Conversion of Cortisone to Cortisol and Prostaglandin F2α Production by the Reproductive Tract of Cows at the Late Luteal Stage In Vivo

Previous in vitro studies demonstrated that bovine endometrium has the capacity to convert inactive cortisone to biologically active cortisol (Cr) and that Cr inhibits cytokine‐stimulated prostaglandin F_2α (PGF) production. This study was carried out to test the hypothesis that bovine reproductive tract has the capacity to convert cortisone to Cr in vivo and to evaluate the effects of intravaginal application of exogenous cortisone on uterine PGF secretion during the late luteal stage. The temporal relationships between PGF and Cr levels in uterine plasma were also determined. Catheters were inserted into jugular vein (JV), uterine vein (UV), vena cava caudalis (VCC) and aorta abdominalis (AA) of six cows on Day 15 of the oestrous cycle (ovulation = Day 0) for frequent blood collection. On Day 16, the cows were divided randomly into two groups and infused intravaginally with vaseline gel (10 ml; control; n = 3) or cortisone dissolved in vaseline gel (100 mg; n = 3). Blood samples were collected at ?2, ?1, ?0.5, 0, 0.5, 1, 1.5, 2, 3, 4, 5 and 6 h after treatments (0 h). Intravaginal application of cortisone increased plasma concentrations of Cr between 0.5 and 1.5 h in UV, at 0.5 h in VCC, at 1 h in JV and at 1.5 h in AA. The plasma concentrations of PGF in UV and of PGF metabolite in JV were greater at 0.5 and 1 h in the cortisone‐treated animals than in control animals. The levels of PGF in UV blood plasma decreased after Cr reached its highest levels. The overall findings suggest that the female reproductive tract has the capacity to convert cortisone to Cr in vivo. Based on the temporal changes of PGF and Cr levels in the uterine plasma, a biphasic response in PGF secretion was found to be associated to the Cr increase induced by the cortisone treatment at the late luteal stage in non‐pregnant cows.     

Effects of Matrix Filtration of Low-Quality Boar Semen Doses on Sperm Quality

The aim of this work was to develop a method to enhance the sperm parameters of ejaculates with low sperm quality from Piétrain boars. Seminal doses were filtered through columns of DEAE Sephadex (length 2.5 ± 0.5 cm), CM Sephadex (length 5 ± 0.5 cm), glass wool (length 2 ± 0.5 cm) or glass bead (length 10 ± 0.5 cm), with an exit flow rate of 1 ml/40 s in all cases. For each male, 10 ml of the sperm cell-rich fraction diluted at 1 : 6 were filtered. Sperm quality was assessed before and after filtration. Sperm morphology, sperm motility and sperm concentration were determined using the computer program sca ^® 2002 Production, and sperm viability was evaluated by fluorescence multistaining. Osmotic resistance test and hyperosmotic resistance test were used to determine the osmotic resistance of spermatozoa, whereas l -lactate production estimated the metabolic activity. Results showed a decrease of sperm concentration and osmotic resistance of spermatozoa after filtration in the four matrixes. However, an increase in the frequency of viable spermatozoa with intact acrosome after filtration in glass bead columns and an increase of morphologically normal spermatozoa after filtration in Sephadex CM-50, glass wool and glass bead columns were observed. Despite the decrease in the frequency of progressive motile spermatozoa, l -lactate production and mitochondrial sheath integrity maintained constant after filtration. Our findings indicate that column filtration is an effective method to enhance the sperm quality by selecting viable and morphologically normal spermatozoa without altering DNA, plasma membrane, mitochondrial sheath integrity or inducing premature acrosome reaction.     

OC3Morphometry Characterisation of Catalan Donkey Spermatozoa and Identification of Sperm Morphometric Subpopulations

In order to determine the injure produced in boar spermatozoa through cryopreservation process, we analyzed the expression of the hexose transporters Glut-3 and Glut-5 and the zona pellucida binding protein As-A (P68) in three different steps of the freezing-thawed protocol: at 17°C (fresh BTS-diluted semen, 1 : 2 v/v, step 1), at 5°C (after glycerol addition; step 2), and post-thawing (step 3). All sperm analyses were carried out with immunogold techniques under electronic microscopy. For this study eight healthy post-pubertal Iberian boars were submitted to a collection of twice per week through 3 months, evaluating two ejaculates from each boar. Glut-3 maintains the expression in the acrosome region post-thawing but not along the tail where is reduced. The expression of Glut-5 and As-A is majority located at the post-acrosome region of the spermatozoa at step 1, but in step 2 and step 3 this expression is relocated to sperm tail area. In conclusion, while cryopreservation affects the localization and the expression of Glut-3 and Glut-5, its fertilizing capacity is not significantly reduced. The stabilization of boar semen at 5°C was found to be the most crucial step for sperm survival.     

Effect of Insemination–Ovulation Interval and Addition of Seminal Plasma on Sow Fertility to Insemination of Cryopreserved Sperm

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.     

Antral Follicle Populations and Embryo Production – In Vitro and In Vivo – of Bos indicus–taurus Donors from Weaning to Yearling Ages

Interest in indicus–taurus cattle has been increasing, as these animals are likely to present the best characteristics of Zebu and European bovine breeds. The aim of this study was to compare the embryo production of indicus–taurus donors with high vs low antral follicle counts obtained by ovum pickup/in vitro production (OPU/IVP) and superovulation (SOV)/embryo collection. Braford females at weaning age (3/8 Nelore × 5/8 Hereford, n = 137, 9 ± 1 month old) were subjected to six serial ovarian ultrasonographs and were assigned to two groups according to the number of antral follicles ≥3 mm as follows: G‐High antral follicular count (AFC, n = 20, mean ≥40 follicles) and G‐Low AFC (n = 20, mean ≤10 follicles). When the females (n = 40) reached 24 months of age, they were subjected to both OPU/IVP and SOV/embryo collection. The average number of follicles remained highly stable throughout all of the ultrasound evaluations (range 0.90–0.92). The mean number of COCs recovered (36.90 ± 13.68 vs 5.80 ± 3.40) was higher (p < 0.05) for females with high AFC, resulting in higher (p < 0.05) numbers of total embryos among females with high vs low AFC (6.10 ± 4.51 vs 0.55 ± 0.83). The mean number of embryos per collection was also higher (p < 0.05) for G‐High vs G‐Low (6.95 ± 5.34 vs 1.9 ± 2.13). We conclude that a single ultrasound performed at pre‐pubertal ages to count antral follicles can be used as a predictor of embryo production following IVP and SOV/embryo collection in indicus–taurus females.     

Urinary free metanephrines measurement in dogs with adrenal gland diseases using a new simple liquid chromatography tandem mass spectrometry method

Measurement of urinary metanephrines in spot samples is used for the diagnosis of canine pheochromocytoma (PC). We describe a simple analytical method based on liquid chromatography tandem mass spectrometry (LC-MS/MS) for measuring free metanephrine (MN) and normetanephrine (NMN) in spot urine samples. Using the developed method, we evaluated the stability of urinary free-MN and free-NMN at various storing conditions. In addition, we assessed the feasibility of urinary free-MN and -NMN measurement for diagnosing PC. Urine samples were mixed with stable isotope internal standards and thereafter purified by ultrafiltration. The purified samples were analyzed by LC-MS/MS in the multiple reaction monitoring mode after separation on a multimode octa decyl silyl column. The coefficient of variation of free-MN and -NMN measurement was 7.6% and 5.5%, respectively. The linearity range was 0.5–10 µg/l for both analytes. Degradation was less than 10% for both analytes under any of the storage conditions. The median free-NMN ratio to creatinine of 9 PC dogs (595, range 144–47,961) was significantly higher (P<0.05) than that of 13 dogs with hypercortisolism (125, range 52–224) or 15 healthy dogs (85, range 50–117). The developed method is simple and may not require acidification of spot urine. The results of this preliminary retrospective study suggest that the measurement of urinary free metanephrines is a promising tool for diagnosing canine PC.     

Uterine Responses to Exogenous Oxytocin Before and After Pre-partum Luteolysis in the Cow

The aim of this study was to test the functional status of uterine oxytocin receptors in cows in vivo around parturition. The animals received consecutive, intra‐arterial injections of 800, 1600 and 3200 mU of oxytocin at three different stages: during late gestation (days 260–274), at 12 h and at 24 h after intramuscular injection of a prostaglandin F_2α analogue at day 275 to induce parturition. Cows (n=6) had been provided with myometrial electrodes and a catheter had been installed in the aorta and in a branch of the uterine vein (UV). Regular blood samples were obtained from the UV from 5 min before until 45 min after each oxytocin injection to measure plasma levels of prostaglandin F_2α (PGF_2α) and oxytocin. Uterine electromyographic (EMG) activity was registered continuously during each experiment. The increase of oxytocin levels in UV plasma after intra‐arterial injections was dose dependent (p < 0.02). Pre‐ and post treatment oxytocin levels at 24 h after induction of parturition were significantly increased (p=0.0313). Both during late pregnancy and at 12 h after induction of parturition, oxytocin caused a significant increase in EMG activity (p=0.022). After the 3200 mU dose the increase was significantly higher than with the other 2 doses (p=0.004). After each dose, EMG activity returned to baseline levels within some 15 min. At 24 h after induction of parturition, the pre‐treatment level of EMG activity had increased. Doses of 800 mU and 1600 mU of oxytocin produced a significant (p=0.022) increment of EMG activity, which was of the same magnitude as during the preceding stages; after 3200 mU of oxytocin the response was significantly higher than before (p=0.008). No significant increases of PGF_2α levels in UV plasma could be measured after oxytocin injections at any of the three stages. It is concluded that the myometrium of the pregnant cow responds in vivo to physiological doses of oxytocin. At 24 h after induction of parturition, when luteolysis has occurred and a parturient pattern of parturient myometrial activity has already started to develop, the response is enhanced. Physiological doses of oxytocin did not evoke a spurt release of PGF_2α in uterine venous blood during the peripartal period.