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.     

Prolactin and luteinizing hormone secretion in the pregnant pig.

Four pregnant, primiparous, crossbred gilts and six gilts from the same population that had been ovariectomized (OVX) for approximately 3 wk were placed in individual pens in an enclosed building. Blood samples were collected every 30 min for 12 h from all gilts via an indwelling jugular vein cannula when the pregnant gilts were at d 30, 50, 70, 90, and 110 of gestation. Serum was quantified for LH and prolactin (PRL) by RIA. The OVX gilts served as controls to ensure that any variations in serum LH and PRL concentrations observed in the pregnant animals were not due to environmental factors unrelated to pregnancy. Within the pregnant gilts, mean serum LH concentrations, mean basal serum LH concentration, and mean serum LH peak height were similar on all days; however, number of LH peaks on d 30, 50, and 70 were greater (P < .05) than on d 90 and 110, and number of LH peaks on d 50 was greater (P < .05) than that on d 70. Within the pregnant gilts, mean serum PRL concentration, mean basal serum PRL concentration, and mean PRL peak height were greater (P < .001) on d 110 than on all other days; however, number of PRL peaks were similar among days. Parameters of LH and PRL secretion in the OVX and pregnant gilts varied independently. Results of this study indicated that 1) LH secretion does not vary appreciably throughout pregnancy and 2) PRL secretion does not vary significantly during the first 90 d of pregnancy, after which it increases markedly on or before 110 d.     

Prolactin,Androstenedione and IGF1 Serum Concentrations During Induced Follicular Growth by eCG Administration in the Bitch

The oestrus cycle in the domestic bitch, a monoestrous species, differs considerably from that of other veterinary domestic animals species. In the bitch the combined use of eCG and hCG is effective to induce oestrus predictably and safely (Stornelli et al., Theriogenology, 78, 2012 and 1056). Although several studies were done to describe the hormonal changes during the canine oestrus cycle, to our knowledge none was done to describe the hormonal changes during induced follicular growth after the administration of eCG. The aim of this work was to study prolactin (PRL), insulin‐like growth factor (IGF1) and androstenedione (ANDR) serum concentrations during follicular growth induced by a single dose of eCG administered to late anoestrous bitches. PRL and ANDR concentrations were lower before than after eCG TRT (before eCG vs pro‐oestrus, oestrus and dioestrus; 4.3 ± 1.8 ng/ml vs 6.5 ± 1.6 ng/ml, p < 0.05; 0.08 ± 0.2 ng/ml vs 0.42 ± 0.16 ng/ml, p < 0.05). Conversely, IGF1 concentrations were similar before and after eCG TRT (286.0 ng/ml ±32.2, p > 0.53). Additionally, PRL concentrations were similar before oestrus compared to during oestrus and dioestrus (6.9 ± 1.7 ng/ml, p > 0.19). Furthermore, IGF1 concentrations were higher before and during oestrus compared to first day of dioestrus (286.1 ± 29.8vs 200.4 ± 29.2 ng/ml, p < 0.01). On the contrary, ANDR concentrations were lower before and during oestrus compared to first day of diestrum (0.35 ± 0.17 ng/ml and 0.38 ± 0.15 vs 0.68 ± 0.17 ng/ml, p < 0.05). These results show that treatment with a single injection of 50 IU/kg of eCG in late anoestrous bitches successfully induced changes in follicular growth which were paralleled with changes in PRL, IGF1 and ANDR serum concentration similar to those occurring during a normally occurring oestrous cycle. In addition, our results suggest that IGF1 in the bitch could play an important role in ovarian folliculogenesis.     

Comparison of the use of cabergoline and gonadotrophin to treat primary and secondary anoestrus in bitches

Objective To determine the efficacy and reliability of cabergoline and pregnant mare's serum gonadotrophin (PMSG) for induction of oestrus in bitches with primary or secondary anoestrus. Procedures We studied 39 healthy bitches of various breeds aged 2–6 years and in primary or secondary anoestrus: 20 bitches were administered 5 µg/kg/day cabergoline orally until day 2 after the onset of pro‐oestrus or for a maximum of 42 days, and 19 bitches were administered 20 IU/kg/day PMSG intramuscularly for 5 consecutive days, followed by an additional single injection of 25 IU/kg of human chorionic gonadotrophin on the fifth day. Results The rates of oestrus induction in the primary and secondary anoestrous bitches treated with cabergoline and PMSG were found to be similar. Pregnancy and whelping rates in the cabergoline group were statistically different from the rates in the PMSG group (P < 0.001). Conclusion Cabergoline is more effective and reliable for the induction of a fertile oestrus in bitches with primary or secondary anoestrous.     

Effect of an Injectable Cabergoline Formulation on Serum Prolactin (PRL) and Milk Secretion in Early Postpartum Beagle Bitches

This study was conducted in order to evaluate effects on prolactin (PRL) concentration and mammary milk secretion of an injectable cabergoline formulation administered to five lactating Beagle bitches during early postpartum (PP). Bitches were bled twice daily (from PP day 3 to PP day 12) and then daily (from PP day 13 to PP day 16) to assay serum PRL. On PP day 6, a subcutaneous (SC) injection of 0.1 ml/kg of placebo was administered. On PP day 9, a SC 0.1 ml/kg dose of injectable cabergoline was administered. All bitches were checked for milk production, using a clinical scoring in order to quantify milk expression from each teat. A circadian variation of serum PRL was evident during the 6 days of pre-treatment monitoring. The day after cabergoline injection, an 80% decrease of PRL serum concentration was observed (p < 0.05). The circadian oscillatory pattern of PRL secretion disappeared after administration of cabergoline, and PRL values remained significantly lower than in the previous days for the first 60 h following treatment (p < 0.001). Milk production was drastically reduced when comparing pre-treatment to post-treatment scores (p < 0.001). A single dose of injectable cabergoline caused a significant reduction in serum PRL concentration and a significant reduction in milk flow. The injectable formulation of cabergoline appeared to be safe and well tolerated.     

Induction of oestrus by administering Inhibin antiserum along with equine chorionic gonadotropin in anoestrous bitches

As dogs experience oestrus only once or twice a year, it is necessary to establish an effective method of oestrous induction for efficient breeding. In the present study, we evaluated inhibin antiserum (IAS) on oestrous induction in anoestrous females. Bitches were administered 0.5 ml/kg IAS or a mixture of 50 IU/kg equine chorionic gonadotropin (eCG) and 0.5 ml/kg IAS and 500 IU human chorionic gonadotropin (hCG) administered 7 days after the mixture injection. As a control, bitches received 50 IU/kg eCG, with 500 IU hCG administered 7 days after eCG injection. Blood-tinged vaginal discharge, vulvar swelling, plasma progesterone concentrations and ovarian follicular development were assessed from day 0 to day 14. IAS alone injection did not induce oestrus in bitches at the anoestrous stage. Conversely, vulvar swelling, blood-tinged vaginal discharge and an estimated luteinizing hormone (LH) surge appeared on days 3–7, days 3–6 and days 7–9 after the IAS+eCG mixture injection, respectively, in all five bitches at the anoestrous stage. The average number of developing and ovulated follicles in bitches administered IAS+eCG was 8.8 and 9.6 respectively. A single eCG injection followed by hCG induced oestrous signs, with an average of 8.3 developing follicles and 4.5 ovulated follicles. This study revealed that IAS alone did not induce oestrus, but when IAS was used in combination with eCG, it induced oestrus and promoted a considerable number of ovulations in anoestrous dogs.     

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

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

Changes in luteinizing hormone pulse frequency and prolactin levels in bitches in response to estrus induction by cabergoline-its cases where it is delayed to induce estrus

The effect of estrus induction by cabergoline on gonadotropin and steroid hormone responses was examined in anestrous bitches. Eleven beagles were used in the study; seven were included in the estrus induction group and four were included in the spontaneous estrus group. Cabergoline was orally administered to the estrus induction group at 5 µg/kg once daily for four weeks, or until hemorrhagic discharge was detected. The inter-estrus interval in the estrus induction group was significantly shorter than the previous estrus interval. Bitches that showed proestrus within four weeks of treatment showed increased luteinizing hormone (LH) pulse frequency and, subsequently, increased estradiol (E₂) levels. Prolactin (PRL) levels declined promptly after treatment, except in one bitch that did not show proestrus during the cabergoline treatment period. There was a significant correlation between the time to proestrus induction and the reduction in PRL levels. A positive correlation was found between the LH levels two weeks after cabergoline administration and PRL reduction. This study demonstrates that an abrupt reduction in PRL is likely to be important for initiation of estrus in bitches. A reduction in PRL indirectly leads to an increase in LH pulse frequency, which regulates follicular development in bitches. However, if the period from the end of the previous estrus to the cabergoline treatment is short, it may take some time to show proestrus without increasing E₂ levels, even if the LH level increases after cabergoline administration.     

Opioid inhibition stimulates luteinizing hormone and prolactin secretion in the gilt

Ten gilts on day 6·11 of the estrous cycle (onset of estrus = day 0) were given 115 mg of naloxone (NAL), an opioid antagonist, in saline i.v. (n = 5) or saline Lv. (n = 5). Jugular blood was collected at 15 min intervals for 2 hr before and 4 hr after treatment. Serum LH concentrations were 0.4 ± 0.1 ng/ml before NAL treatment, increased (P<.01) to 4.3 ± 0.7 ng/ml at 15 min following NAL treatment and returned to control concentrations by 75 minutes. Serum PRL concentrations were 5.0 ± 0.1 ng/ml before NAL treatment, increased (P<.05) to 14.8 ± 2.9 ng/ml at 30 min following NAL treatment and returned to control concentrations by 120 minutes. Serum LH and PRL concentrations were 0.5 ± 0.1 ng/ml and 5.2 ± 0.4 ng/ml, respectively, at 15 min following saline treatment and remained unchanged throughout the blood sampling period. Four of the 5 NAL treated gilts responded with an increase in both serum LH and PRL concentrations. The mean of serum progesterone concentrations, quantitated in samples taken every 2 hr, were similar for controls (22.7 ± 1.8 ng/ml) and NAL (26.5 ± 1.4 ng/ml) treated gilts. The gilt which failed to respond to NAL had nondetectable concentrations of serum progesterone and was excluded from analysis. These data indicate that the opioids modulate LH and PRL secretion during the luteal phase of the estrous cycle.     

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.     

Endogenous opioid modulation of luteinizing hormone and prolactin secretion in postpartum ewes and cows

A possible role for endogenous opioid peptides (EOP) in the control of luteinizing hormone (LH) and prolactin (PRL) secretion was studied by injecting the opioid antagonist, naloxone (NAL), into postpartum ewes and cows. Twelve ewes that lambed during the fall breeding season and nursed their lambs were injected iv with NAL (1.0 mg/kg) on d 10, 14, 18, 22 and 26 postpartum. Blood samples were collected at 15-min intervals from 2 h before to 2 h after NAL, and serum concentrations of LH and PRL were quantified. Following treatment on d 10, suckling lambs were removed from 6 of the 12 ewes, creating non-suckled (NS) and suckled (S) treatment groups for subsequent study on d 14 through 26. On d 10, NAL treatment increased LH (P less than .01) but concentrations of PRL were not affected. When averaged across d 14 to 26, post-NAL concentrations of LH were greater (P less than .001) than pre-NAL concentrations (6.5 +/- .7 vs 1.9 +/- .4 ng/ml). In contrast, concentrations of PRL in the post-NAL period were lower (P less than .001) than pre-NAL concentrations (129 +/- 15 vs 89 +/- 10 ng/ml). Compared with S ewes over d 14 to 26, those in the NS group had similar pre-NAL concentrations of LH, tendencies for higher (P less than .10) post-NAL concentrations of LH, lower (P less than .001) mean serum concentrations of PRL (pre- and post-NAL) and similar pre-NAL vs post-NAL differences in serum PRL. Six suckled beef cows on d 24 to 35 were injected iv with either saline or NAL (.5 mg/kg) in a replicated crossover design. Injections of NAL increased serum concentrations of LH (P less than .05), when averaged over all 12 injections in the six cows, but serum PRL was not changed. However, three of six cows did not respond to NAL with increases in serum LH. These non-responding cows were similar to the responding cows in their pre-injection concentrations of LH and PRL, but they tended (P = .10) to have higher serum concentrations of cortisol than responding cows.     

Post‐weaning anoestrus in primiparous sows: LH patterns and effects of gonadotropin injection and boar exposure

Summary

Post‐weaning anoestrus was studied in eighteen primiparous sows, selected from a breed showing a high proportion of anoestrous sows. The sows were studied from late lactation, through weaning at day 29 post‐partum (p.p.), until day 21 post‐weaning (p.w.). Blood samples were taken once daily, and frequently (every ten minutes) on several days before and after weaning.

Out of a total of ten anoestrous sows, three were exposed to a boar and seven were given gonadotropins (PG600) on day 21 p.w.. Serial blood samples were analysed for LH only and daily samples were additionally analysed for oestradio1–170 and progesterone, by validated radioimmunoassay procedures. Analysis of variance of the basal level, pulse frequency, pulse amplitude and mean level of LH showed, retrospectively, that during lactation the basal and mean levels of LH were significantly lower in anoestrous than in oestrous sows (P ≤ 0.05). Furthermore, the post‐weaning basal and mean levels of LH were also significantly lower in anoestrous than in oestrous sows (P ≤ 0.05). However, because of the small number of oestrous animals (n = 3), these results should be interpreted with caution.

Exposure of anoestrous sows to a boar did not result in oestrus and/or ovulation within seven days, but did increase LH pulse frequency. Injection of gonadotropins resulted in an LH surge, oestrus and ovulation in only three sows, but oestradiol levels were increased in six sows. From our experiments and from reports in the literature we conclude that a lowered secretion of LH may play a role in the aetiology of post‐weaning anoestrus in the sow.     

Concentrations of progesterone and luteinizing hormone in the serum of diestrous bitches before and after hysterectomy

Blood was collected from 6 adult bitches (3 pregnant and 3 nonpregnant) daily for 59 days after the onset of diestrus, and serum progesterone concentrations were determined. The bitches were hysterectomized at various times during diestrus [diestrus day (DD) 5, 15, or 25] to determine whether the uterus has a role in maintaining luteal function in the pregnant or nonpregnant bitch. Concentrations of serum luteinizing hormone (LH) were also measured to determine whether LH concentrations decrease when luteal function abates (experiment 1). All 6 bitches in experiment 1 had progesterone concentrations greater than 1.0 ng/ml during the 2-month period, indicating that neither the pregnant nor nonpregnant uterus was necessary for maintaining luteal function in the bitch. Concentrations of LH appeared to increase in the serum of 5 of the 6 bitches when progesterone concentrations were declining, indicating that cessation of luteal function does not appear to result from a lack of LH. Blood was collected from the 6 bitches used in experiment 1 twice weekly during the 2nd diestrus after hysterectomy (experiment 2) to determine whether alterations in luteal function that might be observed during experiment 1 were due to some aspect of the surgical procedure, rather than to absence of a uterus. Progesterone concentrations in the serum were lower (P less than 0.05) during the 10 days after surgery in the 2 bitches hysterectomized on DD 5 than in the 4 bitches remaining intact during this period (experiment 1).(ABSTRACT TRUNCATED AT 250 WORDS)     

THE INDUCTION OF OESTRUS AND OVULATION IN THE BITCH USING PREGNANT MARE SERUM GONADOTROPHIN AND HUMAN CHORIONIC GONADOTROPHIN

Seventeen bitches (11 anoestrous, 4 prepubertal, 1 pregnant and 1 postpartum) were treated with pregnant mare serum gonadotrophin (110 iu/kg) at weekly intervals up to the occurrence of oestrus or a maximum of 3 treatments. Oestrus was induced in 8/11 anoestrous bitches only. The between bitch ovarian response was very variable. Ovulation occurred in 7/8 PMSG induced oestrous bitches. The 3 oestrous bitches treated with human chorionic gonadotrophin (500 iu) at the start of oestrus had a better ovulatory response than those treated with PMSG alone.     

Expression of the μ Opioid Receptor and Effects of the Opioid Antagonist Naloxone on In Vitro Maturation of Oocytes Recovered from Anoestrous Bitches

The μ-opioid receptor (MOR) is expressed in bovine, human, equine and canine oocytes, and in seasonal breeders, it is expressed with higher intensity during the anoestrous phase. Supplementation of in vitro maturation (IVM) medium with opioid agents, agonists or antagonists, was shown to affect oocyte maturation in several species such as rat, bovine and equine. This study reports the effects of supplementing IVM medium with naloxone (Nx), an opioid antagonist, on nuclear and cytoplasmic maturation rate of oocytes recovered from anoestrous bitches. Cytoplasmic maturation was examined in terms of mitochondrial (mt) distribution. In order to confirm the receptor-mediated action of Nx, in oocytes of anoestrous bitches, MOR expression was analyzed by Western blot. Cumulus–oocyte complexes, recovered from the ovaries of bitches in anoestrous, were cultured in vitro and Nx was added at the concentrations of 1 × 10⁻⁶, 1 × 10⁻⁸ and 1 × 10⁻¹⁰  m . The rate of oocytes resuming meiosis after culture in presence of 1 × 10⁻⁶  m Nx (29%) was significantly higher than that of oocytes of control group (12%; p < 0.05). However, treatment with Nx did not affect mt distribution pattern. In denuded oocytes and in corresponding cumulus cells, a doublet of 65 and 50 kDa was observed. We conclude that, in oocytes of anoestrous bitches, MOR is expressed and Nx significantly improves nuclear maturation rate. Further studies should be performed to elucidate the expression of other opioid receptors, such as δ and κ, and possible interactive effects of their antagonists on canine oocyte maturation.     

Effect of photoperiod and castration on prolactin, testosterone and luteinizing hormone concentrations in male calves

After 8 wk exposure to 8 h of light per day, prolactin (PRL) averaged 18.3 ng/ml of serum in eight male calves. Four calves then received 16 h of light per day; 6 wk later (age 14 wk) PRL averaged 93.8 ng/ml of serum, whereas PRL averaged 36.9 ng/ml of serum in four calves maintained under 8 h of daily light. By wk 20, PRL was not different in calves exposed to 16 or 8 h of daily light, averaging 34.7 and 17.2 ng/ml serum. Testosterone averaged .43 ng/ml of serum at wk 8 but was greater at wk 14 in calves receiving 16 h of light daily when compared with controls receiving 8 h of light (1.92 vs. .97 ng/ml of serum). Testosterone concentrations were not different between photoperiod treatments at wk 20. Luteinizing hormone (LH) concentrations were unaffected by photoperiod. In a second experiment, four male calves were castrated at approximately 2 wk of age while four similar controls were left gonadally intact. After 8 wk exposure to 8 h of light per day, PRL averaged 12.3 ng/ml of serum in all calves. After 6 wk exposure to 16 h of light per day, PRL in serum increased in castrates to 48.0 ng/ml and in controls to 59.8 ng/ml. We conclude that serum concentrations of PRL and testosterone, but not LH, increased in bull calves receiving 16 h of light daily relative to calves receiving 8 h of light, and that the PRL response to photoperiod is independent of the testes. However, 16 h light-induced stimulation of serum concentrations of prolactin is not maintained indefinitely.     

Secretory Patterns of Prolactin in Dogs: Circannual and Ultradian Rhythms

The objectives of the present study were to characterize in dogs circannual and ultradian prolactin (PRL) secretory patterns and also to compare gender differences in the ultradian period of study in the Southern hemisphere. Blood samples were collected at 15‐min intervals for 2.5 h from seven male and seven female dogs and a single monthly sampling, over a 1‐year time span, from six male dogs for the ultradian and circannual studies, respectively. Plasma PRL was measured by a homologous enzyme immunometric assay. The ultradian study evidenced PRL elevations suggesting pulsatile secretion in both genders. Significantly higher mean smoothed baseline (ng / ml [7.02 ± 1.2 vs 1.23 ± 1.0, p < 0.01]) and AUC (ng/ml * 2.5 h [25.2 ± 3.8 vs 4.4 ± 3.8, p < 0.01]) were found in females when compared with males. In the circannual study, plasma PRL concentrations did not statistically differ among the months of the year. When grouped together the 3 months with a longer daylight had significantly higher PRL concentrations than the 3 months with the shortest (2.31 ± 0.37 vs 0.96 ± 0.37, p < 0.01). The correlation between length of daylight and PRL concentrations was 0.24, p < 0.05. It is concluded that PRL does have a circannual rhythmicity and that there are ultradian gender‐related differences in the period under study in these groups of dogs. This study also demonstrates plasma PRL elevations suggesting pulsatile secretion in male dogs.     

Effect of Progesterone Prior to GnRH-PGF2α Treatment on Induction of Oestrus and Pregnancy in Anoestrous Awassi Ewes

An experiment was conducted to examine the effect of progesterone prior to a GnRH‐PGF_2α treatment on oestrus and pregnancy in seasonally anoestrous Awassi ewes. Twenty‐four ewes were randomly assigned to three groups to be pre‐treated with 60 mg medroxyprogesterone acetate sponges (group A), 600 mg progesterone sponges (group B) or blank sponges (group C) for 4 days. All ewes were injected with 100 μg of GnRH 24 h after sponge removal followed, 5 days later, by 20 mg PGF_2α injection. Ewes were exposed to three fertile rams at the time of PGF_2α injection (day 0, 0 h) and were checked for breeding marks at 6‐h intervals for 5 days. Blood samples were collected from all ewes 1 day (day ?10) prior to sponge insertion, at the time of sponge removal (day ?6), 1 day following sponge removal (day ?5, at the time of GnRH injection) and at the time of PGF_2α injection (day 0) for analysis of progesterone. Progesterone concentrations on days ?10 and ?5 were basal and averaged 0.2 ± 0.04 and 0.2 ± 0.2 ng/ml, respectively. Progesterone concentrations on day ?6 were elevated only in group B ewes and were higher (p < 0.0001) than those of groups A and C. Progesterone concentrations on day 0 were higher (p = 0.002) in groups A and B than group C. Oestrous responses occurred only in ewes of groups A and B (p > 0.05). Induced oestrus conception rate was greater (p < 0.01) in group A than groups B and C. Ewes returned to oestrus 17–20 days following day 0 were two of eight, six of eight and three of eight of groups A, B and C, respectively, all of which eventually lambed. The overall lambing rate was 82% in progesterone‐primed ewes compared with only 38% non‐progesterone‐primed ewes (p < 0.05). Progesterone priming apparently sensitizes GnRH‐PGF_2α‐treated seasonally anoestrous ewes and increases their response in oestrus and pregnancy rates.     

Serum Levels of Cardiac Markers NT‐proANP and NT‐proBNP in Brachycephalic bitches at Different Gestational Stages

The aim of this study was to determine serum levels of natriuretic peptide precursors (NT‐proANP and NT‐proBNP) during pregnancy in brachycephalic bitches. Fifteen healthy multiparous bitches were selected for this prospective study. Serum levels of NT‐proANP and NT‐proBNP were measured during anoestrous and at 14, 35, 42, 49 and 56 days (2nd, 5th, 6th, 7th and 8th weeks) of pregnancy. Fourteen animals had normal gestations, and one bitch developed single foetus syndrome. The natriuretic peptide levels of this animal were not included in this study; however, it is important to report that its NT‐proANP levels were four times greater than those of normal patients. There was no significant difference (p = 0.072) in NT‐proBNP levels between anoestrous (0.20 ± 0.10 ng/ml) and the different pregnancy weeks (0.27 ± 0.12 ng/ml). There was a positive correlation (p < 0.0001) between NT‐proANP and gestational age, and the levels of this marker increased significantly (p < 0.0001) during the 6th (0.26 ± 0.06 ng/ml), 7th (0.28 ± 0.04 ng/ml) and 8th weeks (0.29 ± 0.05 ng/ml) when compared to anoestrous (0.18 ± 0.02 ng/ml). NT‐proANP serum levels are correlated with gestational development and may be indicative of cardiovascular adaptation in canine brachycephalic pregnancy.     

Evaluation of a commercially available luteinizing hormone test for its ability to distinguish between ovariectomized and sexually intact bitches

OBJECTIVE: To determine the value of a commercially available test for measurement of luteinizing hormone (LH) concentrations for its use in distinguishing between ovariectomized and sexually intact bitches. DESIGN: Prospective longitudinal cohort study. ANIMALS: 101 sexually intact bitches and 199 ovariectomized bitches PROCEDURE: A commercial test kit was used to assay serum samples for LH concentration. A kappa test was used to assess the consistency of test interpretations. Sensitivity, specificity, predictive values, and consistency of test interpretation in the sample population and in 2 theoretical populations of bitches with differing frequencies of ovarian removal were determined. RESULTS: The test had excellent sensitivity (98%) but moderate specificity (78%) because of a high frequency of false-positive test results. This means that a single high serum LH concentration was not a reliable indicator of ovarian removal. However, a single low serum LH concentration was an excellent indicator that a bitch was sexually intact. The LH test results were positive within 10 days after ovarian removal and remained high in bitches from which ovaries had been removed > 5 years previously. Analysis of kappa values consistently revealed excellent agreement in test interpretation within and among evaluators and over a 6-hour time span. CONCLUSIONS AND CLINICAL RELEVANCE: A single low LH test result confirms the need for ovariectomy in bitches prior to adoption. The test is applicable to bitches that have been ovariectomized prior to puberty and probably applies over the lifespan of the animal.