Idiopathic subfertility in stallions. Clinical approaches to a complex problem

At least 30-40% of stallions in commercial breeding programs are moderately fertile and 8-12% are subfertile (0.5-3% with severe subfertility). From the total reported cases of the subfertility, in 2-20% of the stallions the cause is unknown or was not established. The objective of this work is to present the concept of subfertile stallion based on the current state of knowledge and advanced molecular diagnostic technologies. Low pregnancy rates have been reported in stallions with normal semen quality after conventional evaluation. Acrosome reaction (AR) is necessary for natural fertilization and impaired acrosome reaction (IAR) leads to subfertility or infertility in horses, however, AR test is not included in routine semen analysis. Genome-wide association study identified FKBP6 as a strong candidate gene responsible for this failure. The gene encodes for FK506 binding protein 6 (FKBP6) which is involved in sperm development and functions. We could conclude that the evaluation of the acrosomal status is essential in cases of stallions with good motility, concentration, morphology and viability but unexplained (idiopathic) subfertility or infertility. It is important to highlight the recent increase in reports of fertility problems in stallions related to disorders of genetic origin.(AU)

Relationship of semen quality to inbreeding and gait of Colombian Creole horses / Relação da qualidade do sêmen com endogamia e marcha de cavalos crioulos colombianos

ABSTRACT: High consanguinity among equines has negative effects on semen quality, thus resulting in low motility and high levels of abnormality in the spermatozoa. However, such a relationship has not been studied in Colombian Creole horses, which have been subjected to particular selection practices focusing mainly on their gait. This research assessed the relationship of semen quality to inbreeding and gait of Colombian Creole horses. Semen was collected from 50 horses using the artificial vagina method. Sperm motility and kinematics were assessed with a computerized analysis system (SCA®). Sperm vitality (SV) and abnormal morphology (AM) were assessed via the eosin-nigrosin staining test. Functional membrane integrity (FMI) was assessed via the hypo-osmotic swelling test (HOST). Genealogies and consanguinity analysis was conducted using the Breeders Assistant for Horses program. An average of 3.6 ± 0.4 % was reported for the inbreeding coefficient (Ft). A decrease in sperm motility and kinematics was reported, which was associated with an increase in consanguinity (P < 0.05). Furthermore, differences in consanguinity were found based on gait. Similarly, a relationship between horse gait and semen quality (P < 0.05) was found. Authors concluded that semen quality of Colombian Creole horses has been affected by inbreeding and its relationship with genetic selection based on gait.

RESUMO: A alta consanguinidade entre equinos tem efeitos negativos na qualidade do sêmen, resultando em baixa motilidade e altos níveis de anormalidade nos espermatozóides. No entanto, tal relação não foi estudada em cavalos crioulos colombianos, que tem sido submetidos a práticas de seleção específicas com foco principalmente em sua marcha. O objetivo desta pesquisa foi avaliar o relação da qualidade do sêmen com endogamia e marcha de cavalos crioulos colombianos. O sêmen foi coletado de 50 cavalos pelo método da vagina artificial. A motilidade e a cinética dos espermatozoides foram avaliadas com um sistema de análise computadorizado (SCA®). A vitalidade do esperma (VE) e a morfologia anormal (MA) foram avaliadas por meio do teste de coloração com eosina-nigrosina. A integridade funcional da membrana (IFM) foi avaliada por meio do test hipo-osmótico (HOST). A análise de genealogias e consanguinidade foi conduzida usando o programa Breeders Assistant for Horses. Uma média de 3,6 ± 0,4% foi encontrada para o coeficiente de endogamia (Ft). Uma diminuição na motilidade e cinética dos espermatozoides, que foi associada a um aumento na consanguinidade (P < 0,05). Além disso, diferenças na consanguinidade foram encontradas com base na marcha. Da mesma forma, foi encontrada uma relação entre a marcha do cavalo e a qualidade do sêmen (P < 0,05). Os autores concluíram que a qualidade do sêmen de cavalos crioulos colombianos foi afetada pela endogamia e sua relação com a seleção genética baseada na marcha.

Relationship of semen quality to inbreeding and gait of Colombian Creole horses / Relação da qualidade do sêmen com endogamia e marcha de cavalos crioulos colombianos

High consanguinity among equines has negative effects on semen quality, thus resulting in low motility and high levels of abnormality in the spermatozoa. However, such a relationship has not been studied in Colombian Creole horses, which have been subjected to particular selection practices focusing mainly on their gait. This research assessed the relationship of semen quality to inbreeding and gait of Colombian Creole horses. Semen was collected from 50 horses using the artificial vagina method. Sperm motility and kinematics were assessed with a computerized analysis system (SCA®). Sperm vitality (SV) and abnormal morphology (AM) were assessed via the eosin-nigrosin staining test. Functional membrane integrity (FMI) was assessed via the hypo-osmotic swelling test (HOST). Genealogies and consanguinity analysis was conducted using the Breeders Assistant for Horses program. An average of 3.6 ± 0.4 % was reported for the inbreeding coefficient (Ft). A decrease in sperm motility and kinematics was reported, which was associated with an increase in consanguinity (P < 0.05). Furthermore, differences in consanguinity were found based on gait. Similarly, a relationship between horse gait and semen quality (P < 0.05) was found. Authors concluded that semen quality of Colombian Creole horses has been affected by inbreeding and its relationship with genetic selection based on gait.

A alta consanguinidade entre equinos tem efeitos negativos na qualidade do sêmen, resultando em baixa motilidade e altos níveis de anormalidade nos espermatozóides. No entanto, tal relação não foi estudada em cavalos crioulos colombianos, que tem sido submetidos a práticas de seleção específicas com foco principalmente em sua marcha. O objetivo desta pesquisa foi avaliar o relação da qualidade do sêmen com endogamia e marcha de cavalos crioulos colombianos. O sêmen foi coletado de 50 cavalos pelo método da vagina artificial. A motilidade e a cinética dos espermatozoides foram avaliadas com um sistema de análise computadorizado (SCA®). A vitalidade do esperma (VE) e a morfologia anormal (MA) foram avaliadas por meio do teste de coloração com eosina-nigrosina. A integridade funcional da membrana (IFM) foi avaliada por meio do test hipo-osmótico (HOST). A análise de genealogias e consanguinidade foi conduzida usando o programa Breeders Assistant for Horses. Uma média de 3,6 ± 0,4% foi encontrada para o coeficiente de endogamia (Ft). Uma diminuição na motilidade e cinética dos espermatozoides, que foi associada a um aumento na consanguinidade (P < 0,05). Além disso, diferenças na consanguinidade foram encontradas com base na marcha. Da mesma forma, foi encontrada uma relação entre a marcha do cavalo e a qualidade do sêmen (P < 0,05). Os autores concluíram que a qualidade do sêmen de cavalos crioulos colombianos foi afetada pela endogamia e sua relação com a seleção genética baseada na marcha.

Effect of different concentrations of Trolox® in association with docosahexaenoic acid on equine semen freezing

The aim of this study was to evaluate the association of different concentrations of Trolox® and the addition of a fixed concentration of DHA in the freezing of semen of Mangalarga Marchador stallions. To that end, 16 ejaculates were frozen in the following extenders: E1) BotuCrio® (BC; Control); E2) BC + 50 ngml-1 DHA + 30 µM Trolox® (BCDHA30T); E3) BC + 50 ngml-1 DHA + 40 µM Trolox® (BCDHA40T); E4) BC + 50 ngml-1 DHA + 50 µM Trolox® (BCDHA50T). All the tested extenders were similar in preserving different kinematic parameters, cell functional integrity, compacted DNA, and high and intermediate mitochondrial activity (P>0.05). However, sperm cryopreserved in BCDHA40T showed higher velocities than sperm frozen in the control extender (P<0.05). The 30 µM concentration of Trolox® was worse for sperm motility and the 50 µM concentration of Trolox® did not adequately preserve the structural integrity of the membranes in an extender containing DHA when compared to the BotuCrio® (P<0.05) extender. The use of Trolox® in freezing extenders containing DHA did not maximize the effect of BotuCrio®, except for in the case of sperm velocity parameters when at a concentration of 40 µM.(AU)

Action of swim-up and caffeine on equine frozen sperm

Cryopreservation of equine semen is crucial to semen commercialization. However, it reduces sperm motility and longevity. Thus, sperm selection methods and addition of motility-activating substances to sperm, such as caffeine, may improve sperm quality of equine frozen semen. The objective of the current work was to evaluate the effects of caffeine on recovery and quality parameters of frozen-thawed sperm subjected to swim-up selection to be used in intracytoplasmic sperm injection (ICSI) in assisted reproductive techniques. Stallion semen were frozen and after thawing different caffeine concentrations were added to the samples performing four treatments control (no caffeine), 3, 5, and 7.5 mM caffeine. Sperm kinematic and motility were assessed by computer-assisted sperm analysis (CASA). Then, the four treated samples were submitted to the swim-up sperm selection, and the number of recovered sperm and morphology were evaluated at four times 20, 40, 60, and 80 min. The swim-up increased the recovery proportion of normal morphology sperm without (80.1±1%) or with caffeine addition (3mM: 81.2±1%, 5mM: 79.9±1% and 7.5 mM 78.9±1%) compared to the thawed semen (70±2%). However, the addition of 5 mM caffeine induced an increase in sperm motility (38.9±2.8 vs. 32.6±3.4%, P<0.05), and sperm recovery after swim-up (7.9x106 vs. 3.4x106 sperm/ml, P<0.05) compared to the control. The addition of 5 mM caffeine to frozen-thawed equine semen before swim-up selection improved sperm motility and increased the sperm recovery rate while not decreasing the percentage of morphologically normal sperm. Thus, caffeine addition to frozen-thawed equine semen before swim-up selection has potential clinical application in improving sperm quality for use in ICSI.(AU)

Equine semen cooled with chemically defined extender containing soy lecithin / Sêmen equino refrigerado com diluidor quimicamente definido contendo lecitina de soja

Equine semen has historically been chilled using milk-based media. However, the use of animal-based components presents several potential concerns, such as variability in formulations, microbial contamination and regulatory issues. We aimed to evaluate the potential of including different concentrations of soy lecithin (LS) in chemically defined Biggers, Whitten and Whittingham (BWW) medium for cooling equine semen to 15°C. Ejaculates were diluted as six different experimental groups: 1) BotuSêmen® (control); 2) BWW; 3) BWW + 1% LS; 4) BWW + 2% LS; 5) BWW + 4% LS and 6) BWW + 6% LS. BWW medium, did not preserve motility, velocity, straightness (STR), linearity (LIN), amplitude of lateral sperm head displacement (ALH), cross flagellar beat frequency (BCF), functional and structural integrity of equine spermatozoa during 24 h of refrigeration when compared to BotuSêmen® (P <0.05). The use of BWW for cooling equine semen was only possible with the addition of LS, being the concentrations equal or higher than 2% better, because they preserved total motility, curvilinear velocity (VCL) and LIN with the same potential of BotuSêmen® (P >0.05). Nevertheless, BotuSêmen® showed superiority in preserving the percentage of sperm progressive motility, average path velocity (VAP), linear progressive velocity (VSL) and BCF during cooling compared to the other extenders (P <0.05). The inclusion of soy lecithin, from 2 to 6% in the BWW medium, allowed maintaining the viability of equine semen cooled at 15ºC for up to 24 hours.

O sêmen equino tem sido historicamente refrigerado usando meios à base de leite. No entanto, o uso de componentes de origem animal causa várias preocupações potenciais, como variabilidade nas formulações, contaminação microbiana e questões regulatórias. Objetivou-se avaliar o potencial de inclusão de diferentes concentrações de lecitina de soja (LS) no meio quimicamente definido BWW - Biggers, Whitten e Whittingham para refrigeração de sêmen equino e armazenamento na temperatura de 15°C. Os ejaculados foram diluídos em seis diferentes grupos experimentais: 1) BotuSêmen® (controle); 2) BWW; 3) BWW + 1% lecitina de soja (LS); 4) BWW + 2% LS; 5) BWW + 4% LS e 6) BWW + 6% LS. O meio BWW, não preservou a motilidade, a velocidade, a retilinearidade (STR), a linearidade (LIN), a amplitude do deslocamento lateral da cabeça (ALH), a frequência de batimento flagelar cruzado (BCF), a integridade funcional e estrutural dos espermatozoides equino durante 24 h de refrigeração quando comparado ao BotuSêmen® (P <0,05). O uso de BWW para refrigeração de sêmen equino só foi possível com adição de lecitina de soja, sendo as concentrações igual ou superior a 2% melhores, pois preservaram a motilidade total, a velocidade curvilinear (VCL) e LIN com mesmo potencial do BotuSêmen® (P >0,05). Ainda assim, o diluidor comercial BotuSêmen® apresentou superioridade em preservar o percentual de espermatozoides progressivamente móveis, a velocidade média da trajetória (VAP), a velocidade linear progressiva (VSL) e a frequência do batimento flagelar cruzado (BCF) durante a refrigeração comparado aos demais diluidores (P <0,05). A inclusão de lecitina de soja, de 2 a 6% no meio BWW, permitiu a manutenção da viabilidade do sêmen equino refrigerado a 15ºC por até 24 horas.

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p<0.05). There was no difference (p>0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.(AU)

The interference of ozone gas in kinects and mitochondrial potential of equine sperm submitted on cryopreservation

The objective of this study was to evaluate the effects of the addition of different concentrations of ozone to quarter horse semen submitted to cryopreservation. Six ejaculates from four stallions were collected and were divided in four experimental groups: a control group (BotuCRIO® extender) and three other groups with BotuCRIO® ozonized at concentrations of 6, 8 and 12 μg of O3/mL. The semen samples were diluted (200 x 106 spermatozoa/mL), filled in straws and frozen. After thawing (37 ºC, 30s), the samples were evaluated at 0, 30 and 60 minutes of incubation regarding sperm kinetics by a computer-assisted sperm analysis (CASA), and plasma membrane integrity (PMI), acrosome integrity (ACi) and mitochondrial membrane potential (MMP) by fluorescent probes. There was a reduction in the kinetic parameters total motility (TM), progressive motility (PM), curvilinear velocity (VCL), straight line velocity (VSL) and average path velocity (VAP) in all groups during the thermoresistance test (TT), a pattern also found in PMI and MMP analyses (p0.05) between the control and treatment (6, 8, and 12 μg of O3/mL) groups, in any of the evaluated times for the kinetic parameters TM, linearity (LIN), straightness (STR), wobble index (WOB), amplitude of lateral head displacement (ALH) and beat cross frequency (BCF). Regarding the VCL, VSL and VAP parameters, the group treated with 6 μg did not differ from the control or from 8 μg, but was higher than 12 μg at 30 and 60 minutes. ACi and PMI did not differ between groups (p>0.05), but PMI was lower in groups 8 μg and 12 μg compared to the control and 6 μg (p<0.05). It was concluded that the addition of ozone does not present beneficial effects for cryopreservation of equine semen at the concentrations used and decreases important parameters of fertility.

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Bird's Nest on the quality of chilled Arabian stallion semen

Abstract The aim of this study was to evaluate the effects of adding different concentrations of edible bird's nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, E-Z mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.

A preliminary study on the effects of E-Z Mixin® and EquiPlus® extenders supplemented with Edible Birds Nest on the quality of chilled Arabian stallion semen

The aim of this study was to evaluate the effects of adding different concentrations of edible birds nest (EBN) which is secreted by swiftlet birds (Aerodramus fuciphagus), into EquiPlus® and E-Z Mixin® extenders on the quality of chilled Arabian stallion semen at various storage times (0, 24 and 48 h). Ten ejaculates were collected from five stallions, and diluted using the two extenders containing 0% (control), 0.12%, 0.24% and 0.24% of EBN + seminal plasma (SP). All the diluted semen samples were then cooled and stored at 5 °C, and examined at 0, 24 and 48 h. Sperm kinetic parameters were assessed using computer assisted sperm analysis (CASA) and viability were assessed using Hoechst33342/PI stain. In both extenders, total motility (TM) and progressive motility (PM) were significantly higher at 0.12% and 0.24% compared to 0.24% + SP at 24 and 48 h. At 0.12%, EZ mixin® treated semen had significantly higher TM and PM than EquiPlus® at 24 and 48 h. At 0.12% and 0.24%, average path velocity (VAP), straight-line velocity (VSL) and curvilinear velocity (VCL) were significantly higher in E-Z mixin® treated semen compared to EquiPlus® at 24 and 48 h. Comparisons between the two extender types at different concentrations of EBN showed no significant difference in lateral head amplitude (ALH), linearity (LIN), straightness (STR), beat cross frequency (BCF) and viability, irrespective of the storage time. The percentage of viable was significantly higher in E-Z mixin® than EquiPlus® at 0 and 48 h in control and 0.12%. Supplementation of the E-Z mixin® extender with 0.12% and 0.24% EBN concentrations in the absence of SP provided better CASA parameters such as TM, PM, VAP, VSL, and VCL at 24 and 48 h storage time. In conclusion, the results of this study indicated that chilled semen from Arabian stallion that was extended using E-Z mixin® and supplemented with 0.12% and 0.24% EBN concentrations performed better and yielded superior results in sperm kinetic parameters and % viable compared to EquiPlus® at 24 and 48 h storage time.(AU)

Clinical application of Doppler ultrasound in the diagnosis of fertility problems in equines / Aplicações da ultrassonografia Doppler no diagnóstico de problemas de fertilidade em equinos

The use of different ultrasound modalities (color, power and pulsed Doppler) in clinical research has enhanced knowledge of reproductive pathophysiology in equine, in addition to improving the clinical diagnosis of reproductive disorders in both the stallion and the mare. In the stallion, color Doppler ultrasound has improved the diagnostic potential of conventional ultrasound, improving the differential diagnosis of pathologies such as testicular torsion (decrease or absence of blood flow in the cord) and orchitis (increased blood flow in the cord). The function of the testicle is highly dependent on the blood flow it receives; therefore, any vascular alterations can affect both the quality and production of sperm. In recent years there have been a greater number of studies in which Doppler ultrasound is used as a tool in the diagnosis of testicular dysfunction and in the monitoring of medical and surgical treatments. In the mare, ultrasound technology has allowed numerous advances in basic research regarding the vascular hemodynamics of the ovary and uterus. Moreover, it has become an indispensable tool in reproductive practice. Among the clinical applications of this technique in mares are the evaluation of CL (corpus luteum) functionality, being a useful tool in the selection of recipients in embryo transfer programs or the use of power Doppler in the diagnosis of pregnancy as early as 7 days postovulation prior to flushing. Finally, the incorporation of color and pulsed Doppler ultrasound into the examination improves the diagnosis of mares with endometritis due to a pathological increase in uterine blood flow.

Uso de quercetina em meio de refrigeração de sêmen equino a base de leite em pó desnatado / Use of quercetin in equine semen cooling medium based on skimded milk poder

A biotécnica de refrigeração de sêmen equino para o trasporte de material genético está amplamente distribuida. Com a necessidade da manutenção da viabilidade espermática é indispensável a autilização e diluentes para este fim. O objetivo deste estudo foi avaliar se a adição de quercetina, que é um flavonóide antioxidante, em diluentes espermáticos altera a viabilidade dos espermatozoides equinos refrigerados. Foram avaliados cinco ejaculados de um garanhão, por meio da análise de motilidade e vigor espermático, em diferentes períodos de refrigeração em quatro meios de diluição: BotuSêmen®; BotuSêmen® adicionado de 20 µg/mL de quercetina (SIGMA®); Solução aquosa com 10% leite em pó desnatado (Molico®) ou Solução aquosa com 10% leite em pó desnatado (Molico®) adicionado de 20 µg/mL de quercetina (SIGMA®). Por meio dos resultados obtidos no presente estudo, foi possível verificar que não há diferença estatística entre a motilidade espermática de sêmen de garanhão diluído em BotuSêmen® em relação ao diluído solução aquosa com 10% leite em pó desnatado (Molico®) pelo período de refrigeração avaliado (36h).

The biotechnique of equine semen cooling for the transfer of genetic material is widely distributed. With the need to maintain sperm viability, it is indispensable to use and thinners for this purpose. The aim of this study was to evaluate whether the addition of quercetin, which is an anti-oxidant flavonoid, in spermatic diluents alters the viability of refrigerated equine spermatozoa. Five ejaculates of a stallion were evaluated by analysis of motility and spermatic vigor in different cooling periods in four dilution media: BotuSêmen® ; BotuSêmen® added 20 µg/mL of quercetin (SIGMA® ); Aqueous solution with 10% skimmilk powder (Molico® ) or Aqueous solution with 10% skimmilk powder (Molico® ) added 20 µg/mL quercetin (SIGMA® ). Through the data obtained, it was possible to verify that there is no statistical difference between the sperm motility of diluted stallion semen in BotuSêmen® in relation to the diluted aqueous solution with 10% skimmed milk powder (Molico® ) for the evaluated refrigeration period (36h).

Uso de quercetina em meio de refrigeração de sêmen equino a base de leite em pó desnatado / Use of quercetin in equine semen cooling medium based on skimded milk poder

A biotécnica de refrigeração de sêmen equino para o trasporte de material genético está amplamente distribuida. Com a necessidade da manutenção da viabilidade espermática é indispensável a autilização e diluentes para este fim. O objetivo deste estudo foi avaliar se a adição de quercetina, que é um flavonóide antioxidante, em diluentes espermáticos altera a viabilidade dos espermatozoides equinos refrigerados. Foram avaliados cinco ejaculados de um garanhão, por meio da análise de motilidade e vigor espermático, em diferentes períodos de refrigeração em quatro meios de diluição: BotuSêmen®; BotuSêmen® adicionado de 20 µg/mL de quercetina (SIGMA®); Solução aquosa com 10% leite em pó desnatado (Molico®) ou Solução aquosa com 10% leite em pó desnatado (Molico®) adicionado de 20 µg/mL de quercetina (SIGMA®). Por meio dos resultados obtidos no presente estudo, foi possível verificar que não há diferença estatística entre a motilidade espermática de sêmen de garanhão diluído em BotuSêmen® em relação ao diluído solução aquosa com 10% leite em pó desnatado (Molico®) pelo período de refrigeração avaliado (36h).(AU)

The biotechnique of equine semen cooling for the transfer of genetic material is widely distributed. With the need to maintain sperm viability, it is indispensable to use and thinners for this purpose. The aim of this study was to evaluate whether the addition of quercetin, which is an anti-oxidant flavonoid, in spermatic diluents alters the viability of refrigerated equine spermatozoa. Five ejaculates of a stallion were evaluated by analysis of motility and spermatic vigor in different cooling periods in four dilution media: BotuSêmen® ; BotuSêmen® added 20 µg/mL of quercetin (SIGMA® ); Aqueous solution with 10% skimmilk powder (Molico® ) or Aqueous solution with 10% skimmilk powder (Molico® ) added 20 µg/mL quercetin (SIGMA® ). Through the data obtained, it was possible to verify that there is no statistical difference between the sperm motility of diluted stallion semen in BotuSêmen® in relation to the diluted aqueous solution with 10% skimmed milk powder (Molico® ) for the evaluated refrigeration period (36h).(AU)

Utilização de leite com diferentes concentrações de gordura como diluentes para sêmen equino refrigerado / Use of milk with different fat concentrations as extenders for equine cooled semen

Dois experimentos foram realizados para verificar a viabilidade de diluentes para sêmen equino refrigerado. O objetivo do primeiro experimento foi verificar a eficiência do leite UHT integral e semidesnatado, comparado ao leite desnatado. Amostras foram analisadas a fresco e posteriormente diluídas em leite UHT integral, semidesnatado e desnatado. Foram realizados exames de motilidade espermática e vigor, CFDA/PI e HOST pós-diluição (0h), e no sêmen refrigerado a 5 ºC após 24 e 48 horas. Não houve diferença entre os três diluentes, quanto à motilidade espermática (p=0,9880), vigor (p=0,7249), CFDA/PI (p=0,3382) e HOST (p > 0,01). Houve uma diminuição na qualidade do sêmen, no decorrer do tempo (p < 0,01), independente do diluente utilizado. Pode-se afirmar que, na falta de leite UHT desnatado, o semidesnatado e o integral podem ser usados, uma vez que eles não provocam prejuízo na qualidade do sêmen refrigerado armazenado por até 48 horas. No segundo experimento, o objetivo foi verificar a eficácia do leite desnatado em comparação com diluentes comerciais (Botusemen® (BS) e Botusemen Special® (BSS)). Logo após a coleta do sêmen, as amostras foram diluídas com leite UHT desnatado (LD), Botusemen® (BS) e Botusemen Special®(BSS). A análise de sêmen seguiu o mesmo protocolo do primeiro experimento. As amostras de sêmen refrigeradas com BSS apresentaram HOST e CFDA/PI (p< 0,001) maiores nas 24h e 48 h o que indica que o diluente BSS promove aumento da longevidade dos espermatozoides em comparação com os outros analisados.

Two trials were performed to verify the viability of extenders equine cooled semen. The objective of the first trial was to verify the efficacy of whole and semi-skimmed UHT milk compared to skim milk. Fresh semen samples were analyzed and later diluted in UHT whole milk, semi skimmed and skimmed milk. Sperm motility and vigor, CFDA / PI and HOST tests were performed post-dilution (0h), and in semen cooled at 5oC after 24 and 48h. There was no difference between the 3 extenders, for sperm motility (p = 0.9880), vigor (p = 0.7249), CFDA / PI (p = 0.3882) and HOST (p> 0.01). There was a decrease in semen quality over time (p <0.01), regardless of the extender used. It can be stated that in the absence of skimmed UHT milk, semi-skimmed and whole milk can be used since they do not impair the quality of refrigerated semen stored for up to 48 hours. In the second trial the objective was to verify the efficacy of the skim milk compared to commercial extenders (Botusemen® (BS) and Botusemen Special® (BSS)). Soon after semen collection, the samples were diluted with UHT skim milk (LD), Botusemen® (BS) and Botusemen Special® (BSS). Semen analysis followed the same protocol as the first experiment. BSS-cooled semen showed higher HOST and CFDA / PI (p <0.001) in 24h and 48h indicating that BSS diluent promotes increased sperm longevity compared to the others analyzed.

Effects of the addition of docosahexaenoic acid and a-tocopherol on quality of equine spermatozoa stored at 5ºC / Efeitos da adição de ácido docosahexaenoico e a-tocoferol na qualidade do espermatozoide equino armazenados a 5ºC

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and α-tocopherol (α-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; α-Toh; DHA/α-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of α-Toh; : 10 ng mL-1 of DHA + 2 mM of α-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p < 0.05) when compared to control (62.1 ± 16.2), DHA/α-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p < 0.05) than the groups treated with DHA (2,036.0 ± 687.0), α-Toh (1,890.8 ± 749.5) and DHA/α-Toh (1,821.1 ± 627.2). In conclusion, α-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.

A composição da membrana plasmática tem impacto na transição de fase do estado cristal líquido para o estado gel das células espermáticas submetidas à refrigeração. A incorporação de ácidos graxos poliinsaturados aumenta sua fluidez e pode contribuir para a motilidade espermática. O objetivo deste estudo foi comparar o efeito da adição de ácido docosahexaenóico (DHA) e α-tocoferol (α-Toh) ao diluidor de refrigeração, isoladamente ou combinado, aos parâmetros do sêmen equino, submetidos à refrigeração por até 72 horas. Foram utilizados dois ejaculados de dez garanhões coletados com vagina artificial e avaliados quanto à motilidade, integridade da membrana plasmática, fragmentação da cromatina, atividade mitocondrial e peroxidação lipídica, de acordo com os seguintes tratamentos: C; DHA; a-Toh; DHA / a-Toh; EtOH 100: e EtOH 140 (correspondentes ao controle; 10 ng mL-1 de DHA; 2 mM de α-Toh;: 10 ng mL-1 de DHA + 2 mM de α-Toh; 100 µL de etanol e 140 µL de etanol, respectivamente). O tratamento com DHA mostrou maior motilidade (68,2 ± 12,3; p < 0,05) quando comparado aos grupos controle (62,1 ± 16,2), DHA/α-Toh (61,3 ± 12,7) e EtOH (58,1 ± 8,6). Na análise de peroxidação lipídica, o grupo controle mostrou 2.506,2 ± 796,4 ng de MDA 108 espermatozoides-1, sendo significativamente maior (p < 0,05) do que os grupos tratados com DHA (2.036,0 ± 687,0), α-Toh (1.890,8 ± 749,5) e DHA / a-Toh (1.821,1 ± 627,2). Em conclusão, α-Toh foi eficaz na diminuição da peroxidação lipídica de espermatozoide equino submetido ao resfriamento, e o DHA melhorou a motilidade espermática e, apesar de ser um ácido graxo poliinsaturado com alta suscetibilidade à peroxidação, reduziu a peroxidação lipídica.

Utilização de leite com diferentes concentrações de gordura como diluentes para sêmen equino refrigerado / Use of milk with different fat concentrations as extenders for equine cooled semen

Dois experimentos foram realizados para verificar a viabilidade de diluentes para sêmen equino refrigerado. O objetivo do primeiro experimento foi verificar a eficiência do leite UHT integral e semidesnatado, comparado ao leite desnatado. Amostras foram analisadas a fresco e posteriormente diluídas em leite UHT integral, semidesnatado e desnatado. Foram realizados exames de motilidade espermática e vigor, CFDA/PI e HOST pós-diluição (0h), e no sêmen refrigerado a 5 ºC após 24 e 48 horas. Não houve diferença entre os três diluentes, quanto à motilidade espermática (p=0,9880), vigor (p=0,7249), CFDA/PI (p=0,3382) e HOST (p > 0,01). Houve uma diminuição na qualidade do sêmen, no decorrer do tempo (p < 0,01), independente do diluente utilizado. Pode-se afirmar que, na falta de leite UHT desnatado, o semidesnatado e o integral podem ser usados, uma vez que eles não provocam prejuízo na qualidade do sêmen refrigerado armazenado por até 48 horas. No segundo experimento, o objetivo foi verificar a eficácia do leite desnatado em comparação com diluentes comerciais (Botusemen® (BS) e Botusemen Special® (BSS)). Logo após a coleta do sêmen, as amostras foram diluídas com leite UHT desnatado (LD), Botusemen® (BS) e Botusemen Special®(BSS). A análise de sêmen seguiu o mesmo protocolo do primeiro experimento. As amostras de sêmen refrigeradas com BSS apresentaram HOST e CFDA/PI (p< 0,001) maiores nas 24h e 48 h o que indica que o diluente BSS promove aumento da longevidade dos espermatozoides em comparação com os outros analisados.(AU)

Two trials were performed to verify the viability of extenders equine cooled semen. The objective of the first trial was to verify the efficacy of whole and semi-skimmed UHT milk compared to skim milk. Fresh semen samples were analyzed and later diluted in UHT whole milk, semi skimmed and skimmed milk. Sperm motility and vigor, CFDA / PI and HOST tests were performed post-dilution (0h), and in semen cooled at 5oC after 24 and 48h. There was no difference between the 3 extenders, for sperm motility (p = 0.9880), vigor (p = 0.7249), CFDA / PI (p = 0.3882) and HOST (p> 0.01). There was a decrease in semen quality over time (p <0.01), regardless of the extender used. It can be stated that in the absence of skimmed UHT milk, semi-skimmed and whole milk can be used since they do not impair the quality of refrigerated semen stored for up to 48 hours. In the second trial the objective was to verify the efficacy of the skim milk compared to commercial extenders (Botusemen® (BS) and Botusemen Special® (BSS)). Soon after semen collection, the samples were diluted with UHT skim milk (LD), Botusemen® (BS) and Botusemen Special® (BSS). Semen analysis followed the same protocol as the first experiment. BSS-cooled semen showed higher HOST and CFDA / PI (p <0.001) in 24h and 48h indicating that BSS diluent promotes increased sperm longevity compared to the others analyzed.(AU)

Influência da orquiectomia na avaliação ultrassonográfica das glândulas anexas de equinos / Influence of orchiectomy on ultrasound assessment of equine accessory glands

A ultrassonografia confere uma avaliação minuciosa do trato reprodutivo de equinos, conferindo relevância no exame andrológico para percepção de higidez ou diagnóstico antecipado de afecções. Portanto, objetivou-se descrever e determinar as mensurações e alterações das glândulas anexas de equinos submetidos à orquiectomia. Garanhões da raça Quarto de Milha (n = 5), com idade de 4-6 anos, foram avaliados por meio de palpação e ultrassonografia transretal previamente à orquiectomia e após, com intervalo de 30, 60 e 90 dias do procedimento. As glândulas bulbouretrais, vesículas seminais e ampolas foram palpadas e delimitadas quanto ao tamanho e higidez. Além disso, procedeu-se o escaneamento e mensuração através da ultrassonografia modo-B, por via transretal. A orquiectomia foi realizada de forma independente ao estudo. Para comparação dos dados utilizou-se o teste T-Student e de Variância (ANOVA). A ultrassonografia mostrou-se eficiente para avaliação e mensuração das ampolas dos ductos deferentes, glândulas vesiculares, bulbouretrais e a próstata de equinos. Considerando as consequências da orquiectomia nas estruturas supracitadas, sugere-se que a mesma não promove hipoplasia das glândulas anexas durante o período de 90 dias após cirurgia. No entanto, apenas as ampolas dos ductos deferentes demonstraram mensurações distintas nesse período, com diminuição significativa associada à orquiectomia.(AU)

Ultrasonography provides a thorough assessment of the reproductive tract of horses, giving relevance to the andrological examination for the perception of health or early diagnosis of conditions. Thus, the objective is to describe the assessment and measurement of the attached glands of horses submitted to orchiectomy. Quarter Horses (n = 5), aged 4-6 years, were assessed by palpation and transrectal ultrasound prior to orchiectomy and after 30, 60 and 90 days after the procedure. Bulbourethral glands, seminal vesicles and ampoules were palpated and delimited in terms of size and health. In addition, scanning and measurement was carried out using B-mode ultrasound, linear probe, frequency of 7.5 Hz, by transrectal approach. Orchiectomy was performed independently from the study. To compare the data, was used T-Student and Variance test (ANOVA). The ultrasonography proved to be efficient for evaluating and measuring the ampoules of the vas deferens, vesicular gland, bulbourethral and the prostate of horses. Considering the consequences of orchiectomy on the aforementioned structures, it is suggested that it does not promote hypoplasia of the attached glands during the period of 90 days after surgery. However, only the ampoules of the vas deferens showed different measurements during this period, with a significant decrease associated with orchiectomy.(AU)

Penectomia parcial como tratamento de paralisia crônica secundária a hematoma peniano em um equino relato de caso / Partial penectomy as treatment to secondary chronic paralysis of penile hematoma in a horse case report

O presente trabalho relata o caso de um equino da raça American Trotter, garanhão, com 10 anos de idade, apresentando lesão edematosa ao longo do corpo do pênis que apresentava desvio ventrocaudal associado a focos de ferimentos abrasivos e disfunção do movimento de retração em direção à cavidade prepucial, com diagnóstico clínico e ultrassonográfico de hematoma peniano generalizado. O tratamento clínico inicial se baseou em terapia anti-inflamatória, massagem, hidroterapia fria alternada com compressas quentes durante 5 dias consecutivos, porém sem melhora significativa da lesão. Optou-se então pela realização da técnica de penectomia parcial com incisão de pele e fáscia no aspecto ventral do pênis em formato triangular seguida de uretroplastia. O animal recebeu alta hospitalar 15 dias após o procedimento, com frequência e aspecto normais de micção, além de retorno à função de exposição e retração do pênis. A opção pela técnica cirúrgica de penectomia parcial mostrou ser uma alternativa eficiente, sendo um procedimento a se considerar no tratamento de hematoma associado à paralisia peniana crônica.(AU)~ipt

The present study reports the case of a 10-year-old American Trotter stallion, presenting edematous lesion along the body of the penis which presented ventrocaudal deviation associated with abrasive injury foci and dysfunction of retraction movement towards the preputial cavity, with clinical and ultrasonographic diagnosis of generalized penile hematoma. Initial clinical treatment was based on anti-inflammatory therapy, massage, cold hydrotherapy alternating with warm compresses for 5 consecutive days, but without significant improvement of the lesion. It was decided to perform the partial penectomy technique with skin and fascia triangular shaped incision in the ventral aspect of the penis followed by urethroplasty. The animal was discharged from the hospital 15 days after the procedure, with normal frequency and aspect of urination, in addition to returning to the function of exposure and retraction of the penis. The option for the partial penectomy surgical technique proved to be an efficient alternative, being a procedure to consider in the treatment of hematoma associated with chronic penile paralysis.(AU)

Effects of the addition of docosahexaenoic acid and a-tocopherol on quality of equine spermatozoa stored at 5ºC / Efeitos da adição de ácido docosahexaenoico e a-tocoferol na qualidade do espermatozoide equino armazenados a 5ºC

Plasma membrane composition has impact on phase transition from liquid crystal to gel state of cooled sperm cell. The incorporation of polyunsaturated fatty acids increases its fluidity and can contribute to sperm motility. The aim of this study was to compare the effect of adding docosahexaenoic acid (DHA) and α-tocopherol (α-Toh) to the cooling extender, singly or combined, to the equine sperm parameters, submitted to cooling, up to 72 hours. Two ejaculates of ten stallions collected with artificial vagina were used, and evaluated for motility, plasma membrane integrity, chromatin fragmentation, mitochondrial activity and lipid peroxidation, according to the following treatments: C; DHA; α-Toh; DHA/α-Toh; EtOH 100: and EtOH 140 (corresponding to control; 10 ng mL-1 of DHA; 2 mM of α-Toh; : 10 ng mL-1 of DHA + 2 mM of α-Toh; 100 µL of ethanol and 140 µL of ethanol respectively). DHA treatment showed higher motility (68.2 ± 12.3; p < 0.05) when compared to control (62.1 ± 16.2), DHA/α-Toh (61.3 ± 12.7) and EtOH (58.1 ± 8.6) groups. In lipid peroxidation assay, the control group showed 2,506.2 ± 796.4 ng of MDA 108 spermatozoa-1, being significantly higher (p < 0.05) than the groups treated with DHA (2,036.0 ± 687.0), α-Toh (1,890.8 ± 749.5) and DHA/α-Toh (1,821.1 ± 627.2). In conclusion, α-Toh was effective in diminishing lipid peroxidation of equine sperm subjected to cooling, and DHA improved sperm motility and, in spite of being a polyunsaturated fatty acid with high susceptibility to peroxidation, reduced lipid peroxidation.(AU)

A composição da membrana plasmática tem impacto na transição de fase do estado cristal líquido para o estado gel das células espermáticas submetidas à refrigeração. A incorporação de ácidos graxos poliinsaturados aumenta sua fluidez e pode contribuir para a motilidade espermática. O objetivo deste estudo foi comparar o efeito da adição de ácido docosahexaenóico (DHA) e α-tocoferol (α-Toh) ao diluidor de refrigeração, isoladamente ou combinado, aos parâmetros do sêmen equino, submetidos à refrigeração por até 72 horas. Foram utilizados dois ejaculados de dez garanhões coletados com vagina artificial e avaliados quanto à motilidade, integridade da membrana plasmática, fragmentação da cromatina, atividade mitocondrial e peroxidação lipídica, de acordo com os seguintes tratamentos: C; DHA; a-Toh; DHA / a-Toh; EtOH 100: e EtOH 140 (correspondentes ao controle; 10 ng mL-1 de DHA; 2 mM de α-Toh;: 10 ng mL-1 de DHA + 2 mM de α-Toh; 100 µL de etanol e 140 µL de etanol, respectivamente). O tratamento com DHA mostrou maior motilidade (68,2 ± 12,3; p < 0,05) quando comparado aos grupos controle (62,1 ± 16,2), DHA/α-Toh (61,3 ± 12,7) e EtOH (58,1 ± 8,6). Na análise de peroxidação lipídica, o grupo controle mostrou 2.506,2 ± 796,4 ng de MDA 108 espermatozoides-1, sendo significativamente maior (p < 0,05) do que os grupos tratados com DHA (2.036,0 ± 687,0), α-Toh (1.890,8 ± 749,5) e DHA / a-Toh (1.821,1 ± 627,2). Em conclusão, α-Toh foi eficaz na diminuição da peroxidação lipídica de espermatozoide equino submetido ao resfriamento, e o DHA melhorou a motilidade espermática e, apesar de ser um ácido graxo poliinsaturado com alta suscetibilidade à peroxidação, reduziu a peroxidação lipídica.(AU)

Penectomia parcial como tratamento de paralisia crônica secundária a hematoma peniano em um equino – relato de caso / Partial penectomy as treatment to secondary chronic paralysis of penile hematoma in a horse – case report

O presente trabalho relata o caso de um equino da raça American Trotter, garanhão, com 10 anos de idade, apresentando lesão edematosa ao longo do corpo do pênis que apresentava desvio ventrocaudal associado a focos de ferimentos abrasivos e disfunção do movimento de retração em direção à cavidade prepucial, com diagnóstico clínico e ultrassonográfico de hematoma peniano generalizado. O tratamento clínico inicial se baseou em terapia anti-inflamatória, massagem, hidroterapia fria alternada com compressas quentes durante 5 dias consecutivos, porém sem melhora significativa da lesão. Optou-se então pela realização da técnica de penectomia parcial com incisão de pele e fáscia no aspecto ventral do pênis em formato triangular seguida de uretroplastia. O animal recebeu alta hospitalar 15 dias após o procedimento, com frequência e aspecto normais de micção, além de retorno à função de exposição e retração do pênis. A opção pela técnica cirúrgica de penectomia parcial mostrou ser uma alternativa eficiente, sendo um procedimento a se considerar no tratamento de hematoma associado à paralisia peniana crônica.~ipt

The present study reports the case of a 10-year-old American Trotter stallion, presenting edematous lesion along the body of the penis which presented ventrocaudal deviation associated with abrasive injury foci and dysfunction of retraction movement towards the preputial cavity, with clinical and ultrasonographic diagnosis of generalized penile hematoma. Initial clinical treatment was based on anti-inflammatory therapy, massage, cold hydrotherapy alternating with warm compresses for 5 consecutive days, but without significant improvement of the lesion. It was decided to perform the partial penectomy technique with skin and fascia triangular shaped incision in the ventral aspect of the penis followed by urethroplasty. The animal was discharged from the hospital 15 days after the procedure, with normal frequency and aspect of urination, in addition to returning to the function of exposure and retraction of the penis. The option for the partial penectomy surgical technique proved to be an efficient alternative, being a procedure to consider in the treatment of hematoma associated with chronic penile paralysis.