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Simple Summary: Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most commonly used classes of drugs in both human and veterinary medicine. However, many clinical side effects have been observed, especially when treatment has been prolonged. While the anti-inflammatory efficacy and safety of repeated administration of firocoxib (Previcox®), which is a selective NSAID COX-2 inhibitor, has been evaluated for short-term use (one to fourteen days), its clinical relevance for longer-term use is not known. As a preliminary study, healthy, adult male and female horses (n = 7) were treated with daily oral firocoxib (0.11–0.14 mg/kg) for 40 days concomitant with the collection of blood samples encompassing treatment to assess hematological and biochemical endpoints. While this preliminary or pilot study was not without inherent limitations, treatment resulted in some hematological or biochemical effects that returned to pre-treatment or baseline values post-treatment without any clinically relevant adversity. Thus, the apparent favorable outcomes with the extended use of firocoxib provide a basis for future studies to reproduce these results with other more comprehensive study designs that may involve prolonged administration to healthy as well as injured or diseased horses to evaluate efficacy and the use of other clinically relevant endpoints. Non-steroidal anti-inflammatory drugs (NSAIDs) represent one of the most commonly used classes of drugs in both human and veterinary medicine. However, many clinical side effects have been observed, especially when treatment has been prolonged. While the anti-inflammatory efficacy and safety of repeated administration of firocoxib (Previcox®), which is a selective NSAID COX-2 inhibitor, has been evaluated for short-term use (one to fourteen days), its clinical relevance for longer-term use is not known. As a preliminary study, healthy, adult male and female horses (n = 7) were treated with firocoxib for 40 days concomitant with the collection of blood samples encompassing treatment to assess hematological and biochemical endpoints. Daily oral administration of firocoxib was performed with one 57 mg tablet/animal (0.11–0.14 mg/kg), which was crushed and mixed with feed. Blood samples were collected one day before treatment (D0 or basal sample), during (D10, D20, D30, and D40), and after treatment (D55 and D70). Results indicated some hematological and biochemical effects were significantly reduced (p < 0.05) towards the end of treatment on D40 relative to pre-treatment or baseline values on D0. Post-treatment, all values returned to pre-treatment values within 30 days without any apparent clinical adversities. In conclusion, while these preliminary results are favorable for prolonged use of firocoxib in horses, future studies are required to evaluate the efficacy of prolonged use accompanied with other clinically relevant endpoints in healthy as well as injured or diseased animals. [ABSTRACT FROM AUTHOR]

Cholesterol-loaded cyclodextrin (CLC) grants resistance to sperm during cryopreservation but may compromise sperm capacitation and fertility (Zahn et al. Theriogenology, 2002;58(2):237:240). Stimulants of sperm kinetics like pentoxifyllin (PTX) may increase sperm energy production and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al. Anim.Reprod.Sci. 2017;179:27-34). The present study aimed to stimulate semen motility by adding PTX after thawing of semen previously cryopreserved with different concentrations of CLC. Mangalarga Marchador stallions (n=12; 4-14 years) were assigned to four groups: (G1) untreated control, (G2) inclusion of cholesterol-loaded cyclodextrin (CLC) at concentrations of 1mg, (G3) 1.5mg and (G4) 2mg. Two straws of each stallion per group were thawed and divided into two aliquots, pentoxifylline (2 mg/mL, 7.18 mM) homogenized in BotuCrio®, Botupharma, Brazil (PTX: G1, G2, G3 e G4) was added to one and not to other aliquot (No-PTX: G1, G2, G3 and G4). Sperm kinetics was analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®) immediately after thawing (T0) or at 40 minutes (T40), 80 minutes (T80) and 120 minutes (T120) after thawing after incubation at 37°C. For statistical analysis, ANOVA F-test was performed.Intracellular hydrogen peroxide production was estimated by flow cytometry with dichlorodihydrofluorescein diacetate. Fertility was tested by deep horn insemination 0 to 6h after ovulation: No-PTX G1, No-PTX G3 and PTX G3. PTX reduced sperm velocity parameters at all times (p<0.05) compared to the groups without addition of pentoxifylline (No-PTX G1, G2, G3 and G4). Egg yolk based semen extenders may interfere with PTX cell penetration as they reduce permeability of the cell membrane. Regarding the integrity of the sperm plasma membrane and production of hydrogen peroxide, the use of pentoxifylline conferred an increase in sperm possibly under oxidative stress in the control group, with injured plasma membrane and with intracellular hydrogen peroxide (G1 PTX). When PTX was added to the group with highest concentration of cholesterol (G4 PTX), the intactness of the plasma membrane was improved, but with intracellular hydrogen peroxide. Mitochondrial potential and plasma membrane destabilization were also evaluated (n.s.). Pregnancy rates were 50% (2/4) for No-PTX G1, 67% (2/3) for No-PTX G3 and 67% (2/3) for PTX G3. In conclusion, addition of PTX did not interfere with mitochondrial potential, membrane destabilization and conception rate but was detrimental to cell longevity and to sperm kinetics. [ABSTRACT FROM AUTHOR]

Doppler ultrasound is a relevant method of monitoring testicular hemodynamics and widely used in andrological evaluations in humans. Testicular blood flow in men increases with age, while the pediatric phase is characterized by physiological testicular hypo-vascularization (Dudea et al., Medical Ultrasonography. 2010;12:43-51). Doppler ultrasound is rarely used in stallions which explains the lack of information and benchmarks. Variation in blood flow of adult stallions of different ages has, however, been reported (Pozor and McDonnell, Theriogenology. 2004;61:799-810) and should be further explored. The objective of the present study was to evaluate the blood flow pattern of the testicular artery in normal stallions (anatomically positioned testicle) of different ages. Both testicles of 30 Mangalarga Marchador stallions were evaluated by spectral Doppler and subjected to three different groups: Young (30 months-4 years, n=10), Adults (6-9 years, n=10) and Old (10-22 years, n=10). For the spectral mode, resistance and pulsatility indexes (RI and PI, respectively), systolic peak velocity (SPV), end-diastolic velocity (EDV) and timed average mean maximum velocity (TAMMV) were measured from the supra-testicular (Supra) and capsular (Cap) arteries.Stallions were evaluated during the reproductive season and were restrained without sedation. For statistical analysis, overall effect and pairwise testing with Šidák correction was performed. The effect of the predictor variables presence of torsion, age group and side were assessed using mixed models, Pearson correlations were also applied to assess the association between pairs of variables. Results showed higher SPV Supra in Old than in Young stallions (P<0.041) and, when testis were compared, higher PI Supra in left testicles for Old stallions was observed when compared to right testicles for Young and Adult stallions (P<0.001). These findings suggest lower testicular blood flow in Old stallions possible related to degenerative processes resulting in increased blood flow resistance. Higher PI in the left testicles may be related to early development of left testicles and epididymis (Nishikawa, 1959, Tokyo: Shiba Tamuracho Minatoku) and a lesser descent into the scrotum (Bergin et al., Biol. Reprod. 1970;3:82-92). The difference in perfusion between testes may induce more pronounced degenerative processes in the left testis due to its greater exposure to intra-abdominal temperatures. [ABSTRACT FROM AUTHOR]

Currently, the majority of the equine ART laboratories collect germinal vesicle (GV)-stage oocytes to perform ICSI, and the gonadotrophins used for in vitro maturation are mainly porcine, bovine, or human. In this study, the effect of two different concentrations of equine FSH (eFSH) in the maturation medium was evaluated. Cumulus-oocytes complexes (COCs) were collected via ultrasound-guided transvaginal follicle aspiration and randomly allotted to the groups. The control group was our standard maturation medium (BMM - TCM199-Earle's salts, 10% FBS, and additives) with human gonadotrophins added (Lazzari.et.al. Journal of Equine Veterinary Science. 2020; 89:103097); experimental groups were composed of BMM with eFSH added (0.01 and 0.05 IU/mL). After holding, COCs were transferred to maturation medium and cultured for 36h. ICSI was performed, and presumptive zygotes were cultured. The cleavage rate and number of cells were evaluated on D3, blastocyst formation and embryo quality, classified on A-B-C grade scale (adapted from Lewis.et.al. Reproduction, Fertility and Development. 2023; 35: 338-351), were evaluated from D6-10. Differences were analyzed by Fisher's Exact Test (significance level P<0.05). There was no difference (P>0.05) in degenerated, GV-stage, or MI oocytes after maturation for control, 0.01, and 0.05, respectively. The number of oocytes that reached the (meiosis) MII-stage was lower (P<0.05) for 0.01 U/mL (56.5%, 35/62)than the control (73.2%, 52/71), and also tended to be lower (P=0.0985) compared to 0.05 U/mL (71.2%, 47/66). There was no difference in cleavage or cell number among groups (Table 1). Group 0.01 U/mL had a higher (P<0.05) blastocyst rate (45.7%, 16/35) than control and 0.05 U/mL groups (28.8%,15/52; 25.5%, 12/47; respectively). No difference was found in embryo quality among groups. Although the 0.01 U/mL group showed a lower MII rate, it also showed numerically less 2-cell stage embryos, more 4-8 and 8-16 cells stage embryos, and more cleaved embryos. Additionally, the 0.01 U/mL group tended to have more high-quality embryos (grade A) than the control group and showed a higher total blastocyst rate compared to the other groups. Additional studies are required to establish an ideal maturation protocol using this new equine FSH. [ABSTRACT FROM AUTHOR]

Motility stimulants increase sperm energy production and their addition after semen thawing will increase motility (Stephens et al., J. Equine Vet. Sci. 2013;33:615-621), and stimulate metabolic pathways responsible for sperm capacitation (Guasti et al., Anim. Reprod. Sci. 2017;179:27-34). Semen of 12 Mangalarga Marchador stallions was cryopreserved with addition of different concentrations of cholesterol-loaded cyclodextrin (CLC): G1 (control – without cholesterol), G2 (1mg of cholesterol), G3 (1.5mg of cholesterol) and G4 (2mg of cholesterol). To run the experiment, two straws from each stallion per experimental group were thawed and divided into two aliquots of 400µL. This was supplemented with 100µL of Fert Talp, a Tyrode's albumin lactate pyruvate medium with (FT) or without (No-FT) heparin, a motility stimulant. This subdivided the four groups into eight. Sperm kinetics were performed analyzed by CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®). Sperm longevity was analyzed by using a thermoresistance test at 37°C (TTR). Statistical analysis was done by ANOVA F-test. Complementary to sperm kinetics, the production of intracellular hydrogen peroxide was estimated by flow cytometry using Dichlorodihydrofluorescein diacetate (DCFDA®). Results were classified as: Q1 [Plasma membrane (PM) injured without intracellular hydrogen peroxide]; Q2 (PM injured with intracellularhydrogen peroxide); Q3 (intact MP with intracellular hydrogen peroxide); Q4 (intact PM without intracellular hydrogen peroxide) and Tukey test was performed for statistical analysis. Immediately after thawing, there was no difference between FT and No-FT (p>0.05). After incubation at 37°C, the addition of FT showed higher values for RAP (percentage of rapid sperm, %), VAP (average path velocity, μm/s) and LIN (linearity of the curvilinear trajectory, %) after 80 minutes of incubation and also superior rates of RAP, VSL (straight line velocity, μm/s), VAP and LIN after 120 minutes of incubation (p<0.05), demonstrating satisfactory results both with regard to sperm kinetics and longevity. It can be concluded that the use of FT was beneficial for sperm velocity. These findings suggest that the addition of this motility stimulant after thawing may improve fertility because sperm velocity – VCL (curvilinear velocity, μm/s), VSL and VAP are the most reliable parameters to assess sperm quality (Verstegen et al. Theriogenology, 2002;57:149-179). Significant differences were also observed for Q4 (intact PM, without hydrogen peroxide), where the G2 FT group demonstrated superiority when compared to all groups not supplemented with FT (p>0.05), providing satisfactory plasmatic membrane results against oxidative stress. [ABSTRACT FROM AUTHOR]

Testicles are supported by spermatic cord and show some mobility within the scrotum. Excessive testicle mobility can result in rotation around its vertical axis, which may result in cord strangulation and reduction of blood perfusion (Filho and Oliveira, ABCMED. 2012). Depending on duration and severity of torsion, a reduction in diastolic velocity or retrograde diastolic flow may happen and lead to the absence of arterial flow, as a result of increased resistance to blood flow (Dogra et al., Journal of Clinical Ultrasound. 2001;29:105-108). Retrograde diastolic blood flow has been detected in stallions with 180° spermatic cord torsion (Pozor and McDonnel, Theriogenology. 2004;61:799-810). The objective of the present study was to evaluate testicular artery blood flow in stallions with 180° spermatic cord torsion in comparison to stallions with testis not affected by spermatic cord torsion. Both testis of 38 Mangalarga Marchador stallions were evaluated by spectral Doppler ultrasound. Animals with normal testis were divided into two experimental groups according to their age to verify if there would be any differences between groups: Normal Young, 30 months-4 years old (NY, n=10) and Normal Adults, 6-9 years old (NA, n=10). Similarly, animals with unilateral 180° spermatic cord torsion were divided into: Young with torsion, 30 months-4 years old (TY, n=10) and Adults with torsion, 6-9 years old (TA, n=8).For Doppler evaluations, spectral mode, resistance and pulsatility indexes (RI and PI, respectively), systolic peak velocity (SPV), end-diastolic velocity (EDV) and timed average mean maximum velocity (TAMMV) were measured at the supratesticular (Supra) and capsular (Cap) arteries. Stallions were evaluated during the reproductive season and were restrained without sedation. For statistical analysis, overall effect and pairwise testing with Šidák correction was performed. The effect of the predictor variables presence of torsion, age group and side were assessed using mixed models, Pearson correlations were also applied to assess the association between pairs of variables. For young stallions, when normal testis (n=20) were compared to rotated testis (n=15) and contralateral (n=5), rotated testis showed lower Supra RI than normal testis (P<0.001). For adult stallions, contralateral testis (n=7) Supra EDV were lower than normal testis (n=20) (P<0.001). Findings showed blood flow changes due to torsion in rotated testis and its contralateral. Therefore, these parameters should be considered to assess damage caused by torsion of the spermatic cord because of impaired testicular perfusion. Findings reinforce the theory of a reduced blood perfusion not only for the rotated testicles, but also for the contralateral one. [ABSTRACT FROM AUTHOR]

Cholesterol loss occurs in equine semen during cryopreservation and induces early capacitation and reduced viability in the female reproductive tract (Blanch et al., Reproduction in Domestic Animals. 2017;47(6):959-64). In order to increase cell resistance and longevity, the addition of cholesterol has shown satisfactory results. However, attention to the amount of cholesterol added is important as high concentrations may inhibit sperm capacitation (Contreras et al, Theriogenology. 2022;189:1-10). The object of the present study was to test the effect of cholesterol inclusion on equine semen characteristics, thus, addition of cholesterol-loaded cyclodextrin (CLC) was performed as described by Purdy and Graham (Cryobiology. 2004;48:36-45). Twelve healthy Mangalarga Marchador stallions, aged 4 to 14 years were assigned into four groups for the same ejaculate in a cross-over experimental design: without cholesterol (G1; control) or inclusion of CLC at concentrations of 1mg/120 × 106 sperm per mL (G2), 1.5mg/120 × 106 sperm per mL (G3) and 2mg/120 × 106 sperm per mL (G4).. After centrifugation, the pellet was resuspended in a freezing egg yolk-based extender (Botu-CrioTM, Botupharma, Brazil) at a final concentration of 100 × 106 sperm/mL. Semen samples were packed into 0.5 mL straws. Sperm kinetics were performed using CASA (Sperm Class Analyzer, SCA® v.4.0 Microptic®) and longevity wasevaluated by thermoresistance test at 37°C. For statistical analysis, the ANOVA F-test was performed. Evaluations were carried out at four times: T0 (immediately after thawing); T40 (after 40 minutes); T80 (after 80 minutes); and T120 (after 120 minutes). At T0, mitochondrial potential (probe JC-1, Sigma-Aldrich®) and plasma membrane destabilization (probe M540, Sigma-Aldrich®) were evaluated by flow cytometry (BD FACS Verse TM®) and Tukey test was performed for statistical analysis. At T0, higher total motility (TM) and progressive motility (PM) parameters were observed in G3 than in G1 (p<0.05). Progressive linear velocity (VSL) was higher in G3 when compared to G2 and in G4 (p<0.05). However, no difference was observed between G1 and G3. Mitochondrial potential and plasma membrane stability were similar in all experimental groups at T0 (p>0.05). After incubation at 37°C, at T40, T80 and T120, MT, MP and RAP parameters did not differ among groups. However, parameters related to cellular velocity (VCL, VSL and VAP) were higher in G3 than in G2 at T40 (VCL), T80 (VCL, VSL and VAP) and T120 (VCL and VAP). G4 showed superior VCL and VSL values at T80 than G2 (p<0.05). Therefore, 1.5mg and 2.0mg of CLC inclusion resulted in higher sperm kinetics, in addition it did not interfere with mitochondrial potential and the stability of plasma membrane and may therefore be a good option for cryopreservation of equine semen. [ABSTRACT FROM AUTHOR]

In the present study, the effects of four different electrolyte solutions on the packed cell volume (PCV), plasma volume and plasma concentrations of total protein, sodium, potassium, chloride, total magnesium, and ionized calcium in healthy and experimentally dehydrated horses were evaluated. Four crossbred horses, two males and two females were used. In experiment 1 (E1Des) the animals were experimentally dehydrated, while in the second experiment (E2Hig) healthy equines were used. In both experiments the animals were subjected to the following treatments: SE - isotonic electrolyte solution; SEGlu - glucose-enriched SE; SEMalt - maltodextrine-enriched SE and SEMg - magnesium sulphate-enriched SE. The electrolyte solutions used in all treatments were administered at 15mL kg-1 h-1 during 12h through small-bore nasoesophagus tube by continuous flow. The treatments with the electrolyte solutions resulted in an increase of plasma volume and a decrease in total plasma protein, hematocrit, while the electrolytes remained within the reference values. [ABSTRACT FROM AUTHOR]