Transcriptomics Reveal Molecular Differences in Equine Oocytes Vitrified before and after In Vitro Maturation.

In the last decade, in vitro embryo production in horses has become an established clinical practice, but blastocyst rates from vitrified equine oocytes remain low. Cryopreservation impairs the oocyte developmental potential, which may be reflected in the messenger RNA (mRNA) profile. Therefore, this study aimed to compare the transcriptome profiles of metaphase II equine oocytes vitrified before and after in vitro maturation. To do so, three groups were analyzed with RNA sequencing: (1) fresh in vitro matured oocytes as a control (FR), (2) oocytes vitrified after in vitro maturation (VMAT), and (3) oocytes vitrified immature, warmed, and in vitro matured (VIM). In comparison with fresh oocytes, VIM resulted in 46 differentially expressed (DE) genes (14 upregulated and 32 downregulated), while VMAT showed 36 DE genes (18 in each category). A comparison of VIM vs. VMAT resulted in 44 DE genes (20 upregulated and 24 downregulated). Pathway analyses highlighted cytoskeleton, spindle formation, and calcium and cation ion transport and homeostasis as the main affected pathways in vitrified oocytes. The vitrification of in vitro matured oocytes presented subtle advantages in terms of the mRNA profile over the vitrification of immature oocytes. Therefore, this study provides a new perspective for understanding the impact of vitrification on equine oocytes and can be the basis for further improvements in the efficiency of equine oocyte vitrification.

Lipid metabolites, interleukin-6 and oxidative stress markers in follicular fluid and their association with serum concentrations in mares.

The application of trans-vaginal ovum pick up (OPU) and intracytoplasmic sperm injection (ICSI) is well established for commercial in vitro embryo production in horses. These assisted reproductive techniques are especially applied during the non-breeding season of the mare. However, little is known about how the health of the oocyte donor may affect the biochemical composition of the follicular fluid (FF) in small and medium-sized follicles routinely aspirated during OPU. This study aimed to investigate associations between systemic and FF concentrations of interleukin-6 (IL-6), total cholesterol, triglycerides, non-esterified fatty acids (NEFA), reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), and oxidative stress index (OSI) during the non-breeding season in mares. At the slaughterhouse, serum and FF of small (5-10 mm in diameter), medium (> 10-20 mm in diameter), and large (> 20-30 mm in diameter) follicles were sampled from 12 healthy mares. There was a strong positive association (P < 0.01) between the concentration of IL-6 in serum and those measured in small (r = 0.846), medium (r = 0.999), and large (r = 0.996) follicles. Serum concentrations of NEFA were positively correlated (P < 0.05) with those measured in small (r = 0.726), medium (r = 0.720), and large (r = 0.974) follicles. Values of total cholesterol and OSI in serum and medium follicles were significantly associated (r = 0.736 and r = 0.696, respectively). The serum concentrations of all lipid metabolites were markedly higher than those measured in FF of small- and medium-sized follicles. Values of IL-6 and OSI did not change significantly between serum and all follicle classes (P ≥ 0.05). To conclude, changes in the blood composition associated with inflammation, oxidative stress, and disturbed lipid metabolism of mares may lead to an inadequate oocyte microenvironment, which could affect oocyte quality and the success rate of OPU/ICSI programs. Further research should indicate whether these changes may ultimately affect in vitro oocyte developmental capacity and subsequent embryo quality.

An updated insight on testicular hemodynamics: Environmental, physiological, and technical perspectives in farm and companion animals.

In all organs, control of blood flow is important but might be particularly critical for testicular functions. This is because of the very low oxygen concentration and high metabolic rate of the seminiferous tubules, the physiological temperature of the testis, and its location outside the abdominal cavity. Many factors affect the characteristics of TBF in farm and companion animals, such as environment (thermal and seasonal effects) and physiological (species, breeds, age, body weight, and sexual maturity). Thermal environment stress has detrimental effects on spermatogenesis and consequently has more serious impacts on both human and animal fertility. Numerous studies have been performed to assess TBF in different animal species including bulls, rams, bucks, alpacas, stallions, and dogs with varied results. Hence, assessment of TBF by Doppler ultrasonography is of great importance to estimate the effect of high environment temperature on testicular functions. Also, differences observed in the TBF may result from different technical aspects such as the identification of the segment of the testicular artery to be examined. In the current review, we focused on the imperative roles of TBF in various animal species. Besides, we discussed in detail various factors that could affect TBF. These factors can significantly modify the TBF and thus should be considered when establishing reference values in farm animals for better clinical diagnosis. The information provided in this review is valuable for researchers and veterinarians to help them a better understanding of testicular hemodynamics for the proper evaluation of breeding soundness examination in males.

Extracellular vesicles in low volume uterine lavage and serum: novel and promising biomarker for endometritis in Arabian mares.

BACKGROUND: Extracellular vesicles (EVs) are a promising biomarker and play a vital role in cell-cell communication. This study aimed (I) to identify and characterize EVs from low volume uterine lavage (LVL) and serum in mares with endometritis, compared to healthy controls and (II) to measure serum levels of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2). Mares were divided into 30 sub-fertile (endometritis) and 20 fertile (controls). Serum and LVL was collected for EV isolation, and determination of serum levels of inflammatory mediators. Characterization and visualization of EVs were done by electron microscopy, dynamic light scattering and flow cytometry. RESULTS: Serial ultracentrifugation of LVL and use of a commercial kit for serum were strategies for EVs isolation. Mares with endometritis released higher amounts of larger size EVs. The EVs from mares with endometritis differentially expressed CD9 and CD63, compared to controls. Mares suffering from endometritis evoked higher levels of inflammatory mediators. CONCLUSIONS: Thus, EVs could be used for a better understanding the regulatory mechanisms associated with developing endometritis in mares.

Ageing affects plasma steroid concentrations and testicular volume, echotexture and haemodynamics in rams.

The objective was to determine the effect of ageing on plasma steroid concentrations and testicular volume, echotexture and haemodynamics in Ossimi rams. Twenty-four rams were allocated, by age, into three groups: young (1 year; 32.00 ± 0.78 kg, n = 6); mature (2.5 years; 48.00 ± 1.02 kg, n = 9) and aged (7.5 years; 65.00 ± 1.10 kg, n = 9). Aged rams had the highest (p Ë‚ 0.05) plasma testosterone concentrations 3.90 ± 0.75 ng/ml (three-fold the other groups), whereas in young rams, plasma estradiol-17ß concentrations (5.85 ± 0.34 pg/ml) were approximately one-quarter (p Ë‚ 0.05) of other groups. Regarding pulsed-wave Doppler indices values of the (resistive index RI; and pulsatility index PI) were lower (p Ë‚ 0.05) in the mature (0.32 ± 0.04 and 0.36 ± 0.03, respectively) and aged (0.51 ± 0.03 and 0.77 ± 0.06) rams compared to young rams (0.62 ± 0.03 and 0.98 ± 0.07). In the young group, testicular pixel intensity was highest (p Ë‚ 0.05) but values of coloured area/pixels were lowest (p Ë‚ 0.05) compared to the other groups. In conclusion, these parameters indicate that aged rams (7.5 years) are likely to be suitable for breeding ahead of the younger aged groups (1 and 2.5 years) examined, but a final decision would need to be tempered by consideration of other parameters in the breeding soundness examination (BSE), such as physical soundness and semen characteristics. Of the parameters studied, testicular pixel intensity could be most readily incorporated in a BSE for deciding a ram's breeding efficiency.

Melatonin improves testicular haemodynamics, echotexture and testosterone production in Ossimi rams during the breeding season.

The objective was to determine effects of a single parenteral dose of melatonin on testicular blood flow indices, testicular echogenicity and plasma testosterone concentrations in rams during the physiological breeding season. We hypothesized that melatonin enhances testicular blood flow, echogenicity and plasma testosterone concentrations during the breeding season in rams. During the breeding season, 12 sexually mature Ossimi rams were randomly allocated to either a melatonin group (n = 8) that received 18 mg of melatonin in 1 ml of corn oil (injected SC) or a control group (n = 4) that received 1 ml corn oil only. Blood collection and ultrasonographic assessment of the testes and supratesticular arteries were conducted immediately before treatment (W0) and once weekly for 6 weeks after melatonin injection (W1-W6). Mean plasma testosterone concentrations were greater (p < .05; at least 1 ng/ml) in the melatonin-treated group compared to the control group from W4 to W6 after treatment. A decrease (p < .05) in both resistive index (RI) and pulsatility index (PI) began 1 week after melatonin injection (W1) and persisted until the end of the experiment, with mean RI and PI values in the melatonin group lower (p < .05) than those in the control group on W3 and W4. Furthermore, plasma testosterone concentrations in melatonin-treated rams were inversely correlated to both RI and PI (r = -.7 and -.6, respectively, p < .01). Testicular echogenicity decreased (p < .05) 1 week after melatonin injection (W1) and remained lower (p < .05) in the melatonin-treated group compared to the control group until the end of the study (W6). In conclusion, melatonin administration significantly altered testicular blood flow and echogenicity and increased plasma testosterone concentrations in Ossimi rams during the breeding season.

Exogenous oxytocin administration improves the testicular blood flow in rams.

The current research aimed to examine the effects of single-dose oxytocin administration on testicular blood flow measurements peak systolic velocity (PSV), end-diastolic velocity (EDV), resistive index (RI) and pulsatility index (PI) and plasma steroid (testosterone and oestradiol-17ß) concentrations in rams. In the treated group, eight mature Ossimi rams during the breeding season were injected intravenously (iv) with 20 IU oxytocin, while the other eight male rams were administered normal saline (2 ml) iv as a control group. Venous blood samples and testicular blood flow in the left and right testes were examined immediately before (0) and 5, 30, 60 and 120 min after injections. The control group did not show significant changes in the variables examined (p > .05), except for the EDV (p < .05). In the treated group, the administration of oxytocin led to a significant decrease in RI and PI values starting 5 min after oxytocin administration until 60 min after its application (p < .05). The plasma concentrations of testosterone and oestradiol-17ß raised from 5 to 30 min after oxytocin injection (p > .05) and then declined significantly until 120 min (p < .05). In conclusion, oxytocin is a potent testicular vasodilator affecting the testicular vascular tone and steroid concentrations in rams.

Alterations in the Expression Profile of Serum miR-155, miR-223, miR-17, miR-200a, miR-205, as well as Levels of Interleukin 6, and Prostaglandins during Endometritis in Arabian Mares.

So far the intimate link between serum microRNA (miRNA) and uterine inflammation in mares is unknown. We aimed (I) to investigate expression profile of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 (II) and to measure concentrations of interleukin 6 (IL-6), and prostaglandins (PGF2α and PGE2) in serum of mares with healthy and abnormal uterine status (endometritis). This study was conducted on 80 Arabian mares: young (4-7 years), and old (8-14 years). Mares were divided into 48 sub-fertile (endometritis) and 32 fertile (control) at stud farms. Serum was collected for measuring IL-6, PGF2α, and PGE2, as well as miRNA isolation and qRT-PCR. Concentrations of IL-6, PGE2, and PGF2α were higher in mares with endometritis compared to control. Age of mares had a remarkable effect on IL-6, PGE2, and PGF2α concentrations. Relative abundance of eca-miR-155, eca-miR-223, eca-miR-17, eca-miR-200a, and eca-miR-205 was higher in both young and old mares with endometritis. We noticed that eca-miR-155, eca-miR-223, eca-miR-200a, and eca-miR-205 revealed higher expression level in old than young mares with endometritis. This is the first study that has revealed the changes in cell free miRNA and serum inflammatory mediators during endometritis, and these findings could be used for a better understanding the pathophysiology mechanisms of endometritis in equine.

Hemodynamic changes in cytokines, chemokines, acute phase proteins and prostaglandins in mares with subclinical endometritis.

Apparently healthy mares with conception failure or embryonic loss may have subclinical endometritis (SE). Our objective was to document evidence of systemic immune responses against SE in mares. In apparently healthy (control) mares as well as those with chronic endometritis (ChE) or subacute suppurative endometritis (SSE), both considered classes of SE, serum concentrations of cytokines (IL-1ß, IL-6, IL-10 and TNF-α), chemokines (IL-8), acute phase protein (SAA), and plasma concentrations of prostaglandins (PGF2α and PGE2) were measured using validated enzyme linked immunoassays (EIA). Mixed-breed mares of known reproductive history, were used. Based on an endometrial cytological examination, mares were allocated into the following groups: healthy (control), ChE, and SSE (12, 26 and 11 mares, respectively). Serum concentrations of IL-6, IL-8 and IL-10 and plasma PGF2α concentrations were increased (P < 0.01) in mares with SSE compared to healthy mares. Furthermore, serum concentrations of IL-6 (P < 0.05) and IL-10 (P < 0.05) were elevated in the ChE group compared to the healthy group whereas serum SAA (P < 0.01) and plasma PGE2 (P < 0.05) were higher in healthy mares compared to ChE and SSE mares. However, serum concentrations of IL-1ß and TNF-α were not significantly different among groups. In conclusion, there were two novel findings: (1) development of serum testing for a set of biochemical markers has promise to explore the pathogenesis of inflammation in mares with SE; and (2) none of the individual biomarkers studied, excluding IL-8, was a significant predictor of SE. However, serum IL-6:IL-10 and IL-6:TNF-α concentrations, as well as plasma PGE2:PGF2α ratios, may yield a novel diagnostic marker for chronic subclinical endometritis.

Seasonal variation in testicular blood flow dynamics and their relation to systemic and testicular oxidant/antioxidant biomarkers and androgens in rams.

The environmental temperature increased during summer and decreased during winter to the limits that might negatively affect animal and human reproduction. The responses of Egyptian rams to either hot or cold climatic conditions were studied in six mature rams subjected to weekly testicular Doppler ultrasonographic examination, blood sampling, seminal plasma collection and semen evaluation. The maximum environmental temperature and the relative humidity were used to classify the climatic condition according to the heat stress equation of sheep into hot months where temperature-humidity index (THI) was >26 (31.67 ± 0.54), and cold months where THI was <22 (18.39 ± 0.41). Testosterone, estradiol, superoxide dismutase (SOD), glutathione peroxidase (GPX) and lipid peroxide product (malondialdehyde, MDA) were measured in both blood and seminal plasma, while catalase (CAT) and reduced glutathione (GSH) were measured in blood and seminal plasma, respectively. Results revealed that, during the hot months, rams displayed significantly decreased testicular blood flow, increased seminal plasma MDA, decreased seminal plasma (SOD, GPx and GSH) and blood CAT antioxidant enzymes. The present study evidenced two novel findings: (a) the marked decrease in testicular blood flow volume, that is remarkable increase in both resistive index (RI) and pulsatility index (PI) values, during hot months could be negatively affected both seminal plasma enzymatic activities and seminal attributes, and (b) the SOD and GPx activities in seminal plasma of such animals were suitable predictive markers for seminal attribute evaluation.