Effect of early administration of progesterone on the function of the corpus luteum of llamas.

The aim of the present study was to evaluate if early administration of progesterone immediately after ovulation affects corpus luteum lifespan in llamas. Female llamas (n = 16) were induced to ovulate by Buserelin injection in the presence of an ovulatory follicle (Day 0). On Day 2, ovulation was confirmed and animals were randomly divided into two groups: treated animals (n = 8) received an intravaginal device containing 0.3 g of progesterone from Day 2 to Day 6 post-induction of ovulation and control group (n = 8) received a device with 0 g of progesterone. Blood samples were collected daily to determine plasma progesterone concentration and transrectal ultrasonographies were performed from Day 7 to Day 12 post-induction of ovulation. Mean maximum diameter of the corpus luteum was significantly lower and was reached before in the treated group than in the control group. The mean highest plasma progesterone concentration and the day that concentration was achieved were similar between groups. However, mean plasma progesterone concentration was significantly higher in the treated group than in the control group on Days 3 and 4 and lower on Days 8 and 9 post-induction of ovulation. The day that plasma progesterone concentration returns to 1 ng/ml differed between groups, occurring earlier in the treated group. In conclusion, the early increase of plasma progesterone concentration during the luteal phase, promoted the premature activation of the luteolytic process affecting corpus luteum function in llamas as it was previously reported in other species.

Current knowledge about the processes of luteolysis and maternal recognition of pregnancy in camelids.

Camelids have many unique reproductive features that considerably differ from those of other domestic species. Females are induced ovulators with subsequent development of a corpus luteum (CL) with a short lifespan. Plasma progesterone concentration starts to increase on day 4, peaks on day 8-9 and, in non-pregnant animals, basal concentration is reached around day 10-11 post-induction of ovulation. Luteolytic pulses of prostaglandin F2α (PGF2α ) are firstly detected on day 7 or 8 (approximately on day 5-6 after ovulation), with maximal luteolytic peaks observed between days 9 and 11 post-mating, in coincidence with a high endometrial expression of cyclooxygenase 2, a limiting enzyme in prostaglandins synthesis. Unlike other species, oxytocin seems not to be involved in the luteolytic process in these species. The CL is the main source of progesterone secretion, and its function is required to support pregnancy. Despite constant research efforts, aspects of reproduction and maternal recognition of pregnancy in camelids remain not fully understood. A transient decrease and subsequent recovery in plasma progesterone concentration are observed after day 9 post-mating in pregnant animals in association with a pulsatile release of PGF2α and a transitory decrease in CL vascularization. Thus, embryo recognition should occur between days 8 and 12 post-mating. In camels, conceptus tissues exhibit aromatizing activity with the capacity to synthesize large amounts of oestradiol. Similarly, llama blastocysts secrete oestradiol-17ß during the preimplantation stage, with a higher production during the elongation period. An increase in the endometrial expression of oestrogen receptor α is also observed on day 12 post-mating. All these evidences suggest that oestrogen could be the signal released by the embryo at the time of its recognition in camelids. Besides, nearly 98% of pregnancies are carried out in the left horn. A decrease in the endometrial expression of mucin 1 and 16 genes has been reported, suggesting that these changes are crucial for successful embryo implantation; however, no differences have been observed between horns. Thus, maternal recognition of pregnancy in camelids is a particularly complex process that must occur in a concise time to allow the rescue of the CL and embryo survival.

Effect of short-term artificial light and transvaginal progesterone device on first ovulation in late transitional mares.

In study I, plasma progesterone concentrations were evaluated in anoestrous mares that received an intravaginal progesterone release device (IPRD) for 10 days. Mares were divided into 3 groups based on the dosage of progesterone (0 g, n=3; 1.38 g, n=5; and 1.9 g, n=5). No statistical differences were found in plasma progesterone concentrations between the two doses tested. In study II, the effects of a protocol based on a short program of artificial light combined with an IPRD containing 1.38 g of progesterone on oestrous behaviour and onset of ovulation were evaluated. IPRDs were inserted into 31 late transitional mares (10 days of treatment). The mares were divided into a control group (n=9, IPRD with 0 g of progesterone) and two treatment groups (T1, n=10, IPRD with 0 g of progesterone and artificial light; T2, n=12, IPRD with 1.38 g of progesterone and artificial light). The percentages of mares in heat within the first 14 days after treatment were 100%, 70%, and 100% in the control, T1, and T2 groups, respectively (P=0.097), and their ovulation rates were 44%, 60%, and 100%, respectively (P≤0.01). In conclusion, a protocol based on artificial light and an IPRD containing 1.38 g of progesterone for 10 days could be considered to advance the first ovulation of the year in late transitional mares, as it ensures a higher rate of ovulation within the first 14 days after treatment.

Effect of hCG administration on Day 7 post-mating on accessory corpus luteum development and progesterone concentration in llamas.

A high embryo loss rate has been reported in llamas. As strategies that lead to an increase in plasma progesterone (P4) concentration might improve fertility, the aim of the present study was to evaluate if the administration of hCG on Day 7 post-mating is useful to develop an accessory corpus luteum (CL), increasing plasma P4 concentration. Twenty (n = 20) female llamas, ranging between 5 and 10 years of age and four (n = 4) males of proven fertility, ranging between 8 and 10 years of age were included in the study. Accessory CL developed in all treated llamas after hCG administration and plasma P4 concentration was significantly greater in treated than in control females (P˂0.0001). The diameter and vascularization of the original CL were not affected by treatment in pregnant llamas. However, in treated non-pregnant llamas, corpus luteum diameter was greater than in the control group from Day 14 post-mating until the end of the study (P˂0.001). In treated llamas, the accessory CL was detected throughout the study in pregnant and non-pregnant females, but its vascularization started to decrease around Day 16 post-mating in non-pregnant animals (P˂0.05). In conclusion, hCG treatment on Day 7 post mating was useful to induce an accessory CL and increase plasma P4 concentration in llamas. Thus, this treatment could be considered as a useful strategy to improve pregnancy rates in llama herds.

Plasma IGF1 and 17ß-Estradiol Concentrations During the Follicular Wave in Llamas.

The aim of this study was to characterize the temporal association between follicular waves and circulating concentrations of 17ß-estradiol (E2) and IGF1 in llamas. Follicular waves could be clearly divided in three phases: growth, plateau and regression; with a mean duration of 18.8 ± 0.32 days. All follicular waves showed overlapping, so that as one dominant follicle was regressing, another one was growing. E2 plasma concentration showed a wavelike pattern, similar to that followed by the dominant follicle; reaching its maximum concentration at the end of the growth phase and decreasing at the end of the plateau phase. IGF1 also showed variations during the follicular wave. It tended to increase during the growth phase and decreased toward Days 14 and 16. IGF1 reached its maximum concentration before E2 did (5 ± 0.8 vs. 7.2 ± 0.5 days after wave emergence) and before the maximum follicular diameter was attained (10.2 ± 0.46 days after wave emergence). Both hormones started to rise again in coincidence with the development of a new follicular wave. The observed profiles allow to suggest that IGF1 could have a role on folliculogenesis and ovarian steroideogenesis in llamas, as reported for other species.

Estradiol-17ß Injection Induces Ovulation in Llamas.

This study aimed to investigate the effect of three different doses of estradiol-17ß on ovulation and subsequent luteal development and function in llamas. Twenty-three llamas were examined daily by transrectal ultrasonography until the detection of an ovulatory follicle (≥8 mm). Thereafter, animals were divided into five groups: Control (n = 3; treated with 1.6 ml of saline solution), GnRH group (n = 6, treated with an intravenous injection of 8.4 µg Buserelin), and estradiol groups that received 0.6 mg (E1, n = 4), 1 mg (E2, n = 4), or 1.6 mg (E3, n = 6) of estradiol-17ß intravenously. Detection of ovulation was based on ultrasonographic visualization of disappearance of the largest follicle and subsequent presence of a newly formed corpus luteum (CL) and progesterone concentration exceeding 1 ng ml-1. Daily blood samples were collected to determine plasma progesterone concentration. Ovulation rate was 0% for control and E1 groups, 25% for E2 group, and 100% for GnRH and E3 groups. Differences in the mean CL diameter between GnRH and E3 groups were not statistically significant. Plasma progesterone concentration was similar between groups during the different days in ovulated animals. However, the day that the plasma progesterone concentration was above 1 ng ml-1 and the day that the highest plasma progesterone concentration was achieved differed among E3 and GnRH groups, occurring later in females treated with estradiol. In conclusion, an injection of estradiol-17ß is capable of inducing ovulation in llamas and the response depends on the dose used. Most of the animals required the highest tested dose (1.6 mg) to induce the ovulatory process. Although the CL diameter in females induced to ovulate with estradiol was similar to that in llamas induced to ovulate with a GnRH analog, the rise in plasma progesterone concentration above 1 ng ml-1 and the peak progesterone concentration were attained 1 day later in the estradiol treated females.

Administration of progesterone BioRelease LA inhibits follicular growth in llamas (Lama glama) regardless of follicle diameter at the start of treatment.

The aims of the study were twofold: first, the comparison of the pharmacokinetics parameters of two doses of Progesterone BioRelease® LA, (BioRelease Technologies, Lexington, KY, USA) one of 300 mg and other of 150 mg and their effects on ovarian dynamics in llamas. Based on the results from the first study, the aim of the second study was to evaluate the effect of the doses of 150 mg of progesterone on follicular activity considering the stage of the largest follicle at the beginning of treatment. The results in Study 1 showed that both doses of the formulation induced plasma progesterone concentrations higher than 1 ng/ml during the first 6 days of treatment in all females, progesterone concentrations steadily decline until Day 5 following by a slowly decrease. The total amount of progesterone released during treatment was higher in Group 300 than in Group 150 (p = 0.045). Mean maximum concentrations were 14.9 ± 2.24 and 14.3 ± 2.16 ng/ml for Group A versus Group B (p = 0.58), and they were registered on Day 1.5 ± 0.22 and 1.7 ± 0.34 days, respectively (p = 0.10). None of the animals of Group A showed progesterone concentration below 1 ng/ml during all studied period. The treatment applied in Study 2 was efficient in inhibiting the ovarian follicular dynamics and to start a superestimulatory treatment. The use of progesterone Biorelease® LA of 150 mg in comparison with the dose of 300 mg could be more effective in the use of synchronization protocols in llamas for AI or prior to the application of an ovarian superstimulatory treatment.

Antiproliferative Effects of Oxytocin and Desmopressin on Canine Mammary Cancer Cells.

Neoplasms of the mammary gland represent the most frequent tumor type in the female dog, and according to the histologic criteria, approximately 50% of them are malignant. In the most aggressive cases of mammary cancer, surgery is not enough to warrant a favorable outcome, and adjuvant therapies are needed to improve the patient's overall survival. The aim of the present study was to evaluate the effects of two peptides on proliferation of a canine mammary cancer cell line derived from a simple carcinoma. The cell line CMT-U27 was grown in 96-well plates, at two cell densities (4 × 103 and 8 × 103 cells/well). Cultures were treated with oxytocin (OT) or desmopressin at five concentrations (10, 50, 100, 500, and 1000 nM). After 72 h of incubation, cell proliferation was determined by the MTT assay. Results showed that with 4 × 103 cells/well, OT at 50, 500, and 1000 nM was growth inhibitory for the cells, being statistically significant at 1000 nM. On the contrary, no antiproliferative effect was observed with 10 or 100 nM. At 8 × 103 cells/well, OT showed a significant antiproliferative effect only with the highest concentration (1000 nM). Desmopressin at 4 × 103 cells/well decreased cell viability at concentrations of 50, 100, 500, and 1000 nM (statistically significant with the highest concentration), while no effect was observed with 10 nM. With 8 × 103 cells/well, this peptide reduced cell growth at 100, 500, and 1000 nM. In conclusion, we suggest that these peptides may be potential and promising compounds for the treatment of dogs with simple carcinomas of the mammary gland. In vivo studies are required to confirm this hypothesis.

Plasma and ocular prednisolone disposition after oral treatment in cats.

OBJECTIVE: To evaluate the plasma and aqueous humor disposition of prednisolone after oral administration in cats. METHODS: Six cats were administered with a single oral dose of prednisolone (10 mg). Blood and aqueous humor samples were serially collected after drug administration. Prednisolone concentrations in plasma and aqueous humor were measured at 0.25, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, and 5.0 h after administration by a high-performance liquid chromatographic analytical method developed and validated for this purpose. RESULTS: Mean ± standard error (SE) of maximum plasma prednisolone concentration (300.8 ± 67.3 ng/mL) was reached at 1 h after administration. Prednisolone was distributed to the aqueous humor reaching a mean peak concentration of 100.9 ± 25.5 ng/mL at 1.25 h after administration. The mean ± SE systemic and aqueous humor exposure (AUC) was 553.3 ± 120.0 ng h/mL and 378.8 ± 64.9 ng h/mL, respectively. A high AUC(aqueous humor)/AUC(plasma) ratio was observed (0.68 ± 0.13). The mean half-life time of elimination in plasma and aqueous humor was 0.87 ± 0.16 h and 2.25 ± 0.44 h, respectively. CLINICAL SIGNIFICANCE: The observed high ratio between aqueous humor and plasma prednisolone concentrations indicates that extensive penetration of prednisolone to the anterior segment of the eye may occur. This is the first step that contributes to the optimization of the pharmacological therapeutics for the clinical treatment of uveitis.

Therapeutic benefit of melatonin in experimental feline uveitis.

Uveitis is a frequent ophthalmic disorder which constitutes one of the main causes of blindness in domestic cats. The aim of this report was to analyze the effect of melatonin on experimentally induced uveitis in cats. Bacterial lipopolysaccharide (LPS) was injected intravitreally into one eye from intact cats, while the contralateral eye was injected with vehicle. Melatonin was orally administered every 24 hr to a group of ten cats, from 24 hr before until 45 days after intravitreal injections. Eyes were evaluated by means of clinical evaluation, intraocular pressure (IOP), blood-ocular barrier integrity (via measurement of protein concentration and cell content in samples of aqueous humor [AH]), electroretinogram (ERG), and histological examination of the retinas. In LPS-treated eyes, several clinical signs were observed until day 45 postinjection. The treatment with melatonin significantly decreased clinical signs and prevented the reduction in IOP induced by LPS. In LPS-injected eyes, melatonin significantly preserved the blood-ocular barrier integrity, as shown by a decrease in the number of infiltrating cells and protein concentration in the AH. Mean amplitudes of scotopic ERG a- and b-waves were significantly reduced in eyes injected with LPS, whereas melatonin significantly prevented the effect of LPS. At 45 days after injection, LPS induced alterations in photoreceptors and at the middle portion of the retina, whereas melatonin preserved the retinal structure. These results indicate that melatonin prevented clinical, biochemical, functional, and histological alterations induced by LPS injection. Thus, melatonin might constitute a useful tool for the treatment of feline uveitis.

Embryo transfer technique: Factors affecting the viability of the corpus luteum in llamas.

The aim of the present study was to evaluate the effect of the embryo transfer (ET) maneuvers on plasma progesterone concentrations in recipient Lama glama females and the relationship between the site the embryo was transferred to and corpus luteum (CL) localization. Experiment I (effect of transcervical threading): adult non-pregnant, non-lactating llama females were randomly assigned into two groups: control group (without cervical threading, n=10) and group A (with cervical threading, n=10). In both groups, CL activity was evaluated through measurement of progesterone plasma concentrations. In group A, on Day 6 after inducing ovulation with buserelin, the cervix was threaded to evaluate the effect of the maneuver on CL viability. No significant differences were observed in mean progesterone concentrations between groups (P>0.05). Experiment II (effect of depositing PBS): females (n=66) were randomly assigned into six groups (n=10 per group and control group: n=6) to evaluate the effect of depositing PBS in different sites in the uterus in relation to the localization of the CL: group 'Left-Ipsilateral': transcervical placing of PBS in the left uterine horn (CL in left ovary); group 'Left-Contralateral': transcervical placing of PBS in the left uterine horn (CL in right ovary); group 'Right-Ipsilateral': transcervical placing of PBS in the right uterine horn (CL in right ovary); group 'Body-Left': transcervical placing of PBS in the uterine body (CL in left ovary); group 'Body-Right': transcervical placing of PBS in the uterine body (CL in right ovary) and control group. Corpus luteum activity was evaluated in all groups by measuring plasma progesterone concentrations. On Day 6 post-buserelin, the corresponding maneuver was carried out according to the group. No significant differences were found for the mean plasma progesterone concentrations between groups (P>0.05). Experiment III (effect of ET on CL viability): females (n=22) were used as embryo donors and 50 females as recipients, in order to evaluate if placing the embryo in different areas of the uterus influences CL viability. Recipients were randomly divided into five groups, according to the place in the uterus where the ET was conducted with respect to the ovary where ovulation occurred: group 'Left-Ipsilateral': ET in the left uterine horn (CL in left ovary); group 'Left-Contralateral': ET in the left uterine horn (CL in right ovary); group 'Right-Ipsilateral': ET in the right uterine horn (CL in right ovary); group 'Body-Left': ET in the uterine body (CL in left ovary) and group 'Body-Right': ET in the uterine body (CL in right ovary). Corpus luteum activity was evaluated in all groups by measuring plasma progesterone concentrations. Embryos were recovered by flushing the uterus on Day 8 after the first mating of the donor and transcervical ET was carried out in recipients 6 days after buserelin administration. Pregnancy rates were: group 'Left-Ipsilateral': 50%; group 'Left-Contralateral': 20%; group 'Right-Ipsilateral': 30%; group 'Body-Left' and 'Body-Right': 10%. No significant differences (P=0.4728) were detected between the pregnancy rates in the five groups. Threading the cervix and transcervical placing of PBS either in the uterine horns or the body did not affect plasma progesterone concentrations in the llama, indicating that the different embryo transfer maneuvers do not interfere with CL viability. To improve pregnancy rates it could be suggested that ET in the left uterine horn with an ipsilateral CL, is the most desirable option.

Oestradiol-17beta plasma concentrations after intramuscular injection of oestradiol benzoate or oestradiol cypionate in llamas (Lama glama).

BACKGROUND: Llamas (Lama glama) are induced ovulators and the process of ovulation depends on dominant follicular size. In addition, a close relationship between behavioural estrus and ovulation is not registered in llamas. Therefore, the exogenous control of follicular development with hormones aims to predict the optimal time to mate. Oestradiol-17beta (E2) and its esters are currently used in domestic species, including camelids, in synchronization treatments. But, in llamas, there is no reports regarding the appropriate dosages to be used and most protocols have been designed by extrapolation from those recommended for other ruminants. The aim of the present study was to characterize plasma E2 concentrations in intact female llamas following a single intramuscular (i.m.) injection of two oestradiol esters: oestradiol benzoate (EB) and oestradiol cypionate (ECP). METHODS: Twelve non pregnant and non lactating sexually mature llamas were i.m. injected on day 0 with 2.5 mg of EB (EB group, n = 6) or ECP (ECP group, n = 6). Blood samples were collected immediately before injection, at 1, 6, 12, 24 h after treatment and then daily until day 14 post injection. Changes in hormone concentrations with time were analyzed in each group by analysis of variance (ANOVA) using a repeated measures (within-SS) design. Plasma E2 concentrations and area under the concentration-time curve (AUC) values were compared between groups by ANOVA. In all cases a Least-Significant Difference test (LSD) was used to determine differences between means. Hormonal and AUC data are expressed as mean +/- S.E.M. RESULTS: Peak plasma E2 concentrations were achieved earlier and were higher in EB group than in ECP group. Thereafter, E2 returned to physiological concentrations earlier in EB group (day 5) than in ECP group (day 9). Although plasma E2 profiles differed over time among groups there were no differences between them on AUC values. CONCLUSIONS: The i.m. injection of a single dose of both oestradiol esters resulted in plasma E2 concentrations exceeding physiological values for a variable period. Moreover, the plasma E2 profiles observed depended on the derivative of oestradiol administered. This basic information becomes relevant at defining treatment protocols including oestrogens in llamas.

Characterization of uveitis induced by use of a single intravitreal injection of bacterial lipopolysaccharide in cats.

OBJECTIVE: To investigate the use of a single intravitreal injection of bacterial lipopolysaccharide (LPS) to experimentally induce uveitis in cats. ANIMALS: 7 young male European shorthair cats that were considered physically and ophthalmologically healthy. PROCEDURES: In each cat, LPS was injected intravitreally into 1 eye; the contralateral eye was injected with the preparation vehicle. During a period of 45 days, both eyes were evaluated by means of clinical evaluation; assessment of the integrity of the blood-aqueous humor barrier (determined via measurement of protein concentration and cell content in samples of aqueous humor); functional analysis (via electroretinography); and following euthanasia, histologic examination of the retinas. RESULTS: In LPS-treated eyes, several clinical signs were observed until day 45 after injection. Compared with vehicle-treated eyes, intraocular pressure was significantly lower and protein concentration and the number of infiltrating cells were significantly higher in LPS-treated eyes. Mean amplitudes of scotopic electroretinographic a- and b-waves were significantly reduced in eyes injected with LPS, compared with findings in eyes injected with vehicle. At 45 days after injection, LPS-induced alterations in photoreceptors and the middle portion of the retina were detected histologically. CONCLUSION AND CLINICAL RELEVANCE: Results indicated that a single intravitreal injection of LPS in eyes of cats induced clinical, biochemical, functional, and histologic changes that were consistent with the main features of naturally occurring uveitis. This technique may be a useful tool in the investigation of new treatment strategies for uveitis in cats.

Circadian rhythm of intraocular pressure in cats.

OBJECTIVE: To evaluate the rhythm of intraocular pressure (IOP) in healthy domestic cats with no evidence of ocular disease and to analyze the influence of photoperiod, age, gender and ocular diseases on diurnal-nocturnal variations of cat IOP. ANIMALS: All animals were Domestic Short-haired cats; 30 were without systemic or ocular diseases, classified as follows: 12 male intact adult cats, five intact adult female, five adult spayed female, and eight male cats; the latter were less than 1 year of age. In addition, five adult cats with uveitis and three adult cats with secondary glaucoma were included. PROCEDURE: IOP was assessed with a Tono-Pen XL at 3-h intervals over a 24-h period in 12 healthy adult male cats kept under a photoperiod of 12-h light/12-h darkness for 2 weeks. Eight animals from the same group were then kept under constant darkness for 48 h, and IOP was measured at 3-h intervals for the following 24 h. In addition, IOP was assessed at 3 p.m. and 9 p.m. in five intact females, five spayed females, and in eight young cats, as well as in five adult cats with uveitis and three glaucomatous cats. RESULTS: Consistent, daily variations in IOP were observed in animals exposed to a light-dark cycle, with maximal values during the night. In cats exposed to constant darkness, maximal values of IOP were observed at subjective night. Differences of IOP values between 3 p.m. and 9 p.m. (diurnal-nocturnal variations) persisted in intact females, spayed females, and young animals, as well as in uveitic and glaucomatous eyes. CONCLUSIONS: The present results indicate a daily rhythm of cat IOP, which appears to persist in constant darkness, suggesting some level of endogenous circadian control. In addition, daily variations of cat IOP seem to be independent of gender, age, or ocular diseases (particularly uveitis and glaucoma).