The specific inhibition of the cardiac electrogenic sodium/bicarbonate cotransporter leads to cardiac hypertrophy.

Two alkalinizing mechanisms coexist in cardiac myocytes to maintain intracellular pH: sodium/bicarbonate cotransporter (electroneutral isoform NBCn1 and electrogenic isoform NBCe1) and sodium/proton exchanger (NHE1). Dysfunction of these transporters has previously been reported to be responsible for the development of cardiovascular diseases. The aim of this study was to evaluate the contribution of the downregulation of the NBCe1 to the development of cardiac hypertrophy. To specifically reduce NBCe1 expression, we cloned shRNA into a cardiotropic adeno-associated vector (AAV9-shNBCe1). After 28 days of being injected with AAV9-shNBCe1, the expression and the activity of NBCe1 in the rat heart were reduced. Strikingly, downregulation of NBCe1 causes significant hypertrophic heart growth, lengthening of the action potential in isolated myocytes, an increase in the duration of the QT interval and an increase in the frequency of Ca2+ waves without any significant changes in Ca2+ transients. An increased compensatory expression of NBCn1 and NHE1 was also observed. We conclude that reduction of NBCe1 is sufficient to induce cardiac hypertrophy and modify the electrical features of the rat heart.

Anti-Müllerian hormone in queens: Serum concentrations and total ovarian follicle population.

The objectives of this study were: a) To report anti-Müllerian hormone (AMH) serum concentrations in neonatal, pre and postpubertal female cats. b) To establish the relationship between serum AMH with either age and estrous cycle c) To correlate the total number of different ovarian follicle types with AMH in adult queens. A single blood sample was collected from 10 neonates (including 5 male), 15 prepubertal and 48 postpubertal female cats to measure AMH. Eight, 10, and 18 of this latter group were in follicular (FP), luteal phase (LP), and anestrus (AN), respectively. The total number of each follicle type was histologically counted using the Gougeon and Chainy (1987) formula in a subgroup of 10 adult queens. Overall AMH mean of these the female cats was 6.31 ± 0.54 ng/mL. The neonatal females had lower AMH serum concentrations than their male littermates (2.56 ± 0.49 vs. >23 ng/mL; P < 0.01). Concentrations were also higher in prepubertal than in neonatal and postpubertal cats (11.79 ± 1.36 vs. 2.56 ± 0.49 vs. 4.87 ± 0.38 ng/mL; P < 0.01). Queens below 12 mo of age had the highest AMH levels (10.41 ± 1.16; P < 0.01). Age was inversely correlated with AMH (r = -0.5; P < 0.01). Animals in FP had lower AMH concentrations than AN females (2.51 ± 0.33 vs. 5.46 ± 0.76 ng/mL; P < 0.05). No difference in the total number of each follicle type were found between either ovary (P > 0.05). A high correlation was only found between small antral follicles and AMH concentrations (r = 0.85; P < 0.01). It was concluded, that AMH can provide an indirect, reliable marker for the assessment of ovarian follicle size and functionality. Age as well as pubertal state should be considered when evaluating AMH concentrations in this species.

Failure of a single dose of medroxyprogesterone acetate to induce uterine infertility in postnatally treated domestic cats.

In mice and sheep, neonatal administration of progesterone or progestins inhibited development of uterine glands. The aims of the present study were (1) to describe uterine gland development on postnatal Days 6 to 8 and (2) to evaluate the effects of a single postnatal administration of a progestin on reproduction and adult uterine glands morphology and function in domestic cats. Necropsy was performed on three 1-week-old female cats which had died unrelated to this study. Ten female kittens were randomly assigned within the first 24 hours of birth to: medroxyprogesterone acetate 10 mg/animal subcutaneously (MPA; n = 6) or placebo (PLC; n = 4) and followed up until puberty when they were mated. Twenty-four days after the end of estrus, ovulation and pregnancy were diagnosed by serum progesterone measurement and ultrasonography, respectively. Then, all the cats were ovariohysterectomized. After necropsy or surgery, the excised organs were histologically evaluated. Seven queens ovulated (4 of 6 MPA and 3 of 4 PLC; P > 0.1) and were pregnant (P > 0.1). Four MPA cats presented endometrial hyperplasia and one of them developed a pyometra. The 1-week-old females presented uterine glands in the stage of budding and incipient penetration of the glandular epithelium into the underlying stroma. The MPA-treated queens revealed that the area occupied by uterine glands per square-micrometer (0.55 ± 0.2 vs. 0.49 ± 0.2; P > 0.1) and the height of the glandular epithelium (µm; 24.5 ± 6.7 vs. 24.4 ± 7.2; P > 0.1) did not differ from those of the PLC group. Neither significant gross nor microscopical differences were also found for ovaries (P > 0.1). It is concluded that 1-week-old kittens had an incipient stage of uterine gland development and that a single postnatal supraphysiological dose of MPA did not alter uterine adenogenesis in this species. Furthermore, this treatment seemed to predispose to uterine disease without prevention of fertility.

In vitro and in vivo assessment of skim milk with and without egg yolk on canine spermatozoa incubated at 4ºC

The aims of this study were to assess and compare the effect of skim milk with (MEY) and without (SMI) egg yolk on canine spermatozoa incubated at 4ºC in vitro and to evaluate the efficacy of MEY in vivo. Also, the effect of semen cooled storage before freezing was also evaluated in vitro. The ejaculates of 10 dogs were collected, pooled, centrifuged, and divided into 4 aliquots and diluted in one of the following 4 diluents: Prostatic fluid (PRO), commercial diluent (COM), SMI, or MEY. Extended samples were stored at 4°C and evaluated daily for 6 days. Percentage of total (P < 0.01) and progressive (P < 0.01) motility, intact acrosomes (P < 0.05), and positive endosmosis (P < 0.01) decreased over time in the diluents, with COM and PRO having as the best and the worst performances, respectively. Furthermore, MEY motility differed from PRO (P < 0.01) and SMI (P < 0.01) but not COM. Acrosome integrity was higher in MEY when compared to SMI (P < 0.05). Sixteen pairs of dogs were randomly assigned to either fresh undiluted semen (n = 8) or to 24-h cooled MEY diluted semen (n = 8). Seven (87.5%; P > 0.1) bitches from each group became pregnant and whelped normally. MEY extended semen samples were cooled for 2, 24, or 48 h at 4°C, and a second dilution was performed prior to freezing and thawing. Post-thaw total and forward sperm motility decreased with increasing cooled storage (P < 0.05), although no significant differences in total or forward motility, normal acrosomes, positive endosmosis, live spermatozoa, or positive endosmosis were found between 2 and 24 h storage. These in vitro and in vivo results show that MEY can be considered a simple, inexpensive, and efficient diluent for canine semen chilling. Furthermore, MEY could be successfully frozen after 1 day of cooled storage.(AU)

In vitro and in vivo assessment of skim milk with and without egg yolk on canine spermatozoa incubated at 4ºC

The aims of this study were to assess and compare the effect of skim milk with (MEY) and without (SMI) egg yolk on canine spermatozoa incubated at 4ºC in vitro and to evaluate the efficacy of MEY in vivo. Also, the effect of semen cooled storage before freezing was also evaluated in vitro. The ejaculates of 10 dogs were collected, pooled, centrifuged, and divided into 4 aliquots and diluted in one of the following 4 diluents: Prostatic fluid (PRO), commercial diluent (COM), SMI, or MEY. Extended samples were stored at 4°C and evaluated daily for 6 days. Percentage of total (P 0.1) bitches from each group became pregnant and whelped normally. MEY extended semen samples were cooled for 2, 24, or 48 h at 4°C, and a second dilution was performed prior to freezing and thawing. Post-thaw total and forward sperm motility decreased with increasing cooled storage (P < 0.05), although no significant differences in total or forward motility, normal acrosomes, positive endosmosis, live spermatozoa, or positive endosmosis were found between 2 and 24 h storage. These in vitro and in vivo results show that MEY can be considered a simple, inexpensive, and efficient diluent for canine semen chilling. Furthermore, MEY could be successfully frozen after 1 day of cooled storage.

Echocardiographic and Doppler assessment of maternal cardiovascular function in normal and abnormal canine pregnancies.

The objective was to verify maternal hemodynamic differences between normal and abnormal pregnancies in dogs. Brucella-negative pregnant bitches (n = 31) were retrospectively classified into abnormal (which had either their pregnancy interrupted between Days 52 and 60 or perinatal death of more than 50% of the litter; n = 14) and normal (which had delivered healthy puppies at term; n = 17). These dogs were evaluated with echocardiography every 10 days from Days 0 to 60 of gestation (Day 0 = estimated day of LH peak). Systolic blood pressure was also assessed. At Day 50 of gestation, left ventricular free wall in systole increased in the normal but not in the abnormal group (P < 0.01). In contrast, end systolic stress (P < 0.01) and systolic blood pressure (P < 0.01) diminished only in normal animals. We concluded that signs of altered maternal cardiovascular adaptation to pregnancy may be predictors of obstetrical complications in dogs.

Electrocardiographic changes in normal and abnormal canine pregnancy.

The aim of this study was to describe the canine electrocardiographic changes in the course of normal and abnormal pregnancy. Twenty-three Brucellosis-negative pregnant bitches were retrospectively classified as normal (n = 12) or abnormal (n = 11). A control group of non-pregnant dioestrous bitches (n = 10) was also included. Normal pregnant females delivered healthy puppies at term while abnormal animals interrupted their pregnancy between days 52-60 (from estimated luteinizing hormone peak) or presented perinatal litter death higher than 60%. All the bitches were electrocardiographically evaluated every 10 days from day 0 to day 65 of the oestrous cycle, to parturition or abortion. Percentage heart rate change increased 31.3% from day 40 to 60 in normal gestation while it decreased -1.8% in dioestrous bitches, although it did not change in the abnormal group (p < 0.01). In the abnormal pregnant group but not in the others, percentage QRSa change fell to -34% on day 60 (p < 0.01). At the same time point, percentage QRSd change was 6.2% vs -4.9% in normal gestations and dioestrous animals, respectively (p < 0.05). Corrected QT interval augmented from day 40 onwards up to 9.9% and 4.3% in the normal pregnant and dioestrous groups, respectively, while it remained unchanged in abnormal gestations (p < 0.05). It is concluded that during normal canine pregnancy, some electrocardiographic parameters begin changing from day 40 onwards, and that pathological gestations differ from normality from day 30. The use of electrocardiography in canine obstetrics might contribute to identify abnormal outcomes before they become clinically evident.

Doppler ultrasonographic assessment of maternal and fetal blood flow in abnormal canine pregnancy.

The aim of this study was to describe the changes of uterine artery, umbilical artery and fetal abdominal aorta, renal and internal carotid arteries blood flow in abnormal canine pregnancy. Twenty-two, Brucella-negative pregnant bitches were retrospectively classified into abnormal (which had either interrupted their pregnancy between days 52 and 60 or had perinatal death >60% of the litter; n=11) and normal (which had delivered healthy puppies at term; n=11). In all the animals, color and pulsed-wave Doppler examinations of uterine artery were conducted every 10 days from Day 20 to 50 from estimated luteinizing hormone peak. Doppler ultrasonography was also conducted in the fetuses to assess umbilical artery, abdominal aorta, renal and internal carotid arteries from Day 40 to 60 of gestation. Throughout the study, resistance index (RI) of uterine, umbilical and fetal renal arteries decreased up to -15% compared to -36% (P<0.01), -11% compared to -23% (P<0.05) and 2% compared to -13% (P<0.05), respectively in the abnormal and normal bitches. Fetal abdominal aorta and internal carotid did not differ between groups (P>0.05). It is concluded that in dogs, uterine artery, umbilical artery and fetal renal artery RI differ between normal and abnormal gestation being useful for the prediction of adverse obstetric outcome.

The GnRH antagonist acyline prevented ovulation, but did not affect ovarian follicular development or gestational corpora lutea in the domestic cat.

Two experiments were conducted to investigate the effects of the GnRH antagonist acyline (330 microg/kg, given sc) on ovarian follicular development and ovulation, as well as on pregnancy maintenance in domestic cats. In the first experiment, seven queens in proestrus (total of 24 proestrus periods), were randomly assigned to treatment with either acyline (ACY; n=17) or a placebo (PLC; n=7). All queens were mated with a fertile tomcat. In the ACY and PLC groups, cessation of estrus occurred (mean+/-SEM) 7.0+/-1.3 and 7.0+/-1.7 d after treatment (P>0.1), ovulation occurred in 2 of 17 and all seven estrus periods (P<0.05), and pregnancy rates were 1 of 16 and 7 of 7 (P<0.05), respectively. In the ACY and PLC groups, intervals from treatment to the onset of the ensuing proestrus were 18.4+/-1.7 and 120+/-17.2 d. In the second experiment, 14 pregnant queens were randomly allocated, according to their mating date, to treatment with acyline in early pregnancy (from 20 to 25 d, n=3), mid pregnancy (from 26 to 45 d; n=4), late pregnancy (> 45 d; n=3), or injection of a placebo in early (n=1), mid (n=2), or late pregnancy (n=1). Ultrasonographic assessments of the uterus were done every second day for 2 wk post treatment, and serum progesterone (P(4)) concentrations were determined before treatment, and at 7 and 14 d after treatment. No pregnancies were prematurely terminated and post-treatment P(4) concentrations did not differ among treatment groups (P>0.1). In conclusion, in the domestic cat, GnRH withdrawal by acyline prevented ovulation when given in early follicular phase (proestrus), but did not significantly affect luteal function during pregnancy.

Comparison of two doses of the GnRH antagonist, acyline, for pregnancy termination in bitches.

Gonadotropin-releasing hormone (GnRH) antagonists are particularly useful when a rapid inhibitory effect on the gonadal axis is required. The aim of this study was to test the efficacy and clinical safety of a low and high dose of the third generation GnRH antagonist, acyline, on pregnancy termination in female dogs. The effect of the antagonist on the progesterone (P(4)) serum concentration was also described. Twenty-one mid-pregnant bitches were randomly assigned to a single subcutaneous (SC) dose of a placebo (PLACE; n = 7), a low (ACY-L; 110 microg/kg; n = 6) or high (ACY-H; 330 microg/kg; n = 8) dose of acyline. The animals were followed up for 15 days. All ACY treated but no placebo-treated animals terminated their pregnancy by abortion (p < 0.01). The ACY-L and ACY-H groups interrupted their pregnancy 7 +/- 1.9 and 6.4 +/- 1.3 days after treatment, respectively (p = 0.7). A significant interaction between treatment and day was found (p < 0.01) for P(4) serum concentrations when PLACE was compared with both ACY groups. No difference was found for this hormone between both ACY groups (p > 0.05) where P(4) diminished throughout the study. The decreasing rate varied among animals and was closely related to the time of abortion when P(4) reached basal concentrations. In PLACE animals, gestation progressed normally and P(4) did not change throughout the study (p > 0.05). None of the bitches presented side effects. It was concluded that acyline safely terminated mid-pregnancy by permanently decreasing P(4) serum concentrations.

An experimental model to study resistance index and systolic/diastolic ratio of uterine arteries in adverse canine pregnancy outcome.

The aim of this study was to describe the changes in the resistance index (RI) and systolic/diastolic ratio (S/D) of the uterine arteries during mid-pregnancy abortion induction in the dog. Sixteen 30-35 day pregnant bitches were randomly assigned to either a pharmacological protocol to interrupt gestation (n = 8) or were used as untreated control group (n = 8). Doppler assessments of uterine arteries blood flow were carried out before the initiation of the protocol and then every other day up to abortion (treated group) or parturition (control group). All treated bitches aborted 6 +/- 1.2 days after initiation of the treatment (while none of the non-treated bitches aborted). Pre-treatment RI and S/D did not differ between groups (p > 0.2) while average post-treatment indexes were (mean +/- SD): 0.62 +/- 0.1 vs 0.53 +/- 0.1 (p < 0.01) and 2.96 +/- 0.9 vs 2.23 +/- 0.3 (p = 0.01), for the treated and non-treated group respectively. Correlations between days to abortion and RI or S/D were 0.75 (p < 0.01) and 0.79 (p < 0.01) and, -0.78 (p < 0.01) and -0.73 (p < 0.01) for the treated and non-treated groups respectively. In the treated group, correlations between serum progesterone (P(4)) concentrations and RI and S/D were -0.76 (p < 0.01) and -0.59 (p < 0.01) respectively. It is concluded that, during induction of abortion, RI and S/D of uterine arteries progressively increased while P(4) decreased.