Catastrophic scapular fracture in a stallion

A 4-year-old Thoroughbred stallion suffered a fracture of the left thoracic limb while racing. Because of the poor prognosis, the horse was euthanized Follow-up questions: • Morphologic diagnosis? • Etiopathogenesis? • Risk factors?

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)

Pontos críticos na produção de embriões equinos com enfoque na doadora / Critical points in the production of equine embryos with a donor approach

Inúmeras são as vantagens da utilização da técnica de transferência de embriões (TE) em equinos. No entanto, alguns fatores podem afetar a taxa de recuperação embrionária: dia da colheita do embrião; condição uterina e idade da doadora; manejos reprodutivo, sanitário e nutricional; garanhão/qualidade do sêmen; habilidade do técnico; condições climáticas, número de ovulações e adaptação da doadora. O objetivo deste artigo é revisar os fatores que podem exercer influência sobre a taxa de recuperação embrionária tendo impacto direto nos índices reprodutivos de um programa de TE.(AU)

There are countless advantages of using the embryo transfer (ET) technique in horses. However, some factors may affect the embryonic recovery rate: day of embryo collection; uterine condition and donor age; reproductive, health and nutritional management; stallion/semen quality; skill of the technician; climatic conditions, number of ovulations and adaptation of the donor. The objective of this article is to review those factors that can influence the embryonic recovery rate, having a direct impact on the reproductive rates of an ET program.(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)

Nutracêuticos no desempenho reprodutivo de garanhões / Nutraceuticals on the stallion breeding performance

O termo nutracêutico vem se tornando comum na reprodução animal e descreve produtos derivados dos alimentos, que podem fornecer benefícios adicionais a saúde, além dos valores básicos encontrados na dieta. Entre os vários nutracêuticos utilizados na reprodução, temos o ômega-3, ômega-6, vitaminas do complexo B, L-carnitina, ß-caroteno e antioxidantes. Portanto, objetivou-se apresentar as principais substâncias utilizadas como nutracêuticos e antioxidantes, seja utilizado de forma oral ou incorporado a diluentes de sêmen, com a finalidade de melhorar a qualidade seminal de garanhões, e consequentemente incrementar taxas de prenhez.(AU)

Nutraceuticals have become remarkably popular in animal reproduction and describe products derived from foods, which may provide additional health benefits, beyond the basic value found in diets. Among the main nutraceuticals used in male reproduction, there are omega-3 and 6, B-complex vitamins, L-carnitine, ß-carotene, and antioxidants. The aim of this review is to present the main substances used as nutraceuticals and antioxidants, either used orally or in combination with semen extenders, focusing on the improvement of seminal quality, and pregnancy rates.(AU)

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)

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.

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)

Chronic progressive lymphoedema in a Friesian horse: clinical aspects and diagnostic approach - case report / [Linfedema progressivo crônico em um cavalo Friesian: aspectos clínicos e abordagem diagnóstica - relato de caso]

Chronic Progressive Lymphoedema (CPL) is a disabling disease of draft horses that affects lymphatic system function typically in the distal limbs. Deformities of distal limbs, especially of the pastern, with fibrosis and skin nodular lesions are consequences of disease progression. A 15-year-old Friesian stallion presented for evaluation with history of forelimb enlargement and nodule formations distal to the carpus for four years. Simple radiographs showed soft tissue nodular lesions and venous contrasted radiography showed intense enlargement of II common digital palmar vein of both forelimbs. Tissue culture tests revealed Streptococcus equi and Proteus mirabilis isolation and skin scraping test identified Chorioptes bovis mites. Histological examination revealed perivasculitis and lymph vessels distention. History, clinical and histological findings, and complementary exams suggested CPL diagnosis. We were unable to find previously published cases describing this disease in Brazil, where the increasing number of draft horses requires attention to this problem. The correct and early diagnosis substantially delays disease progression. Therefore, we highlight the need for nationwide propagation of these data to ensure better diagnosis and early treatment of future CPL cases.

O linfedema crônico progressivo (LCP) é uma enfermidade incapacitante de cavalos de tração que afeta o sistema linfático e se manifesta especialmente na região distal dos membros. A progressão da doença resulta em deformações na região distal dos membros, especialmente na quartela, com fibrose e lesões nodulares na pele. Foi atendido um garanhão da raça Friesian, de 15 anos de idade, com histórico de aumento de volume, nodulações e deformações na região distal dos membros torácicos, com início há aproximadamente quatro anos. Ao exame radiográfico simples, foram identificadas nodulações na pele e no subcutâneo e, ao exame venoso contrastado, identificou-se marcante dilatação II veia digital palmar comum em ambos os membros torácicos. Foi isolado Streptococcus equi e Proteus mirabilis ao cultivo tecidual e identificou-se sarna do tipo corióptica ao raspado de pele. No exame histopatológico, foram identificadas perivasculite e dilatação de vasos linfáticos. Os achados clínicos, histórico, de raça do animal e os exames complementares direcionaram o diagnóstico para o LCP. A pesquisa da literatura nacional não revelou descrições clínicas dessa enfermidade, mas, devido ao aumento do número de animais de tração no país nos últimos anos, a descrição dessa enfermidade se torna importante. O diagnóstico correto e precoce é necessário para retardar a progressão da doença. Nesse sentido, ressalta-se a relevância da divulgação nacional desses achados, a fim de contribuir para o diagnóstico precoce e permitir o retardo da evolução da doença.

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.

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.

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.

Characterization of the seminal bacterial microbiome of healthy, fertile stallions using next-generation sequencing

Abstract High-throughput sequencing studies have shown the important role microbial communities play in the male reproductive tract, indicating differences in the semen microbial composition between fertile and infertile males. Most of these studies were made on human beings but little is known regarding domestic animals. Seminal bacteria studies made in stallions mostly focus on pathogenic bacteria and on their impact on reproductive technology. However, little is known about stallion commensal seminal microflora. That ultimately hinders our capacity to associate specific bacteria to conditions or seminal quality. Therefore, the aim of this study was to characterize the seminal microbial composition of 12 healthy, fertile stallion using next-generation sequencing. Hypervariable region V3 was chosen for bacterial identification. A total of nine phyla was detected. The most abundant ones were Bacteroidetes (46.50%), Firmicutes (29.92%) and Actinobacteria (13.58%). At family level, we found 69 bacterial families, but only nine are common in all samples. Porphyromonadaceae (33.18%), Peptoniphilaceae (14.09%), Corynebacteriaceae (11.32%) and Prevotellaceae (9.05%) were the most representative ones, while the Firmicutes phylum displayed the highest number of families (23, a third of the total). Samples showed high inter-subject variability. Findings previously described in other species notably differ from our findings. Families found in human such as Lactobacillaceae, Staphylococcaceae and Streptococcaceae only represented a 0.00%, 0.17% and 0.22% abundance in our samples, respectively. In conclusion, Porphyromonadaceae, Prevotellaceae, Peptoniphilaceae and Corynebacteriaceae families are highly represented in the seminal microbiome of healthy, fertile stallions. A high variation among individuals is also observed.

Conduzindo um garanhão de forma segura para cobertura ou colheita de sêmen / Handling a breeding stallion to in-hand natural service or semen collection

Cavalos de alto valor genético, morfológico e funcional adquirem valorização comercial, tornando-se garanhões que, frequentemente ultrapassam valores superiores a milhões de reais. Várias pesquisas abordam diluentes para sêmen fresco e congelado, melhoria da qualidade seminal, infertilidade, mas pouco se pesquisa e publica a respeito da contenção e condução do garanhão para colheita de sêmen/cobertura. Embora, na maioria das vezes não seja o médico veterinário que conduz esses animais para a sala de colheita de sêmen, é importante ter conhecimento do manejo desses animais, já que acidentes, às vezes fatais, são relatados em diversos lugares do Brasil e do mundo. Portanto, objetivou-se com esta revisão abordar os principais métodos de contenção e condução de um garanhão para a área de cobertura ou colheita de sêmen no intuito de minimizar riscos e acidentes.(AU)

Few horses become stallions, due to that, they have high genetic merit and commercial value; Usually, more than millions of reais. Lots of research has been addressed to extenders for fresh and frozen semen, improvement of seminal quality, infertility; However, not that much has been researched and published about the restraint and handling a breeding stallion to in-hand natural service or semen collection. Although, most of the time it is not the veterinarian who handles these animals to the breeding environment. It is essential to know how to do in a safety way, since accidents, sometimes fatal, have been reported in different places in Brazil and the world. Therefore, this review aimed to address the restraint methods and handle a breeding stallion to a breeding-Shed room.(AU)

Conduzindo um garanhão de forma segura para cobertura ou colheita de sêmen / Handling a breeding stallion to in-hand natural service or semen collection

Cavalos de alto valor genético, morfológico e funcional adquirem valorização comercial, tornando-se garanhões que, frequentemente ultrapassam valores superiores a milhões de reais. Várias pesquisas abordam diluentes para sêmen fresco e congelado, melhoria da qualidade seminal, infertilidade, mas pouco se pesquisa e publica a respeito da contenção e condução do garanhão para colheita de sêmen/cobertura. Embora, na maioria das vezes não seja o médico veterinário que conduz esses animais para a sala de colheita de sêmen, é importante ter conhecimento do manejo desses animais, já que acidentes, às vezes fatais, são relatados em diversos lugares do Brasil e do mundo. Portanto, objetivou-se com esta revisão abordar os principais métodos de contenção e condução de um garanhão para a área de cobertura ou colheita de sêmen no intuito de minimizar riscos e acidentes.

Few horses become stallions, due to that, they have high genetic merit and commercial value; Usually, more than millions of reais. Lots of research has been addressed to extenders for fresh and frozen semen, improvement of seminal quality, infertility; However, not that much has been researched and published about the restraint and handling a breeding stallion to in-hand natural service or semen collection. Although, most of the time it is not the veterinarian who handles these animals to the breeding environment. It is essential to know how to do in a safety way, since accidents, sometimes fatal, have been reported in different places in Brazil and the world. Therefore, this review aimed to address the restraint methods and handle a breeding stallion to a breeding-Shed room.

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.

Complete avulsion of the hoof capsule and subsequent testicular degeneration in a criollo stallion

Background: Complete avulsion of the hoof in horses, also known as exungulation, is not a commonly reported injury and usually leads to euthanasia due to the great amount of tissue loss, intense pain, secondary complications, expensive and lengthy treatment. It can involve deep structures and cause different complications leading to chronic lameness. In stallions affected by such injury, the reproductive tract and performance may also be affected. The aim of this study was to report a case of complete avulsion of the right front hoof in a Criollo stallion and subsequent bilateral testicular degeneration. Case: A 10-year-old Criollo stallion was referred to the Veterinary Clinical Hospital of the Federal University of Pelotas (HCV- UFPel) with a complete avulsion of the left front hoof. At admission, the stallion had clinical parameters compatible with intense pain and blood loss. Evaluation of the wound demonstrated that the distal end of the third phalanx (P3) was exposed but no fracture was detected on radiological evaluation. No other structure was apparently affected. Initially, anti-inflammatory (phenylbutazone) and opioid (morphine) was given for pain control and supportive fluid therapy was started to restore hydration. Antibiotic (Sulfamethoxazole with trimethoprim) was administered for 10 days. Continued therapy with phenylbutazone, pentoxifylline, omeprazole and supplementation with methionine, biotin and zinc was also given. Local treatment was carried out by cleaning the wound, applying an antimicrobial ointment and dressing it with a bandage. Wound management was adapted according to the evolution and healing process. The stallion was kept in stall rest during its hospitalization time. In the second month after the injury, accumulation of liquid in the scrotum was observed. Clinical and ultrasound evaluation lead to a presumptive diagnosis of testicular degeneration. The stallion was discharged after three months when the wound was almost healed and the hoof had started to grow. Six month later, a follow up by the referring vet showed that the hoof was almost completely grown and the x-ray assessment demonstrated a dorsal rotation and resorption of the distal end of the third phalanx. Discussion: The stallion of this report had a complete avulsion of the hoof capsule caused by trauma. Conservative treatment was established including wound cleaning and dressing to avoid contamination, control of pain and inflammation, antimicrobial care and supplementation to support hoof growth. Time period for wound healing and hoof growth was in agreement with other cases described previously. Bone sequestrum of the distal end of the third phalanx, and detachment of a fragment were observed in this case, followed by bone resorption. The stallion was closely monitored to prevent laminitis in the contralateral limb and no alterations were detected during the treatment period. Testicular degeneration was observed, probably caused as a consequence of hoof avulsion and due to a long period of stall rest. Degenerative alterations in testicles interfere with thermoregulation and spermatogenesis, affecting semen quality and reproductive performance. Rotation of the third phalanx was also observed six months later caused by the hoof loss. In conclusion, the patient of this report had a complete regrowth of the hoof capsule although a long intensive treatment was necessary to achieve this result. As a consequence, testicles degeneration may happen impairing its function as a stallion.(AU)

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)