed by larger doses within the range of 0.6 to 2.5 mg/bitch by selecting dose rates on the basis of individual responsiveness and body weight. The long-term daily bromocryptine dose did not exceed 0.6 mg/bitch and 2.5 mg/bitch in small and large sized bitches, respectively. Gradual habituation and individual dose rates have almost completely eliminated the unwanted side effect of emesis. The period between treatment and onset of estrus varied but the average was 19 days. After the onset of estrus bromocryptine administration was usually continued for another 3 to 6 days. Occurrences of estrus, ovulation and pregnancy were monitored by cytological evaluation of vaginal epithelium, rapid ELISA for plasma progesterone and ultrasonography, respectively. Samples for progesterone were taken on Days 7, 9, 12 and 15 and sonograms of ovarian follicles and of fetuses were taken on Days 0, 22 and 35. The bitches involved in the study either regular or irregular cycles. Bromocryptine treatment induced estrus in all of the bitches including 40 of 48 (83%) with ovulation within a regular estrus and 6 of 48 (12.5%) that showed estrus but did not ovulate. Mating or artificial insemination of bitches in their fertile periods twice at two day intervals resulted in an 83% pregnancy rate (40 cases) and 39 (97.5%) of them gave birth to puppies. However, the average litter size was small with 4.8 +/- 1.6 pups.     

Effect of cyproterone acetate,d-norgestrel and progesterone on cells of the pars distalis of the adenohypophysis in the beagle bitch

Summary The effects of short-term (8 weeks) treatment with different doses of cyproterone acetate (CPA), d-norgestrel (d-N) and progesterone on cells of the pars distalis, as revealed by the immunoperoxidase technique, were studied in cycle-synchronized beagle bitches (first anoestrus). Pituitary glands from non-treated primiparous beagle bitches at the 6th and 9th week of pregnancy were also included. For immunochemical staining specific antisera to the following hormones were used: canine GH, canine PRL, porcine ACTH, bovine TSH, bovine LH and human FSH. Morphological features of high secretory activity in GH cells were evident even after the human oral contraceptive doses of CPA and d-N, and after a dose as low as 0.1 mg/kg/day subcutaneously (s.c.) of progesterone. In contrast, PRL cells did not show any significant treatment-related effects except in those animals which received the highest dose of d-N (0.5 mg/kg/day per os). In this group, as well as in all pregnant bitches, hyperplasia and hypertrophy of PRL cells were found. In the animals treated with the highest doses of CPA (4.0 mg/kg/day per os) and progesterone (42.5 mg/kg/day s.c.) as well as in pregnant bitches, ACTH/MSH and TSH cells showed marked atrophy and regressive changes. Similar morphological signs of depressed secretory activity were also observed in the cells shown to contain FSH and/or LH as a result of treatment with the highest dose of progesterone and at the 9th week of pregnancy. These structural responses indicate that quantitative and/or qualitative differences may exist between progesterone, the synthetic progesterone derivative CPA and the nortestosterone type progestagen d-N with regard to their effect on pituitary hormone secretion in the beagle bitch.Abbreviations ACTH Adrenocorticotropin - FSH Follicle Stimulating Hormone - GH Growth Hormone - LH Luteinizing Hormone - MSH Melanocyte Stimulating Hormone - PRL Prolactin - TSH Thyrotropin - CRH Corticotropin Releasing Hormone - TRH Thyrotropin Releasing Hormone The authors are grateful to Dr. Christel Schöbel and Mrs. P. Kurth for carrying out the experimental work on animals, to Mrs. B. Schilk and Miss U. Tüshaus for their excellent technical assistance, and to Dr. P. Günzel for his advice and encouragement     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

Hormonal changes in spontaneous and aglépristone-induced parturition in dogs

To increase our understanding of the endocrine changes associated with parturition in dogs, plasma concentrations of progesterone (P4), 15-ketodihydroprostaglandin F(2alpha) (PGFM), estradiol-17-beta (E2beta), cortisol, ACTH, prolactin (PRL), LH, and FSH were measured in six spontaneously whelping bitches and in six bitches in which parturition was induced with the progesterone-receptor blocker aglépristone on day 58 of pregnancy. Expulsion of pups in the induced group took place in the presence of P4 concentrations that were still elevated. PGFM concentrations increased before parturition in both groups, but levels were lower in the induced bitches. PGFM levels reached a maximum in both groups during parturition and quickly decreased in the spontaneously whelping group after parturition, but remained elevated in the induced group. In both groups, cortisol concentrations reached similar maximum levels during the last 30 h before the onset of expulsion. During the 3 days postpartum, cortisol concentrations were higher in the induced group. The highly variable ACTH concentrations did not differ significantly throughout the study within or between groups. In both groups, E2beta concentrations decreased and PRL concentrations increased between the late gestational period and the 30-h period before parturition. Concentrations of both LH (spontaneously whelping group) and FSH (both groups) decreased between late gestation and the postpartum period. The results of this study illustrate the hormonal changes around parturition in the bitch, and reveal that aglépristone-induced parturition is associated with still incomplete luteolysis, an altered PGFM profile, and elevated postpartum cortisol concentrations as compared with spontaneously whelping dogs.     

Suppression of luteal function in dogs by luteinizing hormone antiserum and by bromocriptine

Beagle bitches were treated with equine anti-LH serum (ALHS) or the dopamine agonist bromocriptine at selected times during the 2-month luteal phase of the ovarian cycle or pregnancy. After a single injection of ALHS (10 ml, i.m.) at Day 42 of pregnancy (N = 2) or the ovarian cycle (N = 3), progesterone was reduced (P less than 0.05) to 7-24% of preinjection values within 1-2 days, and by 4-8 days returned to levels not different from those in control bitches treated with normal horse serum. Injections of bromocriptine (0.1 mg/kg, i.m.) daily for 6 days caused abrupt declines in progesterone which lasted 6-8 days in bitches treated at Day 8 or 22 of pregnancy (N = 5). In bitches treated at Day 42 of pregnancy (N = 3) or in non-pregnant cycles (N = 4) the bromocriptine treatment caused declines (P less than 0.05) in progesterone which were permanent, extensive (less than 2 ng/ml), and therefore abortive. The declines in progesterone in response to immunoneutralization of LH and to prolactin-lowering doses of a dopamine agonist demonstrate that normal luteal function in dogs requires both LH and prolactin.     

Conceptus ecobiometry and triplex Doppler ultrasonography of uterine and umbilical arteries for assessment of fetal viability in dogs

The objectives were to: 1) evaluate blood flow in the uterine (UA) and umbilical (Uma) arteries in the pregnant bitch, by measuring the resistive index (RI) and pulsatility index (PI); 2) to note the presence or absence of the early diastolic notch and diastolic flow in the UA and Uma flow waveforms, respectively; and 3) perform conceptus ecobiometry for fetal growth assessment during pregnancy. Six healthy bitches were examined on approximately Days -44, -42, -36, -31, -28, -25, -21, -18, -14, -8, -4, and -2 of pregnancy (whelping = Day 0). Triplex Doppler and B-mode ultrasonography were used to assess blood flow and conceptus ecobiometry. All pregnancies ended with a normal whelping and birth of live puppies. Prior to whelping, all conceptus dimensions increased significantly, whereas RI and PI of both the Uma and UA decreased significantly. For the UA, RI and PI were (mean ± SEM) 0.95 ± 0.02 and 2.75 ± 0.41, respectively, on Day -44, and were 0.60 ± 0.01 and 0.99 ± 0.03 on Day -4. For the Uma, RI and PI were 0.99 ± 0.01 and 2.42 ± 0.03 on Day -31, and were 0.62 ± 0.01 and 1.15 ± 0.02 on Day -4. The complete disappearance of the early diastolic notch in the UA, and the appearance of diastolic flow in the Uma occurred on Days -16 ± 5 and -21 ± 1. The authors concluded that UA and Uma perfusion were important end points to assess fetal vitality in bitches. Furthermore, the current reference values provided a baseline for monitoring normal and abnormal pregnancies in bitches.     

Intravaginal insemination of bitches with fresh and frozen-thawed semen with addition of prostatic fluid: use of an infusion pipette and the Osiris catheter

One hundred fifty-two bitches of seven breeds were vaginally inseminated with fresh or frozen-thawed semen of 10 stud dogs of respective breeds. The semen was supplemented with prostatic fluid before insemination. In experiment 1 bitches of each breed were randomly assigned to three treatment groups, consisting of 29 females (group 1), 33 females (group 2) and 32 females (group 3). In group 1 bitches were inseminated into vagina with fresh semen using a bovine infusion pipette. In group 2 bitches were inseminated into vagina with fresh semen using the Osiris catheter. In group 3 bitches were inseminated with frozen-thawed semen with the Osiris catheter. The number of sperms in each insemination dose was adjusted to 300 x 10(6). In experiment two bitches were randomly assigned to two treatment groups, consisting of 30 females (group A) and 28 females (group B). In group A bitches were inseminated with fresh semen, whereas in group B with frozen-thawed semen. Osiris catheter was used in both groups. The total number of sperms was adjusted to provide 250 x 10(6) of progressively motile spermatozoa in each insemination dose. In experiment 1 the pregnancy rates/whelping rates were 86.2/82.8%, 81.8/81.8% and 59.4/59.4% for groups 1, 2 and 3, respectively. The differences between group 1 and 3 were statistically significant (p < 0.05). The litter sizes at birth/litter sizes at weaning were 5.8+/-2.3/5.4+/-2.0, 6.3+/-1.4/5.7+/-1.0 and 3.9+/-1.2/3.5+/-1.5 in groups 1, 2 and 3, respectively. The litter size at birth and at weaning was reduced (p < 0.05) when frozen-thawed semen was used for insemination (group 3). There were not significant (p > 0.05) differences in the litter size between groups 1 and 2. In experiment 2 pregnancy rates/whelping rates and litter sizes at birth/litter sizes at weaning were 86.7/86.7%, 60.7/57.1% (p < 0.05) and 6.1+/-1.6/5.7+/-1.7, 4.0+/-1.4/3.8+/-1.4 (p < 0.05) in groups A and B, respectively. This study shows that results of AI with a fresh semen using a bovine infusion pipette and the Osiris catheter are equivalent. The results of the use of the Osiris catheter for vaginal insemination of frozen-thawed dog semen extended with prostatic fluid after thawing are not encouraging. The pregnancy rate, whelping rate and litter size are reduced when frozen-thawed, prostatic fluid-supplemented semen is vaginally deposited using the Osiris catheter.     

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 (C_max) 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 (T_max.) 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.     

Intra-vesicle administration of D-cloprostenol for induction of abortion in mid-gestation bitches

This study evaluated a new method for mid-gestation termination in the bitch, which consisted of ultrasound-guided administration of a single dose of d-cloprostenol, a PGF(2alpha) analogue, into a single gestational sac. Effects on serum progesterone concentration (P(4)) were also investigated. The study was performed between days 28 and 35 of gestation on 15 privately owned crossbred bitches, randomly divided into two groups: group A comprised 10 bitches treated with 15 microg per head d-cloprostenol diluted in 0.8 ml sterile saline (final volume 1 ml); group B comprised 5 bitches treated with 1 ml of sterile saline solution (0.9% NaCl), administered in the same way. In all bitches of group A, fetal death was successfully induced within 5 days (mean: 3.1 days, S.D. 1.2) with no clinical or behavioural complications. Mild adverse effects were observed in two bitches, each weighing less than 10 kg, including salivation, defecation and hyperventilation, which disappeared within 15 min. None of the subjects in group B aborted within 10 days post-treatment. In group A, P(4) declined 2.8 days before pregnancy termination to a mean value below 30 nmol/l (S.D. 2.9 nmol/l). However, two bitches showed a higher concentration of P(4) throughout the sampling period. Our study demonstrates that intra-vesicle administration of a single low dose of D-cloprostenol is an effective and safe technique for induction of abortion, which offers an additional option for termination of unwanted pregnancy in the mid-gestation bitch.     

Luteal function in the hysterectomized bitch following treatment with prostaglandin F2α (PGF2α)

Estrus and ovulation were induced in ten mature, mixed-breed, anestrous bitches (10 to 20 kg) using exogenous gonadotropins. Bitches were bred once, on the second day of estrus. Between 11 and 13 days following estrus, bitches were bilaterally hysterectomized and randomly divided into two treatment groups of five bitches each. Four days following surgery, Group A (treated) was given a single subcutaneous injection of PGF₂α (Prostin F₂ alpha^®) at a dose of 1 mg/kg body weight and Group B (controls) similarly given an equal volume of .9% saline. Blood samples were collected daily by cephalic venipuncture prior to surgery and for 75 days thereafter. Plasma progesterone was monitored by a radioimmunoassay method. Although bitches were teased daily following PGF₂α or saline treatments, estrual behavior was not exhibited. In both the PGF₂α and saline treatment groups, plasma progesterone levels showed a transient decline by 12 hours following injection, although a more dramatic decrease was observed at this time in the prostaglandin-treated bitches. Subsequently, progesterone concentrations tended to increase in both groups at 6 days following treatment, however, not to pre-treatment levels. Within 20 to 32 days following treatment in both groups, plasma progesterone levels declined to <1 ng/ml and remained depressed at least 60 days post-injection. In this study, complete luteal regression was not induced following PGF₂α treatment. Luteal function in both groups, as indicated by plasma progesterone concentrations, was shortened in the absence of the uterus.