Endoscopic transcervical intrauterine artificial insemination in Labrador Retriever bitches

Besides post-thawing reduced semen quality, there are some difficulties in the execution of the endoscopic transcervical intrauterine artificial insemination (AI) with frozen-thawed semen in bitches. Therefore, the aims of the present study were to evaluate behavioral and reproductive parameters (i.e., vaginal cytology and serum progesterone level) to determine time of insemination and to investigate the particularities and difficulties of this technique in bitches using fresh semen. Ten Labrador Retriever bitches were submitted to three endoscopic transcervical intrauterine AIs (with 48 h intervals). Oestrus and ovulation period were established by behaviour evaluation, progesterone assays and vaginal cytology, enabling optimal timing for AI during oestrus. During AI, the following aspects were evaluated: cervical os catheterization difficulty, semen deposition resistance, occurrence of semen backflow, and time required to perform the AI. In this study, it was possible to catheterize the cervical os in all bitches, with different degrees of difficulty, by manipulating the equipment to allow cervical visualization and catheter introduction in the cervical canal. Serial serum progesterone assays enabled estimation of LH surge day, and thus of ovulation. The pregnancy rate was 90%, with a litter size of 5.0 ± 2.6 puppies per bitch. It was concluded that the difficulties in the execution of the endoscopic transcervical intrauterine AI technique in Labrador Retriever bitches were minimized by the equipment manipulation and practical experience.     

Exfoliate vaginal cytology and plasma concentrations of progesterone, luteinising hormone and oestradiol-17sZ during oestrus in the bitch

Oestrus was characterised in nine German shepherd bitches by measuring plasma levels of luteinising hormone (LH), progesterone (P) and oestradiol-17ß (O). Hormone concentration data were compared with the eosinophilic index (EI) obtained from exfoliate vaginal cytology. Maximal values of O and LH occurred before the maximal EI value. The EI peak followed the O peak, with a two day delay. LH increased during the first 24 hours after the maximum 0 value, and P levels were then slightly higher than basal.     

Vaginal cytology and cervicovaginal mucus arborisation in the breeding management of bitches

Fifty bitches were mated on the basis of results of vaginal cytological and cervicovaginal mucus arborisation examination. When these were compared with a control group of 50 bitches mated on days 10 and 12 after the onset of proestrus there was a significant increase in pregnancy rate from 78 to 92 per cent. The litter size and bitch productivity was also increased, although this was not statistically significant. Forty-two per cent of bitches had two peaks of anuclear epithelial cells in the vaginal smear, separated by a mean of 3–6 ± 1–5 (SD) days. For these bitches, the maximum percentage of anuclear cells at the second peak was between 57 and 98 per cent, and the mean interval between the calculated day of the surge in plasma luteinising hormone (LH) and the second peak of anuclear cells was 4-4 ± 2-0 days. In bitches with one peak of anuclear epithelial cells, the maximum percentage of anuclear cells was between 43 and 94 per cent and the mean interval between the calculated day of the LH surge and the peak of anuclear cells was 1–9 ± 2-1 days. Arborisation of mucus collected from the anterior vagina was noted in all bitches; the mean interval between the calculated day of the LH surge and the peak of ferning was 2–3 ± 2-1 days. The collection of exfoliative cells by aspiration from the cranial vagina and the assessment of the anuclear cell index, may allow the identification of the fertile period after the surge of plasma LH. The anuclear cell index offers advantages over other methods of classification of exfoliative vaginal cells. When used in combination with examination of arborisation of cervicovaginal mucus this method can improve the breeding management of bitches.     

Comparison of endocrine changes and ultrasound as means of identifying ovulation in the bitch.

Twenty bitches were monitored through pro-oestrus and oestrus using both circulating plasma hormone levels and ultrasonic examination of the ovaries. Using luteinising hormone (LH) as being the generally accepted optimum indicator of the day of ovulation, comparisons were made of the accuracy of progesterone and ultrasound to identify ovulation. Progesterone agreed with LH in 12 of 20 bitches and was within one day in seven of the other eight. Ultrasound was less accurate in that only four of the 16 estimates agreed, with a further six being within one day. However, if only the bitches which were examined by ultrasound with the latest equipment were included, while only three of 11 coincided, six of the remaining eight were within one day of the LH estimated ovulation. It is concluded that, at present, of the rapid assessments, the measurement of plasma progesterone is a better estimator of ovulation than ultrasound.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Application of vaginal cytology and plasma Srogesterone eterminations to the management of reproduction in the bitch

The use of vaginal cytology and plasma progesterone determinations in the management of 11 bitches presented to our small animal fertility clinic are described. Nine bitches were mated or artificially inseminated and seven became pregnant. Reproductive patterns detected included failure of ovulation, prolonged pro-oestrus with late ovulation, short pro-oestrus/oestrus with early ovulation, short pro-oestrus/oestrus with late ovulation and normal pro-oestrus/oestrus with late ovulation.     

Plasma LH and progesterone levels before and after ovulation and observation of ovarian follicles by ultrasonographic diagnosis system in dogs

Recently, canine frozen semen has been attracting attention for breeding purposes, and methods of judging ovulation and optimum timing for insemination have become important. As methods of predicting the canine ovulation, vaginal smear, plasma sex hormone levels and ultrasonographic diagnosis system (US) have been investigated in combination, but a standard technique has not yet been established. Therefore, we investigated a method of predicting canine ovulation in dogs by US, and by measuring plasma LH and progesterone (P) levels three times a day. Ovulation could be observed by detecting irregularly shaped ovarian follicles by US in six of 11 dogs (54.5%). In these dogs, the time between the LH peak and ovulation was 24-48 hr, 38.0 hr on average. The P level on the ovulation day was 1.88-2.81 ng/ml, 2.34 ng/ml on average. A value of 1.88 ng/ml was detected in one dog, but the other five dogs showed P levels of 2 ng/ml or higher. The P level on the day before ovulation was 0.8-1.56 ng/ml, 1.12 ng/ml on average. Assuming that ovulation occurred two days after the LH peak in the 11 experimental dogs, the P level was 2.12-4.06 ng/ml, 2.78 ng/ml on average. The period of a high LH level, not less than 10 ng/ml, continued for 12 hr around the LH peak. Based on these findings, to predict ovulation using US and LH level, it would be necessary for the tests to be performed several times a day. In contrast, it was shown that the day on which a plasma P level of 2 ng/ml or higher was detected by the test performed once a day corresponded to the ovulation day.     

Endotoxin induces delayed ovulation following endocrine aberration during the proestrous phase in Holstein heifers

The effect of endotoxin on follicular growth and on secretion of LH, estradiol-17β, progesterone and cortisol during the proestrous phase in cattle was investigated. Holstein heifers were treated with PGF₂ at 11–13 d after ovulation to induce luteolysis. At 42 hr after PGF₂ treatment, heifers were administered either lipopolysaccharide (LPS; Escherichia coli, O111:B4, 5 μg/kg, n = 6) or saline (control; n = 6) by i.v. bolus injection. Ovarian structures were monitored daily by transrectal ultrasonography, and blood samples were collected at various times for hormonal analysis. The duration from PGF₂ treatment to ovulation was significantly longer in the LPS group (8.0 ± 1.3 d) than in the control group (4.2 ± 0.2 d). LPS significantly reduced the pulse frequency of LH for 6 hr after the administration, and increased the mean concentration and pulse amplitude of LH from 3 to 6 hr after the administration. The plasma concentrations of progesterone and cortisol were transiently increased after LPS administration. The plasma concentration of estradiol-17β was significantly decreased at 24 hr after LPS administration compared to that in the controls. Five of six LPS-treated heifers exhibited no preovulatory LH surge until 120 hr after PGF₂ treatment and the remaining heifer exhibited the surge at 108 hr after PGF₂ treatment, while the LH surge was observed at 54–78 hr after PGF₂ treatment in control heifers. These results suggest that endotoxin disrupts progression of the proestrous phase of cattle, interrupting the preovulatory estradiol rise and thus delaying the LH surge and the subsequent ovulation.     

Shrinkage in the horizontal dimensions of the vulva (vulvar shrinkage) as an indicator of standing heat in the beagle

During the proestrous and estrous periods in 12 beagles, the following parameters were measured daily: the horizontal dimensions of the vulva, vaginal cytology, and serum luteinizing hormone (LH) and progesterone concentrations. Measurements of serum LH concentrations allowed for the identification of the LH surge and the optimal time for artificial insemination (AI). Nine out of the 12 beagles became pregnant through AI and completed a gestation. Shrinkage in the horizontal dimensions of the vulva (i.e., vulvar shrinkage) was primarily observed prior and subsequent to the LH surge. In six of the nine (66.7%) beagles that became pregnant, vulvar shrinkage was observed on the days in which the LH surge was confirmed, and the rate of vulvar shrinkage tended to be greater at higher serum LH concentrations. Further vulvar shrinkage was identified in all nine beagles within two days of the LH surge. An increase in the serum progesterone concentration was observed after the LH surge in each of the beagles that became pregnant, together with clinical signs of estrous behavior (i.e., standing heat) as well as a change in vulva condition from swollen to soft. This demonstrates that vulvar shrinkage is induced in response to the onset of the LH surge and that the LH surge can be predicted through the measurement of the horizontal dimensions of the vulva, vaginal cytology, and the assessment of serum progesterone concentrations in beagles.     

Optimal timing for canine artificial insemination with frozen semen and parentage testing by microsatellite markers in superfecundency

Parentage testing was performed in sixteen litters by canine artificial inseminations with frozen semen from different sires on Days 5 and 7 after the LH surge. It became apparent that only 25% of dams had superfecundation, but 43.8% of dams were whelped after insemination only on Day 5 after the LH surge and 31.3% of dams after insemination only on Day 7. Of the total 87 puppies, 46% were born after insemination on Day 5 after the LH surge and 54% after insemination on Day 7. This result strongly suggested that canine artificial insemination with frozen semen could be sufficiently successful also on Days 5 and 7 after the LH surge.     

Plasma concentrations of immunoreactive (ir)-inhibin, gonadotropins and steroid hormones during the ovulatory cycle of the duck

The present study was undertaken to determine changes in circulating levels of immunoreactive (ir)-inhibin, FSH, LH, estradiol-17beta, progesterone, and testosterone during the ovulatory cycle of Shao ducks. Serial blood samples were taken from two groups of laying ducks for measurement of ir-inhibin, gonadotropins, and steroid hormones at 2 h intervals for 24 h. Plasma concentrations of ir-inhibin did not change significantly during the ovulatory cycle. The highest level of plasma ir-inhibin was observed 6 h prior to ovulation, which coincided with a decreased level of plasma FSH. One FSH surge was found 12 h after ovulation. Estradiol-17beta, progesterone, and testosterone were also determined during the ovulatory cycle. Two peak values were detected for estradiol-17beta 8 h before ovulation and 4 h after ovulation, while progesterone started to increase 4 h before ovulation and reached a peak at ovulation. The highest level of plasma testosterone was detected around the time of ovulation. These results suggest that inhibin may be involved in the control of FSH secretion during the ovulatory cycle. In addition, both LH and progesterone are of importance in the ovulation process of Shao ducks.     

Updates on Reproductive Physiology, Genital Diseases and Artificial Insemination in the Domestic Cat

Reproduction in the domestic cat is characterized by large individual variations in the female oestrous cycle and in male semen quality. The female cat reproduction is strongly influenced by season, and new data suggest that it is possible that season also affects male fertility. Repeated periods of oestrus and spontaneous ovulation may lead to degenerative endometrial changes causing infertility. For artificial insemination (AI), induction of ovulation is necessary in the absence of the mating stimuli. The large variation in the relationship between follicular growth and timing of expression of oestrus complicates, however, timing of ovulation induction. Stress may lead to progesterone secretion by the adrenal glands and possibly have a negative impact on early pregnancy. The large individual variation in semen quality makes fertility evaluation and feline semen conservation a challenge. For AI, the semen can be deposited in the cranial vagina, the uterus or in the uterine tubes. Intrauterine insemination results in higher pregnancy rates than intravaginal but is more complicated. Surgical intrauterine or intratubal insemination has resulted in the birth of kittens, but is an invasive procedure that is not allowed in all countries. Transcervical intrauterine insemination with frozen-thawed semen has, however, recently resulted in the birth of kittens.     

The correlation between the cytology of the vaginal smear and the time of ovulation in the bitch

The correlation between the vaginal smear and the calculated time of ovulation was studied in 22 bitches, during a total of 24 heat periods. Blood plasma was analysed for levels of oestradiol and progesterone from 6 days before to 6 days after the day of maximum keratinization of the vaginal smear. In all 24 heat periods (100 per cent) maximum keratinization of the vaginal epithelial cells was observed at least 3 days after peak oestradiol levels and in 17 of the heat periods (70.8 per cent) maximum keratinization was seen when the peripheral plasma level of progesterone had increased to >5.44 ± 0.93 ng/ml, which has been calculated as the ovulatory level.     

Effects of Gonadotrophin Releasing Hormone Administration on the Pituitary-Ovarian Axis in Anoestrous vs Ovariectomized Bitches

The aim of this study was to determine the effects of gonadotrophin releasing hormone (GnRH) administration on the plasma concentrations of reproductive hormones in intact and ovariectomized (OVX) bitches. Therefore, blood samples were collected at multiple times before and after the administration of 10 microg/kg GnRH (Fertagyl)) for the determination of the plasma concentrations of luteinizing hormone (LH), oestradiol, progesterone and testosterone in six anoestrus and in six OVX bitches. The mean plasma LH concentrations before and 60 min after GnRH administration were significantly lower in the anoestrous bitches than in the OVX bitches. In both groups GnRH administration resulted in a significant increase in the plasma LH concentration. The highest plasma LH concentrations were found at 10 min after GnRH administration and these values did not differ significantly between the two groups. Only in the anoestrous bitches a significant increase in plasma oestradiol concentrations was found after GnRH administration and these values were significantly higher than those in the OVX bitches. The plasma concentrations of progesterone and testosterone were low (close to or below the limit of quantitation) both before and after GnRH administration and the differences between anoestrous and OVX bitches were not significant. It can be concluded that (i) basal plasma LH concentration is significantly higher in OVX bitches than in anoestrous bitches, (ii) plasma LH concentration increases after GnRH administration in both anoestrous and OVX bitches, (iii) GnRH administration causes a significant rise in plasma oestradiol concentration only if ovarian tissue is present and (iv) measurement of plasma progesterone and testosterone concentrations before and after GnRH administration does not aid in distinguishing between anoestrous and OVX bitches. The results of this study may provide a basis for the diagnosis of remnant ovarian tissue and verification of neuter status in the bitch.     

Effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given GnRH

The effects of plasma progesterone concentrations on LH release and ovulation in beef cattle given 100 microg of GnRH im were determined in three experiments. In Experiment 1, heifers were given GnRH 3, 6 or 9 days after ovulation; 8/9, 5/9 and 2/9 ovulated (P<0.02). Mean plasma concentrations of progesterone were lowest (P<0.01) and of LH were highest (P<0.03) in heifers treated 3 days after ovulation. In Experiment 2, heifers received no treatment (Control) or one or two previously used CIDR inserts (Low-P4 and High-P4 groups, respectively) on Day 4 (estrus=Day 0). On Day 5, the Low-P4 group received prostaglandin F(2alpha) (PGF) twice, 12 h apart and on Day 6, all heifers received GnRH. Compared to heifers in the Control and Low-P4 groups, heifers in the High-P4 group had higher (P<0.01) plasma progesterone concentrations on Day 6 (3.0+/-0.3, 3.0+/-0.3 and 5.7+/-0.4 ng/ml, respectively; mean+/-S.E.M.) and a lower (P<0.01) incidence of GnRH-induced ovulation (10/10, 9/10 and 3/10). In Experiment 3, 4-6 days after ovulation, 20 beef heifers and 20 suckled beef cows were given a once-used CIDR, the two largest follicles were ablated, and the cattle were allocated to receive either PGF (repeated 12h later) or no additional treatment (Low-P4 and High-P4, respectively). All cattle received GnRH 6-8 days after follicular ablation. There was no difference between heifers and cows for ovulatory response (77.7 and 78.9%, P<0.9) or the GnRH-induced LH surge (P<0.3). However, the Low-P4 group had a higher (P<0.01) ovulatory response (94.7% versus 61.1%) and a greater LH surge of longer duration (P<0.001). In conclusion, although high plasma progesterone concentrations reduced both GnRH-induced increases in plasma LH concentrations and ovulatory responses in beef cattle, the hypothesis that heifers were more sensitive than cows to the suppressive effects of progesterone was not supported.     

Effects of different patterns of feed restriction and insulin treatment during the luteal phase on reproductive, metabolic, and endocrine parameters in cyclic gilts

The objectives of the present study were 1) to study potential effects of previous nutritional treatment on developmental competence of early fertilized oocytes in vitro; 2) to study responses to insulin treatment during the period of feed restriction in the late luteal phase which has deleterious effects on subsequent fertility; and 3) to establish the metabolic and endocrine status of gilts during treatment and the subsequent periestrous period. Nineteen trios of littermate gilts were subjected to feed restriction during the first (RH) or second (HR) week of the estrous cycle. A second group of HR gilts received injections of long-acting insulin during their period of feed restriction (HR+I). Intensive sampling was performed in a subgroup of 23 animals on d 15 and 16 of the cycle for analyses of endocrine (gonadotropins and steroid hormones) and metabolic (insulin, IGF-I, leptin, total triiodothyronine [T3], and free T3) variables. Gilts were checked for estrus every 6 h, and time of ovulation was monitored by transcutaneous ultrasonography. Surgeries were performed 12 to 20 h after ovulation, and the early-fertilized oocytes recovered were cultured in vitro under standardized conditions. There was no treatment effect on the developmental competence of fertilized oocytes in vitro; however, ovulation rate was increased in HR+I gilts. No effect of treatment was observed on plasma leptin and IGF-I concentrations on d 15 and 16. However, HR+I gilts had higher (P < 0.05) postprandial insulin and lower (P < 0.05) postprandial total and free T3 on d 15. Plasma concentrations of LH, FSH, and progesterone on d 15 and 16 and plasma estradiol concentrations on d 16 were not affected by previous nutritional or insulin treatment. In the periestrous period, plasma concentrations of LH, FSH, and estradiol were higher (P < 0.05) in RH and HR+I, and the rise in plasma progesterone after the LH surge was lower (P < 0.05), than in HR gilts. No effect of treatment was observed on plasma concentrations of metabolic hormones, except on plasma leptin concentrations, which were higher (P < 0.05) at the time of the LH surge in RH gilts. These results suggest that feed restriction during the late luteal phase may have deleterious effects on ovarian function in the periestrous period, which may be counteracted by insulin.     

Melengestrol acetate blocks the preovulatory surge of luteinizing hormone,the expression of behavioral estrus,and ovulation in beef heifers

We tested the hypothesis that melengestrol acetate (MGA), an orally active progestin, blocks estrus and the preovulatory surge of luteinizing hormone (LH) in beef heifers. Cycling yearling Angus heifers were divided randomly into two groups: MGA-treated (n = 6) and control (n = 5). All heifers received injections of prostaglandin F2alpha (PGF) on d -25, -11, and 0 to synchronize estrus. Following the last PGF injection on d 0, heifers were fed either 0.5 mg MGA in a carrier or the MGA carrier each day for 8 d. At 4-h intervals on d 1 through 6, all heifers were observed for expression of estrous behavior, and blood samples were collected and assayed for LH. Daily blood samples were collected at 0800 on d 1 through 10 and assayed for circulating progesterone concentrations. All control heifers exhibited estrus and a preovulatory surge of LH. In each case, this was followed by increases in circulating concentrations of progesterone indicative of ovulation and normal luteal function. In contrast, none of the MGA-treated heifers exhibited estrus, LH surges, or evidence of ovulation. The results of this experiment show that MGA prevents ovulation in cattle by inhibiting the preovulatory surge of LH.     

Prediction of parturition date in the bitch and queen

Assessment of gestational age and parturition date prediction is a challenging, yet common task in clinical management of the pregnant bitch or queen. Knowing the approximate parturition date is essential for a thorough pregnancy monitoring. Radiographic and ultrasonographic methods are suitable in bitches and queens. In the bitch, moreover, ovulation timing by means of LH or progesterone assay, determination of onset of vaginal dioestrus by means of examination of vaginal cytology, and recognition of impending parturition by monitoring prepartal progesterone levels and body temperature variation are practical methods. A combination of different methods increases the accuracy of parturition date prediction.     

Determination of optimal time for mating by artificial insemination with chilled semen using luteinizing hormone surge as an indicator in beagles.

Artificial insemination (AI) was conducted using the second fraction of semen, which was collected from 15 male dogs, diluted to a total sperm count of 100x10(6) for each insemination with egg-yolk Tris (eyT) citrate acid buffer and incubated at 4 degrees C for 48 hours. Luteinizing hormone (LH) surge was detected to determine the optimal time for mating using canine LH assay kits. Artificial insemination using 100x10(6) sperm was performed on the fourth and sixth days or the fifth and seventh days after the LH surge. The conception rates were 33% (4/12) and 89% (8/9), respectively; the whelping rates also showed similar results. Serum LH and follicle stimulating hormone (FSH) concentrations were measured in nine dogs, and the mean LH concentration (+/- standard deviation) at LH surge was 15.77+/-7.66 ng/ml. The time of the LH surge detected by the canine LH assay kit was very similar to that measured by radioimmunoassay (RIA).     

Use of a GnRH antagonist,antarelix, associated or not with hCG,to control ovulation in cyclic pony mares

The GnRH antagonist antarelix (Teverelix™) was administered to mares (0.01 mg/kg, i.v., twice a day) during the periovulatory period. In Experiment 1, 20 mares were divided into a treated (A3d−) and a control (Control−) group. A3d− mares received antarelix for 3 days from the day when the dominant follicle (F1) reached 32 mm (D0). In Experiment 2, 10 mares were divided into a treated (A6d+) and a control (Control+) group. A6d+ mares received antarelix for 6 days from D0 and hCG was injected in all animals (1600 IU, i.v.) on D1. Pregnancies were determined 13 days after ovulation. In both experiments, antarelix interrupted or totally abolished the LH surge. In Experiment 1, 5/10 of the A3d− mares (with maximum LH concentrations of 11.6 ng/ml at the beginning of treatment) ovulated at the same time as the Control− mares; the other five mares (with LH concentrations under 5.4 ng/ml) ovulated 13.4±0.6 days later. In Experiment 2, all the A6d+ mares ovulated at the same time as the Control+ mares. In treated mares which ovulated during the treatment, progesterone concentrations and fertility did not differ from control mares. These results demonstrate that in mares: (1) a small elevation of endogenous LH can induce ovulation, (2) ovulation can be postponed approximately 13 days after a 3-day antarelix treatment if initiated just before the preovulatory LH surge, (3) ovulation can be induced by hCG on depressed levels of endogenous LH, (4) the inhibition of the post ovulatory LH surge has no effect either on the corpus luteum or on fertility.