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The use of microfluidic technology is increasing in artificial reproduction technologies: With a small amount of semen, it allows for the selection of sperm with the best characteristics of kinetics, morphology and chromatin integrity. The ZyMot Multi (850 μl) is the most popular device of ZyMot Fertility Inc. To date, it was proven to be a valid instrument for sperm selection for in vitro fertilization and intracytoplasmic sperm injection in men. The aim of this study was to test the efficacy of the ZyMot Multi (850 μl) for stallion semen. Frozen-thawed semen from 15 stallions that were previously classified as being of 'good fertility' (GF; n = 8; pregnancy rate ≥ 40%) and 'poor fertility' (PF; n = 7; pregnancy rate 20%), respectively, was used. Each ejaculate was assessed before and after microfluid recovery for kinetics (CASA), membrane integrity (MI) (SYBR14/PI), membrane functionality (MF) (HOS test), acrosome integrity (Spermac Stain Kit), morphology (Spermac stain kit), mitochondrial membrane potential (MMP) (JC-1) and chromatin integrity (aniline blue staining). Sperm concentration was reduced after sperm recovery in both groups, but more markedly in frozen-thawed semen of PF stallions (p .05). Microfluid recovery increased total motility, MI, MF and MMP. While there was a significant increase in the percentage of progressively motile sperm after sperm microfluid recovery, there was a decrease in DAP, DSL, VAP, VSL, LIN, WOB and ALH (p .05). A slight increase (p .05) was detected in beat-cross frequency. The present results suggest that the ZyMot Multi (850 μl) device selects a specific sperm population from any stallion ejaculate with motile sperm and could therefore be a valid tool for in vitro testing with the aim to predict the fertility of frozen-thawed stallion semen.

The presence of bacteria of various origins on horse hoofs enables the onset of infections following trauma or even post-surgical wounds. Thus, the analysis of new antibacterial substances is of fundamental importance. In this study, the antibacterial efficacy of Iron Animals (IA), a stable colloidal suspension of iron oxide, organic acids, and detergents, was tested in vitro and in vivo. In vitro assays were performed to test the unspecific inhibitory effect of IA on both gram-positive and gram-negative bacteria monitoring the microorganism growth by spectrophotometry (optical density OD600) at 37 °C for 24 h. In vivo test consists on the quantification of the bacterial load in colony forming units per gram (CFU/g) of specimens collected from the frog region of the anterior hooves of 11 horses. Sampling followed the application of four disinfectant protocols consisting of two consecutive 3 min scrubs with 50 mL of 10% Povidone-iodine (PI) or 4% Chlorhexidine (CHx), with or without an additional application for 15 min of 10 mL of Iron Animals (PI+IA and CHx+IA). In vitro, IA completely suppressed the bacterial growth of all the tested microorganisms, resulting in effectiveness also against CHx-resistant bacteria, such as Staphylococcus aureus. In vivo, PI emerged as an ineffective protocol, CHx was effective in 18% of cases, but with the addition of IA (CHx + IA) its use emerged as the best disinfectant protocol for horse hoof, achieving the lowest bacterial load in 55% of cases. The addition of IA, after PI or CHx, improves the effectiveness of both disinfectants leading to the highest bactericidal activity in 82% of cases.

To test the hypothesis that delayed/impaired uterine involution could be associated with oxinflammation, we studied the progression of the uterine involution in association with some biomarkers of inflammation and oxidative stress in clinically healthy mares (N = 26) during early postpartum. The examination of the reproductive tract was performed on Days 7 and 21 after foaling. Uterine involution was assessed considering: a) the increase of the gravid uterine horn diameter (GUHD) compared with diameter recorded before pregnancy during the previous breeding season; b) the level of endometrial edema (EE); c) the degree of accumulation of intrauterine fluid (IUFA); d) the status of the cervix (CS). Inflammation and oxidative stress were studied by measuring serum amyloid A (SAA), cortisol, DHEA, AOPP, protein carbonyl groups, malondialdheyde (MDA) and thiols in plasma on Days 7 and 21. By Day 21 after parturition, a significant improvement (P < 0.01) was observed for GUHD and EE; while IUFA increased in six animals. Plasma SAA and DHEA concentrations were higher when the clinical parameters indicated a lower degree of uterine involution. On Day 7, the cortisol/DHEA ratio was lower in animals with higher degree of EE. Plasma AOPP and MDA concentrations were significantly lower (P < 0.05) in animals with the lower GUHD. On Day 21, plasma MDA concentrations were significantly lower (P < 0.05) in animals with the lower IUFA. Our data suggest that a mild condition of inflammation and oxidative stress occur in mares with delayed/impaired uterine involution., Animal science; Animal breeding; Veterinary medicine; Animal behavior; AOPP, Malondialdheyde, Oxinflammation, SAA, Uterine ultrasonography

The aim of this study was to investigate fetal gastrointestinal motility (FGM) of dogs using ultrasonic imaging and its association with vaginal and rectal temperature, serum progesterone concentrations and fetal heart rate. Pregnant bitches were examined after day 54 of gestation and there were determinations of vaginal and rectal temperature and serum progesterone concentrations. The fetal abdomen was evaluated for 30 s using longitudinal and transversal assessments, and FGM was scored as 0 (no peristalsis) or 1 (evident peristalsis). Number of fetuses with a 1 or 0 score were determined for each bitch (number and the percentage of fetuses with FGM). A total of 135 FGM measurements were recorded. There was FGM in 0/3, 0/6, 1/6 (16.7 %), 3/20 (15 %), 5/18 (27.3 %), 18/28 (64.3 %), 12/17 (70.6 %), 14/22 (63.6 %), 6/9 (66.7 %), 4/6 (66.7 %) fetuses from day -9 until 0 preceding parturition, respectively. In the last 5 days before parturition, 63.3 % of fetuses had FGM. Vaginal and rectal temperature were strongly and positively correlated (P 0.001). Vaginal temperature was positively correlated with progesterone concentrations and fetal heart rate (P 0.01), and there was a small negative correlation with FGM (r = -0.331, P 0.05). Due to ease of data collection, the assessment of FGM is a valuable procedure for evaluation of fetal maturity in dogs. Vaginal and rectal temperatures are reliable variables to be assessed during the last week of pregnancy for estimating the time of parturition.

Different additives have been tested in cooled stallion sperm, in order to maintain sperm quality and to ameliorate the decrease in sperm fertility potential. In several species, caffeine and pentoxifylline promote sperm motility by increasing energy production. We evaluate the effects of caffeine and pentoxifylline when added to stallion sperm before or after cooling. Three ejaculates from five stallions each were processed and resuspended in skim milk extender. Caffeine (5 mM), pentoxifylline (3.5 mM), or both additives combined were included to sperm before or after cooling (4°C for 24 hours). Cooled sperm were incubated at 37°C and evaluated at 0, 30, 60, and 120 minutes for motility, morphology, viability (flow cytometry), and membrane functionality (hypo-osmotic swelling test). Results were analyzed by two-factor mixed model for repeated measures and Tukey comparisons. As main effects, the caffeine and pentoxifylline affected significantly motility and kinematic parameters, without interaction between treatment and incubation after cooling. No differences were observed whether the additives were added prior or after cooling. Pentoxifylline added after cooling reduced significantly motility during incubation, but with higher values at 30 minutes. We detected a decrease in morphologically normal sperm (P < .0001), caused by an increase of tail defects (P < .003) in the presence of both additives. Viability and membrane functionality were also significantly impaired by additives. Pentoxifylline when added after cooling improved sperm motility and kinematic parameters for a short period of time. However, sperm characteristic related to fertility potential was compromised after a prolonged exposure to caffeine or pentoxifylline.

GnRH-agonist implants are currently marketed in Europe for use in male dogs but their off label use in female dogs, cats as well as other wild or semidomesticated animals is becoming increasingly popular. Implanted females frequently show a fertile heat after treatment, but the ensuing pregnancy may end spontaneously due to hormonal suppression at the hypothalamic level. This report describes the case of a jill treated with a 4.7 mg deslorelin implant in order to control her cyclicity. A few weeks later she was found to be pregnant and subsequently gave birth to 6 kits. The deslorelin implant did not affect fetal health or pregnancy duration. However, despite normal mammary development with milk production, the jill did not show adequate maternal instinct and did not take adequate care of her kits, 5 of which died during the following weeks. This is the first report of a normal pregnancy following administration of a deslorelin implant in a ferret.

Several studies reveal that coenzyme Q (CoQ) and vitamin E (Vit. E) act against oxidative deterioration, and that CoQ restores the active and antioxidant form of Vit. E. These two antioxidants, acting against lipid peroxidation, seem to be able to improve motility parameters of spermatozoa. The objective of this study is to evaluate the addition of CoQ and Vit. E to semen extender for equine spermatozoa in order to evaluate possible effects on semen motility. First, immediately after collection, semen samples were diluted with 1mM of CoQ and 1mM of CoQ plus 1mM of Vit. E and prepared for frozen storage in liquid nitrogen. After thawing (37 °C/30 s), samples were maintained at 37 °C and subjected to analysis after 0, 2 and 4 h for motility parameters with CASA (Computer-Assisted Sperm Analysis) method. In a second experiment, after the collection, semen samples were diluted with 1mM of CoQ, in presence or absence of seminal plasma where Vit. E is normally present, and prepared for cooled storage at 4 °C. The effects on motility parameters were determined with CASA at 0, 24, 31 and 48 h after collection. During the analysis, samples were kept at 4 °C. The CASA variables were examined with a mixed linear model. No improvement (p > .05) in motility parameters results from the addition of CoQ and Vit. E in frozen or cooled-stored equine semen when compared to control group.

The objective of this study was to determine the long-term effects of the administration of 4.7-mg slow-release deslorelin implant on stallions' plasma testosterone concentrations and sexual behavior. Five control animals received a subcutaneous dose of saline solution, whereas treated animals (n = 11) received a subcutaneous implant of 4.7-mg deslorelin acetate (Suprelorin; Virbac SA, Carros, France). Testosterone plasma concentrations were monitored from April to September every week for the first 30 days, then every 2 weeks until the end of the study (98 days in total). A stimulation test using human chorionic gonadotropin was performed before and 70 days after implant administration. Behavior was assessed by asking owners to fill out a questionnaire before treatment, weekly during the study, and then at 7 months after treatment. In treated stallions, an acute increase in testosterone concentration was evident within 7 days after treatment, which was followed by a gradual decline to below basal values over the next 42 days. Results of this study support that deslorelin is biologically active in the stallion. The answers to the questionnaires showed a significant decrease ( P = 0.03) in the rearing up behavior at week 3 and 4 after treatment. At the end of the study, of 11 owners, 5 asked their stallions to be reimplanted. Lacking side effects, deslorelin could be considered, in some instances, in the treatment of some behavioral problems in stallions, although there are individual variations in response, which deserve further investigations.

The aims of this study were to compare glycerol (G) at customary concentrations and ethylene glycol (EG) as cryoprotectants for stallion semen in a skimmed milk (SM) extender, to test different EG concentrations and to compare the results of manual and computerized analysis with the hypoosmotic swelling (HOS) test. Ejaculates from two stallions were collected over 3 weeks (6 ejaculates per stallion), diluted in a SM based extender, divided into 4 fractions, centrifuged and diluted again to a concentration of 100 x 10(6) mL(-1) progressive motile spermatozoa (PMS) in addition with the cryoprotectant (3% G, 3% EG, 6% EG, 9% EG). Sperm motility was assessed both by microscopy (in raw and frozen-thawed semen immediately after thawing) and with an HTM-IVOS analyzer (Hamilton-Thorne Research, MA, USA), at 0, 1, 4, 6, and 12 h after thawing and storage at 21 degrees C. Raw and frozen-thawed (0 h) semen samples for G and EG at 3% were also submitted to the HOS test with a 100 mOsm sucrose solution and were evaluated to detect the presence of swollen tails. The higher EG concentrations (i.e. 6% EG and 9% EG) significantly reduced the percentage of motile and PMS, immediately after thawing. At the same concentration, i.e. 3%, G resulted in a higher percentage of PMS than EG (36.2 vs. 30%, P < 0.05), but at 12 h after thawing and storage at 21 degrees C, no significant differences were detected between G and EG at 3%. The correlations between progressive motility (assessed by direct microscope observation or measured through the HTM analyzer) and the HOS test results for 3%G and EG were r = 0.61 and r = 0.35, respectively. The HOS test confirmed its suitability as a complementary method of analysis for stallion semen. We conclude that with the SM extender used, EG could substitute G as the cryoprotectant for stallion semen if used at the same or lower concentration.

An accurate clinical examination and in particular a careful collection of both signalment and anamnesis data can be helpful to predict both the result of the artificial insemination and the course of the pregnancy in the mare.

This study was conducted to evaluate clinical efficacy of deslorelin for inhibiting reproduction in the bitch. Ten adult healthy bitches or bitches with mammary neoplasia for which owners were requesting suppression of cyclicity without performing gonadectomy were administered a 4.7- or a 9.4-mg deslorelin implant subcutaneously. The first implant of deslorelin was administered in anoestrus (n = 5) or in dioestrus (n = 5). Treatment was repeated every 5 months for as long as necessary based on the clinical situation of the dog and owner's desires. Some of the bitches implanted in anoestrus came in heat within 4-15 days after treatment, while none of the bitches implanted in dioestrus showed heat during treatment. Suppression of reproductive cyclicity was successfully achieved in 6/10 bitches for 1-4 years. No behavioural and local/general side-effects were observed in any of the treated bitches. The 4.7-mg deslorelin implant may work well for suppression of cyclicity provided that it is administered in dioestrus and at intervals of 4.5 months. A 9.4-mg implant may be more suitable for this use although its efficacy may also be shorter than 12 months. Owner compliance is an important limiting factor.

Equine endometriosis is a multifactorial disease considered to be a major cause of equine infertility. The purpose of this study was to evaluate the reliability of histomorphological grading for biopsy-like samples compared to entire uterine wall samples, to examine the association between the degree of endometriosis with animal age, and to investigate the role of inflammation in endometriosis and the expression of different matrix metalloproteinases in equine endometrium. Histomorphological lesions in 35 uterine samples were examined while comparing biopsy-like samples and entire-wall samples. Seventeen uterine samples were stained with antibodies against MMP-2, MMP-9, MMP-14, and TIMP-2. The morphologic evaluation results of the biopsy-like tissue and entire-wall samples were significantly correlated. Endometriosis in older mares (>12 years of age) was more severe than in young mares (2 ~ 4 years of age), confirming the positive correlation between animal age and disease severity, while inflammation was poorly related to the degree of endometriosis. MMP-2 and MMP-14 were detected in stromal cells, while MMP-9 and TIMP-2 were both found in stromal and glandular epithelial cells. There were no significant differences in MMPs expression between the two groups (young vs. old mares). Additional studies on the activity of MMPs could further define the role of these enzymes in equine endometriosis.