Identification of Apoptotic Bodies in Equine Semen

Apoptosis in the testis is required to ensure an efficient spermatogenesis. However, sometimes, defective germ cells that are marked for elimination during this process escape elimination in the testes, giving rise to ejaculates with increased percentages of abnormal and apoptotic spermatozoa and a high percentage of apoptotic bodies. Apoptosis markers in the ejaculate have been associated with low fertility, either in animals or humans. Therefore, the goal of this study was to investigate whether fresh equine semen contains apoptotic bodies [initially named Merocyanine 540 (M540) bodies] and to study the relationship between the quantity of these bodies and cell concentration, the volume of ejaculate, viability and motility. Moreover, we also studied whether the presence apoptotic bodies in fresh semen was related to the resistance of the stallion spermatozoa to being incubated at 37°C or being frozen and thawed. Fresh equine semen was stained with fluorescent dyes such as M540 and Annexin‐V. Active Caspase 3 was studied in fresh semen through Western blotting and immunofluorescence with a specific antibody. Sperm kinematics was assessed in fresh, incubated and thawed samples using computer‐assisted semen analysis, and viability was evaluated with the LIVE/DEAD Sperm Viability Kit. Overall, our results demonstrate for the first time the presence of apoptotic bodies in equine semen. The quantity of apoptotic bodies was highly variable among stallions and was positively correlated with Caspase 3 activity in fresh samples and negatively correlated with the viability and motility of stallion spermatozoa after the cryopreservation process.     

Colloid Centrifugation Selects Normal Spermatozoa from Polymorphic Bull Ejaculates: A Case Study

Semen from a Western Finncattle bull exhibiting a highly polymorphic spermiogram was processed by colloid centrifugation using Androcoll‐B, a species‐specific silane‐coated silica colloid. In the first experiment, Single Layer Centrifugation (SLC) was used to identify which density colloids were needed to separate different cell populations. Colloids of the two chosen densities were then used in a density gradient resulting in two sperm subpopulations, one containing nearly all normally sized spermatozoa and the other enriched for the macrocephalic spermatozoa. Microcephalic spermatozoa did not appear in either of the selected subpopulations. Using a combination of SLC and DGC with this species‐specific colloid, it was possible to separate the spermatozoa into different subpopulations, that is, a subpopulation containing nearly all normally sized spermatozoa, and another one enriched for the macrocephalic spermatozoa. Thus, colloid centrifugation could be used to select sufficient normal spermatozoa from a highly polymorphic ejaculate for AI, if desired.     

Anti‐Mullerian Hormone Concentration and Antral Ovarian Follicle Population in Murrah Heifers Compared to Holstein and Gyr Kept Under the Same Management

This study was performed to evaluate plasma concentrations of anti‐Mullerian hormone (AMH) and the ovarian antral follicle population (AFP) in different genetic groups. Cyclic heifers (13 Bubalus bubalis [Murrah]; 15 Bos taurus [Holstein] and 10 Bos indicus [Gyr]) were maintained under the same management and were synchronized with two doses of 150 μg IM d‐cloprostenol administered 14 days apart. After the second d‐cloprostenol treatment, heifers had their ovaries scanned daily by ultrasound to define the day of ovulation. On the same day, the AFP was determined and a plasma sample was collected to measure AMH. Murrah heifers had less AFP (25.6 ± 2.1 follicles; p = 0.01) and plasma AMH concentration (0.18 ± 0.03 ng/ml; p < 0.001) than Gyr (60.0 ± 12.2 follicles and 0.60 ± 0.12 ng/ml of AMH); however, data were similar when compared to Holstein (35.9 ± 6.8 follicles and 0.24 ± 0.06 ng/ml of AMH) heifers. Regardless of genetic background, there was a positive relationship between the AFP and plasmatic AMH concentration (Murrah [r = 0.62; p < 0.01], Holstein [r = 0.66; p < 0.001] and Gyr [r = 0.88; p < 0.001]). Also, when heifers were classified according to high‐ or low‐AMH concentration based on the average within each genetic group, high‐AMH heifers had greater (p < 0.0001) AFP than low‐AMH heifers. In conclusion, both Murrah and Holstein heifers presented lower plasma AMH concentration and AFP when compared to Gyr.     

Effect of Overnight Staining on the Quality of Flow Cytometric Sorted Stallion Sperm: Comparison with Tradtitional Protocols

Flow cytometry is considered the only reliable method for the separation of X and Y chromosome bearing spermatozoa in equines. The MoFlo SX DP sorter is highly efficient, allowing the production of foals of the desired sex. However, to achieve acceptable pregnancy rates the currently used protocol requires working with fresh semen obtained close to, or at, the sorting facility. An alternative protocol was tested during two consecutive breeding seasons. Fresh stallion semen was cooled for 20 h, during which staining with Hoechst 33342 took place. On the following day, this sample was flow sorted and compared with spermatozoa from the same ejaculate that had been sexed on the previous day. All sperm parameters evaluated remained unchanged when fresh sorted and refrigerated sorted semen were compared. Pre‐sorting storage at 5°C did not alter sperm velocities nor kinetics, viability or membrane permeability, production of reactive oxygen species, mitochondrial membrane potential or DNA fragmentation index of the sorted sample. The findings open for the possibility of using semen from stallions housed far from the sorting facilities. Processed and stained sperm could be shipped refrigerated on the previous day, sorted and inseminated on the next day.     

Standing Laparoscopic Peritoneal Flap Hernioplasty of the Vaginal Rings does not Modify the Sperm Production and Motility Characteristics in Intact Male Horses

Laparoscopic hernioplasty techniques have been developed in the recent years to avoid the recurrence of inguinal hernias and to spare the testicles for breeding purposes in stallions. However, there have been no previous comprehensive and systematic studies of the reproductive outcomes and prognoses for stallions after inguinal hernioplasty. Therefore, the objective of this study was to assess the possible effects of one of these techniques (standing laparoscopic peritoneal flap hernioplasty) on the sperm production and motility characteristics of six healthy stallions that received this procedure based on 1‐year follow‐ups. There were no significant differences in the measured sperm variables (assessments based on the DSO, MOT, PMOT, VSL, VCL and VAP) during 1‐year follow‐ups.     

Changes in Bull Sperm Kinematics after Single Layer Centrifugation

The objective of this study was to evaluate bull sperm kinematics after centrifugation through a single layer of a colloid [Single Layer Centrifugation (SLC)]. Ejaculates from 20 bulls were extended and stored at 4–6°C for 24 h during transport to the laboratory for SLC through Androcoll‐B, followed by measurement of sperm kinematics in all samples. Total motility (86% and 88% for uncentrifuged and SLC samples, respectively) and progressive motility (84% for both the groups) were similar (p > 0.05). In contrast, straightness (STR) (0.65 vs 0.69), linearity (LIN) (0.32 vs 0.35) and beat cross frequency (BCF) (22.3 vs 23.6 Hz) were significantly higher in the SLC‐selected samples than in the uncentrifuged samples, whereas velocity of the average path (VAP) (95 vs 90 μm/s), curvilinear velocity (VCL) (192 vs 180 μm/s), amplitude of lateral head deviation (ALH) (7 μm vs 6.5 μm) and hypermotility (49% vs 38%) were significantly decreased. The kinematics of the samples with the poorest motility was improved most by SLC. In conclusion, even though SLC had no direct effect on total and progressive motility, it appeared to have a positive influence on several other kinematic parameters that may be important for fertilization after artificial insemination.     

Efficacy of Long Acting Oxytetracycline in the Treatment and Control of Bovine Dermatophilosis

Abstract— Fifty-two cattle on two ranches in St. Kitts were divided into initial treatment and control groups, matched as closely as possible by age, sex and extent of dermatophilosis. Nine unaffected animals were included. The initial treatment group was given long-acting oxytetracycline at the rate of 20 mg.kg^-1. After 4 weeks the control group was similarly treated. The cattle were observed at weekly intervals for a total of 6 weeks. In each group there was a pronounced decrease in the extent of the disease after treatment. This was most apparent in lightly affected animals and on the trunk. Lesions tended to persist in the tick predilection sites and extremities. Complete recovery occurred in 8 (21 per cent) of cattle. The disease tended to recur in treated animals particularly on one of the ranches in which a low standard of husbandry prevailed. On the other ranch improvements in tick control and feeding were associated with a continuing low level of infection. The results show that long-acting oxytetracycline can be used to conveniently halt outbreaks of dermatophilosis and allow time for changes in management designed to reduce recurrence of the disease. Résumé— 52 bovins présents sur 2 ranchs à Saint-Kitts, ont été divisés en groupes d'animaux traités et en groupe d'animaux témoins, répartis aussi minutieusement que possible selon l'âge, le sexe et l'extension de la dermatophilose. Le groupe des animaux traités a reçu de l'oxytétracycline-retard à la dose de 20 mg.kg^-1. Après 4 semaines, le groupe d'animaux témoins fut traité de la même manière. Les bovins furent observés une fois par semaine pendant une durée totale de 6 semaines. Dans chaque groupe, il y eut une baisse sensible de l'extension de la maladie après le traitement. Ce fut le plus manifeste sur des animaux légèrement atteints et sur le tronc. Les lésions ont tendu à persister sur les sites de prédilection des tiques et sur les extrémités. Une guérison complète fut notée sur 8 (21%) des bovins. Les maladies tendaient à récidiver sur les animaux traités, principalement dans l'un des ranchs, où les conditions d'entretien étaient d'un niveau médiocre. Dans l'autre ranch, des améliorations dans l'erradication des tiques et dans l'alimentation ont été associées avec la persistance d'un niveau faible d'infection. Les résultats montrent que l'oxytétracycline retard peut être utilisée pour árrêter commodément les poussées de dermatophilose et laisser du temps pour modifier les méthods afin de récluire la récidive de la maladie. Zusammenfassung— 52 Rinder in zwei Betrieben auf St. Kitts wurden in je eine Erstbehandlungs-und eine Kontrollgruppe aufgeteilt, die mÖglichst ähnlich in Alter, Geschlecht und Ausprägungsgrad der Dermatophilose waren. Neun dieser Tiere waren nicht erkrankt. Die Erstbehandlungsgruppe erhielt ein Langzeit-Oxytetracyclin in der Dosierung von 20mg.kg^-1. Nach 4 Wochen wurde die Kontrollgruppe nach dem gleichen Schema behandelt. Die Tiere wurden über einen Zeitraum von sechs Wochen in wÖchentlichen Abständen untersucht. In beiden Gruppen besserte sich der Schweregrad der Erkrankung nach Therapie deutlich, besonders bei den nur leicht befallenen Tieren und bei Läsionen im Stammbereich. Dagegen neigten die Veränderungen an den Prädilektionsstellen für Zecken und an den Gliedmaßen zur Persistenz. Acht der Tiere (21%) wurden vÖllig wiederhergestellt. Die Erkrankung neigte aber bei den therapierten Tieren zu Rezidiven, vor allem in einem der Betriebe mit niedrigem Pflegeniveau. Nach den vorliegenden Untersuchungen ist die Verwendung von Langzeit-Oxytetracyclin ein brauchbares Mittel, um die Dermatophilose zum Stillstand zu bringen und neue Ausbrüche zu verhindern, bis weitere Änderungen im Management zur Verringerung der Rezidivrate getroffen worden sind. Resumen Cincuenta y dos vacas de 2 explotaciones en St. Kitts se dividieron en un grupo de tratamiento y un grupo de control. Ambos grupos contenian aproximadamente la misma distributión de sexos y la dermatofilosis estaba extendida en igual medida. Se incluyeron 9 animales no afectados. Los animates del grupo de tratamiento inicial recibieron oxitetraciclina de actión retardada a una dosis de 20mg.kg^-1. Cuatro semanas más tarde los animales del grupo de control recibieron el mismo tratamiento. Las vacas fueron observadas semanalmente durante 6 semanas. En cada grupo se produjo una marcada reducción en la extensién de la enfermedad después del tratamiento. Esto fue más marcado en aquellos animales afectados sólo de forma leve en la región del tronco. Las lesiones tendian a persistir en las zonas de predilectión de las garrapatas y en las extremidades. En ocho animales se observó una curatión completa (21%). La enfermedad tendia a recidivar en los animales tratados expecialmente en una de las explotaciones en la cual se mantenían unas condiciones de cría bastante desfavorables. En la otra explotación el control de garrapatas y una mejoria en la alimentatión aparecieron asociadas a un bajo nivel de presentación de la enfermedad. Estos resultados indican que la oxitetraciclina de actión retardada puede utilizarse para controlar focos de dermatofilosis, produciendo remisiones que permiten mejorar el manejo de las explotaciones y evitar recidivas de la enfermedad.     

Plasma indicators of muscle damage in a model of nutritional myopathy in weaner sheep

SUMMARY Subclinical nutritional myopathy was induced in 5-month-old sheep by feeding them a diet low in vitamin E and selenium. Subsequently clinical myopathy was induced by dosing with protected polyunsaturated fatty acids. Plasma activities of creatine kinase (CK), pyruvate kinase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase and aldolase, enzymes of muscle origin, all remained above their reference ranges in clinically affected sheep, but fluctuated widely. Similar fluctuations occurred in subclinically affected animals, resulting in some activities being within the reference ranges and some above, at different times. Plasma malondialdehyde, an indicator of lipid peroxidation, proved of no diagnostic value. Terminal plasma CK activities were significantly correlated with microscopic damage in the vastus lateralis (VL), but not the vastus intermedius (VI) or the tensor fascia lata (TFL) muscles. AST was the most highly correlated with damage in VI and VL. In two clinically affected sheep successfully treated with an oral dose of α-tocopherol actetate all enzymes decreased steadily to within their reference ranges, at rates probably related to their plasma half-lives. These results suggest that measurement of plasma CK activity would be useful in monitoring recovery of treated sheep.