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.

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)

A arte de inseminar éguas com sêmen congelado / The art of inseminating mares with frozen semen

Nos últimos quinze anos, a equideocultura foi uma atividade em destaque com significativo crescimento no Brasil e no mundo. O Brasil é o segundo país no mundo que mais utiliza transporte de sêmen equino, ficando atrás apenas dos Estados Unidos e a utilização do sêmen congelado no país vem expandido a cada dia. O índice de prenhez por ciclo, com sêmen equino congelado é variável e oscila entre 25 e 40%. Adicionalmente, sabe-se que o sêmen de alguns garanhões apresenta baixa viabilidade após o descongelamento. A primeira prenhez obtida com sêmen equino congelado foi relatada há cinco décadas. Segundo Cazalez, 2020, é muito difícil recomendar uma dose inseminante padrão para sêmen congelado em equinos. A maioria dos estudos científicos não consegue controlar o efeito de outros fatores "de confusão" como método de processo, fator égua, fator garanhão, técnica de inseminação etc., tornando difícil uma comparação crítica dos mesmos. Rigby et al. (2001) obtiveram taxas de prenhez similares ao se comparar a inseminação artificial profunda em corno uterino com endoscópio e pipeta flexível.(AU)

In the last fifteen years, equine breeding has been a prominent activity with significant growth in Brazil and in the world. Brazil is the second country in the world that most uses equine semen transport, second only to the United States, and the use of frozen semen in the country is expanding every day. The pregnancy rate per cycle with frozen equine semen is variable and ranges from 25 to 40%. Additionally, it is known that the semen of some stallions has low viability after thawing. The first pregnancy obtained with frozen equine semen was reported five decades ago. According to Cazalez, 2020, it is very difficult to recommend a standard insemination dose for frozen semen in horses. Most scientific studies cannot control for the effect of other "confounding" factors such as processing method, mare factor, stallion factor, insemination technique, etc., making it difficult to critically compare them. Rigby et al. (2001) obtained similar pregnancy rates when comparing deep artificial insemination in the uterine horn with an endoscope and flexible pipette.(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

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.

Causas e Tratamentos de Distúrbios Ejaculatórios em Garanhões / Causes and Treatments of Ejaculatory Disorders in Stallions

A ejaculação é um complexo processo de eventos neurofisiológicos sincronizados em coordenação com vários sistemas e órgãos. Antes do advento da injeção intracitoplasmática de espermatozoides (ICSI), a ejaculação era um passo absolutamente essencial na reprodução. Os distúrbios ejaculatórios por sua vez são caracterizados pela não ocorrência do processo ejaculatório, mesmo que todos os demais parâmetros relacionados ao comportamento sexual apresentem-se normais. As principais causas dessas falhas ou alterações na ejaculação estão relacionadas à sensibilidade dolorosa durante a cópula, ejaculação retrógrada, disfunção psicogênica, obstruções do aparelho reprodutor masculino, urospermia, oligospermia ou azoospermia, além de falhas na contração da musculatura lisa do trato genital ou alterações musculoesqueléticas e neurológicas. O tratamento é dependente da causa primária de cada alteração. O controle da dor, ajustes no manejo, aumento do estímulo antes da coleta associados a tratamentos farmacológicos que atuam nas sinapses neuromotoras penianas tendem a resolver os principais distúrbios ejaculatórios, levando-se em consideração que as respostas a estes tratamentos são variadas de acordo com a individualidade do garanhão com relação a doses, protocolos, vias de administração e combinações de fármacos.(AU)

Ejaculation is a complex process of neurophysiological events synchronized in coordination with various systems and organs. Before the advent of intracytoplasmic sperm injection (ICSI), ejaculation was an absolutely essential step in reproduction. Ejaculatory disorders, in turn, are characterized by the nonoccurrence of the ejaculatory process, even if all other parameters related to sexual behavior are normal. The main causes of these failures or changes in ejaculation are related to painful sensitivity during copulation, retrograde ejaculation, psychogenic dysfunction, obstructions of the male reproductive system, urospermia, oligospermia, azoospermia, in addition to failures in the contraction of the smooth muscles of the genital tract or musculoskeletal disorders and neurological. Treatment is dependent on the primary cause of each change. Pain control, management adjustments, increased stimulus before collection associated with pharmacological treatments that act on penile neuromotor synapses tend to resolve the main ejaculatory disorders, taking into account that the responses to these treatments vary according to the individuality of the stallion with regard to dosages, protocols, routes of administration and drug combinations.(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)

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)

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.

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Abstract Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.

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.

Investigating a method for pharmacologic semen collection in alpacas

Abstract While semen evaluation is standard practice prior to a sale or when infertility is suspected in other species, it is rarely done in camelids due to the difficulties involved in collecting a sample. The reproductive physiology of alpacas differs to that of other domestic animals and is still poorly understood. In the stallion, a technique was developed for semen collection that pharmacologically induces ejaculation without copulation (ex copula). This study investigates whether semen could be reliably collected by ex copula ejaculation in male alpacas. Eleven male Huacaya alpacas were used in this study, and six ex copula treatment protocols were evaluated: (1) saline (control); (2) xylazine only (0.1 mg/kg); (3) xylazine only (0.2 mg/kg); (4) imipramine only (1.0 mg/kg); (5) imipramine (1.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg); and (6) imipramine (2.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg). Each treatment protocol was repeated two to five times. Azoospermic samples obtained from ex copula ejaculation contained numerous epithelial cells but no sperm. A reliable treatment for pharmacologically inducing ejaculation in alpacas remains to be found.

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)

Coenzyme Q-10 improves preservation of mitochondrial functionality and actin structure of cryopreserved stallion sperm

Coenzyme Q-10 (CoQ-10) is a cofactor for mitochondrial electron transport chain and may be an alternative to improve sperm quality of cryopreserved equine semen. This work aimed to improve stallion semen quality after freezing by adding CoQ-10 to the cryopreservation protocol. Seven saddle stallions were utilized. Each animal was submitted to five semen collections and freezing procedures. For cryopreservation, each ejaculate was divided in three treatments: 1) Botucrio® diluent (control); 2) 50 μmol CoQ-10 added to Botucrio® diluent; 3) 1 mmol CoQ-10 added to Botucrio® diluent. Semen batches were analyzed for sperm motility characteristics (CASA), plasma and acrosomal membranes integrity and mitochondrial membrane potential (by fluorescence probes propidium iodide, Hoechst 33342, FITC-PSA and JC-1, respectively), alterations in cytoskeletal actin (phalloidin-FITC) and mitochondrial function (diaminobenzidine; DAB). The 1 mmol CoQ-10 treatment presented higher (P<0.05) amount (66.8%) of sperm cells with fully stained midpiece (indicating high mitochondrial activity) and higher (P<0.05) amount (81.6%) of cells without actin reorganization to the post-acrosomal region compared to control group (60.8% and 76.0%, respectively). It was concluded that the addition of 1 mmol CoQ-10 to the freezing diluent was more effective in preserving mitochondria functionality and cytoskeleton of sperm cells submitted to cryopreservation process.(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.

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.

Diagnosis and management strategies for urospermia in stallions / Diagnóstico e estratégias de manejo na urospermia em garanhões

Urospermia, contaminação do sêmen com urina durante a ejaculação, é considerada a segunda causa mais frequente de disfunção ejaculatoria em garanhões. A presença de urina no sêmen pode afetar drasticamente a fertilidade através do pH alcalino e alta osmolaridade da urina, além de causar uma potencial inflamação uterina após a cobertura ou inseminação artificial. Esta condição pode estar associada a outras doenças como cistite, herpesvírus equino-1 e paralisia periódica hipercalêmica; no entanto, causas idiopáticas são as mais comuns. Por esse motivo, o tratamento da urospermia pode ser frustrante e o prognóstico para a resolução completa dessa condição costuma ser desfavorável. Garanhões que sofrem de urospermia são frequentemente diagnosticados após resultados frustrantes de fertilidade e baixa qualidade do sêmen. O diagnóstico de urospermia é baseado na aparência amarela e odor típico, presença de cristais de urina, pH alcalino, além do aumento da creatinina e níveis de nitrogênio. O tratamento da urospermia é desafiador, já que a maioria dos casos é idiopática, limitando as opções terapêuticas. No entanto, quando uma condição primária é diagnosticada, ela deve ser tratada para tentar resolver a causa da urospermia. Na maioria dos casos, as terapias direcionadas à redução da contaminação da urina incluem a redução da quantidade de urina na bexiga antes da cobertura ou coleta de sêmen, tratamento farmacológico para auxiliar o fechamento do colo da bexiga durante a ejaculação, ou a coleta apenas da porção rica em esperma através da coleta fracionada do ejaculado. O método mais simples e estabelecido para o manejo da urospermia é encorajar o garanhão a urinar antes da coleta de sêmen ou cobertura natural. No entanto, quando a coleta de sêmen livre de urina não é possível, o sêmen contaminado com urina pode ser processado para minimizar os efeitos adversos do pH e da osmolaridade ao esperma. O sêmen contaminado com urina deve ser diluído com diluente à base de leite no intuito de diminuir os efeitos deletérios da urina sobre os espermatozoides. A centrifugação com gradiente de densidade a fim de selecionar os espermatozoides com características superiores também é uma alternativa nesses casos. Além disso, a criopreservação de sêmen contendo baixos níveis de contaminação com urina pode ser realizada.

Urospermia is the contamination of semen with urine during ejaculation. Urospermia is the second most frequent ejaculatory dysfunction of stallions. It can drastically affect fertility mediated by alkaline pH, the high osmolarity of urine, and presumably excessive post-breeding inflammatory response. This condition can be associated with many other diseases (i.e., cystitis, equine herpesvirus-1, and hyperkalemic periodic paralysis); however, idiopathic causes appear to be predominant. For this reason, the treatment of urospermia can be frustrating, and the prognosis for complete resolution of this condition is often poor. Stallions suffering from urospermia are usually diagnosed after poor semen quality and fertility results. Diagnosis of urospermia is based on the yellow appearance, urine smell, urine crystal, alkaline pH, increased creatinine, and urea nitrogen levels. The treatment for urospermia is challenging. Most cases are idiopathic, thereby limiting therapeutic options. However, when a primary condition is diagnosed, it should be treated to solve the cause of urospermia. In most cases, therapies directed at reducing urine contamination include reducing the amount of urine in the bladder before breeding, pharmacological treatments to enhance bladder neck closure during ejaculation, or collecting only the sperm-rich portion of the ejaculate using an open-ended artificial vagina are used to manage stallions with urospermia. The simplest and most established method for managing urospermia is to encourage the stallion to urinate before semen collection or natural breeding. However, when the collection of semen free from urine is not possible, urine-contaminated semen can be processed to minimize the adverse effects of pH and osmolarity on the sperm. Semen contaminated with urine should be immediately extended with a milk-based extender to mitigate the deleterious effects of urine on the sperm. Single-layer gradient centrifugation can also be used to select sperm with superior traits for low urine contamination. In addition, semen cryopreservation can be performed in stallion semen with a low level of urine contamination.

Investigating a method for pharmacologic semen collection in alpacas

While semen evaluation is standard practice prior to a sale or when infertility is suspected in other species, it is rarely done in camelids due to the difficulties involved in collecting a sample. The reproductive physiology of alpacas differs to that of other domestic animals and is still poorly understood. In the stallion, a technique was developed for semen collection that pharmacologically induces ejaculation without copulation (ex copula). This study investigates whether semen could be reliably collected by ex copula ejaculation in male alpacas. Eleven male Huacaya alpacas were used in this study, and six ex copula treatment protocols were evaluated: (1) saline (control); (2) xylazine only (0.1 mg/kg); (3) xylazine only (0.2 mg/kg); (4) imipramine only (1.0 mg/kg); (5) imipramine (1.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg); and (6) imipramine (2.0 mg/kg) followed 10 minutes later with xylazine (0.1 mg/kg). Each treatment protocol was repeated two to five times. Azoospermic samples obtained from ex copula ejaculation contained numerous epithelial cells but no sperm. A reliable treatment for pharmacologically inducing ejaculation in alpacas remains to be found.(AU)

Scrotal thermographic profile and seminal characteristics of Mangalarga Marchador stallions bred in the Atlantic Rainforest biome / [Perfil termográfico escrotal e características seminais de garanhões Mangalarga Marchador criados no bioma Mata Atlântica]

The objective of this study was to determine the scrotal thermographic profile and to verify the influence of temperature and humidity of the humid tropical climate on testicular temperature and seminal quality of Mangalarga Marchador stallions. The thermal profiles of the proximal, middle, and distal zones of the testicles and total surface temperature (TSTT) were recorded using an FLIR E60bx thermal imager. The average air temperature (°C) and relative humidity (%) were obtained 1, 5, 9, 33, and 66 days before semen collection and showed a mean value of 26.5±2.4 and 80.4±6.0 respectively. The scrotal surface temperature was close to 34°C and there was no variation with the age of the stallion, reproductive activity, and characteristics of the ejaculate (P>0.05). The only significant correlations obtained were between TSTT and minor defects (R = 0.41; P<0.05), between TSTT and total defects (R = 0.46; P<0.01), and between TSTT and percentage of morphologically normal sperm (R = -0.46; P<0.05). It was concluded that the Mangalarga Marchador stallions maintained the testicular temperature within favorable conditions for spermatogenesis, demonstrating the efficiency of testicular thermoregulation mechanisms in the Atlantic Forest biome.

O objetivo deste estudo foi determinar o perfil termográfico escrotal e verificar a influência da temperatura e da umidade do clima tropical úmido na temperatura testicular e na qualidade seminal de garanhões Mangalarga Marchador. Os perfis térmicos das zonas proximal, média e distal dos testículos e a temperatura da superfície total (TSTT) foram registrados usando-se um termovisor FLIR E60bx. A temperatura média do ar (° C) e a umidade relativa (%) foram obtidas um, cinco, nove, 33 e 66 dias antes da coleta de sêmen e apresentaram valor médio de 26,5 ± 2,4 e 80,4 ± 6,0, respectivamente. A temperatura da superfície escrotal foi próxima a 34°C, e não houve variação com a idade do garanhão, a atividade reprodutiva e as características do ejaculado (P>0,05). As únicas correlações significativas obtidas foram entre TSTT e defeitos menores (R=0,41; P<0,05), entre TSTT e defeitos totais (R=0,46; P<0,01), e entre TSTT e porcentagem de espermatozoides morfologicamente normais (R=-0,46; P<0,05). Concluiu-se que os garanhões Mangalarga Marchador mantiveram a temperatura testicular dentro de condições favoráveis para a espermatogênese, demonstrando a eficiência dos mecanismos de termorregulação testicular no bioma Mata Atlântica.

Biomarcadores da fisiologia reprodutiva em equinos / Biomarkers of reproductive physiology in equine

Vários estudos encontraram biomarcadores gênicos associados à fertilidade tanto na fêmea como no macho. Este trabalho tem o objetivo de revisar os marcadores gênicos que estão envolvidos na fisiologia da reprodução equina. Diferentes genes foram associados na égua com a fisiologia ovariana e uterina, já no garanhão a presença de genes silenciados na especializada célula espermática vem sendo tema de pesquisas, demonstrando a importância desses genes após a fecundação. Diferente de outras espécies domésticas de interesse zootécnico, os equinos são valorizados como indivíduo, animais de alto padrão genético atingem valores elevados de comércio e reprodução. Os reprodutores equinos, como os homens, recebem tratamento médico para a infertilidade. Assim a identificação de marcadores positivos e negativos referentes à fertilidade, pode ser uma ferramenta para auxiliar a detecção de alterações que comprometam a vida reprodutiva da fêmea e do macho equino.(AU)

Several studies demonstrated genetic biomarkers associated with fertility in both the female and the male. The current study aims to review the genetic markers that are involved in the physiology of equine reproduction. In the mare, different genes have been associated to ovarian and uterine physiology, whereas in the stallion the presence of silenced genes in the specialized sperm cell has been the subject of research, demonstrating the importance of these genes after fertilization. Unlike other domestic species of zootechnical interest, horses are valued as individuals and animals of superior genetic standard reach high values of trade and reproduction. Equine, like men, receive medical treatment for infertility. Thus, the identification of positive and negative markers referring to fertility, can be a tool to assist in the detection of changes that compromise the reproductive life of the female and the male horse.(AU)