Prolonged glucocorticoid administration affects oocyte morphology in cats (Felis catus) undergoing an ovarian stimulation protocol.

While captivity-related stress and the associated rise in baseline glucocorticoid (GC) concentrations have been linked to ovarian quiescence in some felid species, no study has examined the effects of elevated GC on oocyte quality. This study examined the effects of exogenous GC administration on the ovarian response and oocyte quality of domestic cats after an ovarian stimulation protocol. Entire mature female cats were divided into treatment (n = 6) and control (n = 6) groups. Cats in the GC treatment (GCT) group were given 1 mg kg-1 oral prednisolone daily from Day 0-45. All cats (n = 12) were given 0.088 mg kg-1 day-1 progesterone orally from Day 0-37, before treatment with 75 IU eCG im to induce follicular growth on Day 40, followed by 50 IU hCG im 80 h later to induce ovulation. Cats were ovariohysterectomised 30 h after the hCG treatment. Blood samples were collected on Days 0, 10, 30 and 40 (prior to eCG treatment), 80 h after eCG treatment, and on Day 45 for cortisol, glucose, prednisolone, oestradiol, and progesterone analysis. Cortisol concentrations did not differ between treatment groups throughout the study. Mean glucose concentrations were higher in the GCT cats (P = 0.004). Prednisolone was undetectable in all samples. Oestradiol and progesterone concentrations confirmed that the eCG treatment stimulated follicular activity and ovulation in all cats. Following ovariohysterectomy, the ovarian responses were graded (1 = excellent, 4 = poor) and oocytes retrieved from the oviducts. Each oocyte was given a total oocyte score (TOS: using an 9-point scale, 8 = best) based on four parameters: oocyte morphology, size, ooplasm uniformity and granularity, and zona pellucida (ZP) thickness and variation. Ovulation was confirmed in all cats, with a mean of 10.5 ± 1.1 ovulations per cat. Ovarian mass, ovarian response, number of ovulations, and oocyte recovery did not differ between groups. Oocyte diameter did not differ between the groups, but the ZP was thinner in the GCT group (3.1 ± 0.3 µm vs. 4.1 ± 0.3 µm, P = 0.03). The TOS was similar between treatment and control cats, but the ooplasm grade was lower (1.5 ± 0.1 vs. 1.9 ± 0.1, P = 0.01) and there was a tendency for ZP grade to be poorer (0.8 ± 0.1 vs. 1.2 ± 0.2; P = 0.08) in the treatment group. In conclusion, the GC treatment resulted in morphological changes to oocytes collected following ovarian stimulation. Whether these changes would affect fertility warrants further investigation.

Exogenous glucocorticoid treatment affects Sertoli cell load and epididymal sperm quality in domestic cats (Felis catus).

Elevated glucocorticoid (GC) concentrations associated with captivity-related stress have been linked to impaired testicular function and low sperm quality in felids, but direct physiological evidence is lacking. This study assessed the effects of exogenous GC treatment on felid testicular function using the domestic cat (Felis catus) as a model species. Sixteen intact male cats aged 2.4 ± 0.8 years (mean ± SEM) were divided randomly into treatment (n = 8) and control (n = 8) groups. Treatment cats were given 1 mg kg-1 oral prednisolone daily for 50 days. Blood samples were taken on Days 0 (first prednisolone treatment), 2, 4, 7, 10, 20, 30, 40, 50 (prior to neutering) and 60 of the trial. All cats were orchiectomised on day 50, epididymal sperm assessed, and the testes fixed for histological assessment. Testosterone concentrations did not differ between the two groups. While sperm motility was similar between the treatment and control groups, cats given prednisolone had a higher proportion of morphologically abnormal sperm in both the caput (72.5% vs. 59.6%, P < 0.001) and cauda (56.7% vs. 35.8%, P < 0.001) epididymis. Testicular histomorphometric data and total number of germ cells per seminiferous tubule cross section did not differ between groups, nor did the relative abundance of spermatogonia, spermatocytes, and spermatids. Cats given prednisolone had fewer Sertoli cells per tubule cross-section than those in the control group (17.1 ± 0.9 vs. 19.7 ± 0.8, P = 0.04), which was likely related to higher rates of Sertoli cell apoptosis in treatment versus control cats (0.25 ± 0.02 vs. 0.10 ± 0.02 apoptotic Sertoli cells per tubule, respectively; P < 0.001). Sertoli cell load (number of germ cells per Sertoli cell) was also higher in the treatment group than in the control group (11.5 ± 0.8 vs. 9.4 ± 1.2 germ cells per Sertoli cell, respectively; P < 0.001), and was positively correlated with the percentage of morphologically abnormal sperm in the epididymis (r2 = 0.78, P < 0.001). Prednisolone treatment resulted in an increase in the proportion of abnormal sperm in the epididymis, which may be explained by an increased nurturing demand on a reduced Sertoli cell population. These findings provide novel evidence to support the hypothesis that elevated GC concentrations, such as those resulting from captivity-related stress, have the potential to impair testicular function and sperm quality in felids.

Accelerometry and infrared thermography show potential for assessing ovarian activity in domestic cats (Felis catus).

Accurate and reliable monitoring of ovarian activity is challenging in many felids as current methods are either invasive or not amenable to real-time assessments. This 45-day study assessed whether accelerometry and infrared (IR) thermography can be used to address these limitations. Intact female domestic cats (n = 12) were given 0.088 mg kg-1 day-1 altrenogest (progestin) orally for 37 days to suppress follicular growth. On Day 40, cats were given 75 IU eCG im to induce follicular growth and 50 IU hCG im 80 h later to induce ovulation. Cats were ovariohysterectomised 30-31 h after the hCG treatment. Actical® accelerometers were fitted to the cats' collars and activity monitored continuously from the start of the altrenogest treatment until ovariohysterectomy. Infrared images of the perivulvar, perianal, and gluteal area were taken of each cat on Day 30 and daily from Days 36-45 of the study. Perivulvar temperature (PVT), PVT relative to gluteal temperatures (PVT-GT), and PVT relative to perianal temperature (PVT-PAT) were recorded for each image. Blood samples were collected on Days 0, 10, 30, and 40, immediately prior to the hCG treatment, and at the time of ovariohysterectomy. Serum oestradiol and progesterone concentrations indicated complete ovarian suppression by Day 30 and, together with morphological assessment of the ovaries, confirmed the induction of follicular growth and ovulation in all cats. Daily activity counts differed among cats (P < 0.001), so the daily activity counts of each cat were converted to a proportional change from the average daily activity count from Days 30-39 (defined as the 'proportional daily activity'). Proportional daily activity counts increased after the stimulation of follicular growth with eCG, with peak levels (2.03 ± 0.29-fold higher than pre-treatment levels; P = 0.006) occurring three days after the eCG treatment. The PVT-GT showed the greatest sensitivity to detect subtle changes in body temperature, increasing from early to late follicular growth (1.96 ± 0.33 °C increase from Day 41-43; P < 0.001) and decreasing after hCG-induced ovulation (1.24 ± 0.41 °C decrease from Day 43-45; P = 0.01). In conclusion, both accelerometry and IR thermography show potential as non-invasive, real-time methods for assessing ovarian activity in cats, but further research is required to determine if these methods could be used to monitor natural/non-stimulated oestrous cycles.

Monitoring ovarian function and detecting pregnancy in felids: A review.

Reliable detection and monitoring of estrus and pregnancy is essential to the effective ex situ conservation of endangered felids. Here, we review the current methods used to detect estrus and pregnancy in felids and describe the advantages and limitations of each. A total of 194 felid-focused publications were reviewed. The methods used included behavioral assessments (61 publications across 24 species), hormone monitoring (124 studies across 28 species), fecal protein monitoring (two studies in cheetah), ultrasonography (31 publications across seven species), vaginal cytology (22 publications across nine species), and laparoscopy (70 publications across 19 species). Behavior-based assessments of reproductive state are often inconsistent and unreliable in felids; thus hormone measurement is the most frequently used method for monitoring estrous cycles (66% of studies). In non-domestic felids, non-invasive fecal- or urine-based hormone metabolite analyses are preferred to blood assessments (66% of endocrine-based publications). While the measurement of fecal estrogen and progestin metabolites are useful for the retrospective assessment of ovarian activity, their use for real-time detection of estrus is limited. Vaginal cytology, laparoscopy and ultrasonography provide an acute and immediate determination of reproductive state but usually require anesthesia or sedation, which can prevent ovulation and cause abortions; thus, their use for pregnancy diagnosis is limited. Fecal progesterone or prostaglandin F2α metabolites can be used to detect pregnancy in most felids (the exception being fecal progestins for Lynx spp.), but only during mid-to-late gestation. Urinary relaxin measurement is a promising method for earlier pregnancy diagnosis (30-40% duration of gestation) but is presently limited by poor assay sensitivity due to the lack of a feline-specific relaxin antibody. Elevated levels of fecal immunoglobulin J chain have been investigated as a tool to detect pregnancy in cheetah (>80% accuracy), but research is lacking on other species. We conclude that there is no single 'best method' for monitoring ovarian activity and detecting pregnancy in felids, and current best practice would involve a combination of existing methods. Non-invasive methods for detecting estrus and pregnancy in felids (e.g., accelerometry and infrared thermography) should also be considered to augment existing methods.

Cellular Response of Neutrophils to Bismuth Subnitrate and Micronized Keratin Products In Vitro.

The aim of this study was to assess the effect of bismuth subnitrate and micronized keratin on bovine neutrophils in vitro. We hypothesized that recruitment and activation of neutrophils into the teat canal and sinus are the mechanisms of action of bismuth subnitrate and keratin-based teat sealant formulations. To test this, a chemotaxis assay (Experiment 1) and a myeloperoxidase (MPO) assay (Experiment 2) were conducted in vitro. Blood was sampled from 12 mid-lactation dairy cows of variable ages. Neutrophils were extracted and diluted to obtain cell suspensions of approximately 106 cells/mL. In Experiment 1, test substances were placed in a 96-well plate, separated from the cell suspension by a 3 µm pore membrane and incubated for 3 h to allow neutrophils to migrate through the membrane. In Experiment 2, neutrophils were exposed to the test products and the amount of MPO released was measured by optical density. Results showed that neutrophils were not activated by bismuth or keratin products (p < 0.05) in all of the tests performed. These results suggest that the mechanisms of action of bismuth subnitrate and keratin-based teat sealants do not rely on neutrophil recruitment and activation in the teat canal and sinus after treatment.

Aromatase inhibitor treatment with an intravaginal device and its effect on pre-ovulatory ovarian follicles in a bovine model.

BACKGROUND: Letrozole, a non-steroidal aromatase inhibitor, prevents the body from producing its own estrogen. The objectives of the present study were to test the hypotheses that letrozole treatment, initiated prior to selection of the preovulatory dominant follicle, will induce the growth of more than one follicle to a pre-ovulatory size, and will delay ovulation. METHODS: Post-pubertal beef heifers were given two luteolytic doses of PGF (12 h apart) and monitored by ultrasonography for ovulation. Five to eight days later, ovarian follicular wave emergence was synchronized by ultrasound-guided transvaginal follicular ablation (Day 0=wave emergence) and a luteolytic dose of PGF was given 60 and 72 h later. On Day 1, heifers were divided randomly into two groups (n=15/group) and an intravaginal device containing 1 g of letrozole or a blank device (control) was inserted. The intravaginal devices were removed on Day 7, or at the time of ovulation, whichever occurred first. Transrectal ultrasonography and blood sample collection were performed daily from the day of ablation to 12 days after subsequent ovulation. RESULTS: The mean (+/-SEM) interval from device placement to ovulation was longer in letrozole-treated animals compared to controls (6.1+/-0.25 vs 5.1+/-0.26 days, respectively; P<0.01). Single dominant follicles were present in both groups. The day-to-day diameter profiles of the dominant follicles of the ovulatory wave were larger (P<0.05) and the maximum diameters greater in letrozole-treated heifers (14.6+/-0.51 vs 12.4+/-0.53 mm, respectively; P<0.01). The diameter profile of the corpus luteum (CL) that formed after treatment did not differ between groups; however, plasma progesterone concentrations were higher (P<0.01) in heifers treated with letrozole. Estradiol concentrations were reduced following letrozole treatment (P<0.05), although a preovulatory rise of estradiol occurred in both groups. CONCLUSIONS: Administration of letrozole with an intravaginal device during growth of the ovulatory follicle delayed ovulation by 24 h and resulted in the formation of a CL that secreted higher levels of progesterone. A sustained-release intravaginal device may be useful for the development of an aromatase inhibitor-based protocol to control ovulation for herd synchronization and to enhance fertility by increasing circulating progesterone concentrations during the first 7 days post-ovulation in cattle.

A bovine model for examining the effects of an aromatase inhibitor on ovarian function in women.

OBJECTIVE: To test the hypothesis that treatment with an aromatase inhibitor (letrozole) will terminate dominant ovarian follicle growth and result in the emergence of a new follicular wave, regardless of the stage of follicular development at the time of treatment. DESIGN: Prospective study. SETTING: Academic research center. ANIMAL(S): Postpubertal beef heifers. INTERVENTION(S): Randomized trial involving treatment with letrozole on days 1-3, 3-5, 5-7 (day 0 = pretreatment ovulation) or no treatment. MAIN OUTCOME MEASURE(S): Follicular development, corpus luteum (CL) development, hormone profiles, and plasma aromatase inhibitor concentration. RESULT(S): Multiple doses of letrozole lengthened the period of follicular dominance, delayed emergence of the next follicular wave, and resulted in a larger CL regardless of the stage of the follicular wave in which treatments were initiated. No effects on circulating FSH concentrations were detected, but the stimulatory effects on the dominant follicle and CL were associated with increased plasma concentrations of LH in letrozole-treated animals. Plasma P concentrations were numerically higher throughout the luteal phase in letrozole-treated versus control heifers, but differences were not significant. CONCLUSION(S): The results provide rationale for the hypothesis that the mechanism of action responsible for the stimulatory effect of aromatase inhibitors on ovarian function involves an elevation in circulating concentrations of LH rather than FSH.