Dissertations / Theses on the topic 'Stallion sperm'

Centrifugation of stallion semen is an integral part of the cryopreservation procedure, primarily allowing for the concentration of sperm and removal of seminal plasma. In addition, centrifugation is required for maximizing spermatozoal quality in semen from some stallions subjected to cooled transport, because of the detrimental effects of long-term exposure to high levels of seminal plasma. The centrifugation process, however, has potential deleterious effects, including reduction in sperm quality as well as loss of sperm numbers. Since centrifugation plays such a crucial role in semen processing, two experiments were designed to evaluate more efficient centrifugation methods to meet the demands of the equine industry. In Experiment 1, semen was centrifuged in two different tube types (nipple- or conical-bottom), using a cushioned technique (Eqcellsire® Component B) with two different extenders (opaque-INRA96 or clear-HGLL). For Experiment 2, nipple-tube centrifugation was conducted at two different g forces (400 or 600) for 20 min, using three different iodixanol cushion media, Eqcellsire® Component B, OptiPrep[TM], or Cushion Fluid[TM]. Regardless of tube or extender types, centrifugation of semen resulted in sperm recovery rates ≥90%; however, centrifugation in INRA 96 extender yielded higher sperm motility values than did centrifugation in HGLL extender (P < 0.05). Cushion type or g force did not impact post-centrifugation semen quality, based on the laboratory values measured (P > 0.05). These results indicate that cushioned centrifugation of stallion semen in either conical-bottom or nipple-bottom tubes can yield a high sperm harvest, while maintaining sperm function. An optically opaque extender, as is typically used in the equine breeding industry, can be used to achieve this goal. The fertility rate (94%; 131/140) following cushioned semen centrifugation in a commercial program this past year indicates that these laboratory results are transferable to the clinical setting.

During spermatogenesis, histones are largely replaced by transition proteins and protamines in normal stallions. Incomplete nucleoprotein exchange results in the abnormal retention of histones and transition proteins, which is an indicator of poor sperm quality. Equine nucleoprotein exchange has not previously been investigated in detail, so that equine sperm chromatin quality problems, which are often responsible for poor breeding performance of stallions, are not well understood. In order to characterize chromatin remodeling events in stallion spermatogenesis and to identify proteins indicative of sperm chromatin defects, such as excessive amounts of histones, we identified antibodies that recognize equine testis-specific proteins of interest. Immunoblotting of testis and sperm protein lysates and immunofluorescence staining of histological tissue sections were used to identify candidate marker proteins of incomplete sperm chromatin maturation. Results of the study, which represents the first comprehensive characterization of the nucleoprotein exchange during spermatogenesis in the stallion, challenge the paradigm that the main function of histone H4 lysine (hyper-) acetylation (concomitant H4K5 and H4K8 acetylation) is to facilitate nucleosome ejection during spermatid nuclear elongation to allow for transition protein and protamine insertion into the chromatin. That paradigm was based on observations in mice and rats where H4 acetylation in several lysine residues occurs just prior to or during nuclear elongation. In contrast, the equine data presented here show strong acetylation of H4 in K5, K8 and K12 positions immediately after meiosis in round spermatids, independent of nuclear transition protein 1 deposition. Furthermore, results of H4K16 acetylation analyses underline the importance of this mark, which is likely mediated by DNA damage signaling pathways, emphasizing the importance of DNA repair processes for the exchange of nucleoprotein exchange in spermiogenesis and therefore, in extension, for male fertility. In addition, a revised description of the equine spermatogenic cycle is proposed here that is better aligned with human, mouse and rat spermatogenesis. Finally, the testis-specific histone variant TH2B was identified as a potential quantitative marker of equine sperm quality.

Equine spermadhesin HSP-7 is a 14 kDa protein isolated from stallion seminal plasma and present on the surface of spermatozoa. HSP-7 displays carbohydrate and zona-pellucida binding properties, but the physiological role in equine fertilization is not well defined. HSP-7 has 98% amino acid sequence homology with the well-studied boar spermadhesin, AWN. Currently, these two proteins are considered to have the same reproductive function. Immunofluorescence studies presented here show that the stallion and boar spermadhesins are localized to different segments on spermatozoa. The variation in molecular compartmentalization of spermadhesin molecules in different species suggests that these structurally related proteins could be involved in independent events of fertilization. While the variation in HSP-7 abundance was not statistically significant between fertile and subfertile stallions, capacitated spermatozoa displayed a marked increase in HSP-7 when compared to neat sperm (P < 0.05). These results indicate that rather than aiding in capacitation, HSP-7 is exposed with capacitation and may have a more significant role in the acrosome reaction and sperm-oocyte recognition than previously documented.

Orientador: Frederico Ozanam Papa
Resumo: A recuperação de espermatozoides da cauda do epidídimo é uma das principais alternativas nos casos de óbito inesperado, eutanásia, processos obstrutivos ou castração terapêutica. Nessa técnica os espermatozoides não entram em contato com o plasma seminal, não incorporando seus constituintes, que interferem nos processos fisiológicos importantes para fertilização, como a ligação dos espermatozoides ao reservatório da tuba uterina, que aumenta a vida útil do espermatozoide e diminui as chances de poliespermia. Nesse sentido o objetivo do presente trabalho foi comparar a cinética e ligação da tuba uterina aos espermatozoides recuperados da cauda do epidídimo com e sem adição de plasma seminal. Utilizou-se 8 garanhões da raça Minihorse para o resgate de espermatozoides da cauda do epidídimo pela técnica de fluxo retrógrado. Após a colheita foi dividido em 4 grupos, LD apenas com diluente a base de leite desnatado, GO recebeu adição de diluente para congelação a base de gema de ovo, PSB plasma seminal de garanhão com alta fertilidade e capacidade de refrigeração, e PSR plasma seminal de garanhão com fertilidade e capacidade de refrigeração inferiores. Foi avaliado cinética, integridade de membrana espermática e a taxa de ligação à explantes da tuba uterina. Não apresentaram diferença na integridade da membrana nem na taxa de ligação, no entanto, observou-se diferença quanto a cinética espermática. Conclui-se que a adição de plasma seminal de diferentes garanhões interferiu, de for... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Sperm retrieval from the tail of the epididymis is one of the main alternatives in cases of unexpected death, euthanasia, obstructive processes, therapeutic castration. In this technique spermatozoa do not come into contact with the seminal plasma, not incorporating their constituents, which interfere in the physiological processes important for fertilization, such as sperm binding to the uterine tube reservoir, which increases sperm life and decreases the chances of polyspermia. In this sense, the objective of the present study was to compare the kinetics and uterine tubal attachment to the spermatozoa recovered from the tail of the epididymis with and without addition of seminal plasma. Eight minihorse stallions were used to retrieve spermatozoa from the tail of the epididymis by the retrograde flow technique. After harvesting was divided into 4 groups, LD only with diluent the skim milk base, GO received addition of diluent for freezing the egg yolk, PSB seminal stallion plasma with high fertility and cooling capacity, and PSR seminal plasma fertility and lower cooling capacity. Kinetics, spermatic membrane integrity and the rate of binding to the explants of the uterine tube were evaluated. There was no difference in membrane integrity or binding rate, however, a difference was observed in spermatic kinetics. It was concluded that the addition of seminal plasma of different stallions interfered, differently in spermatozoa kinetics of the tail of the epididymis increased t... (Complete abstract click electronic access below)
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Um número significativo de garanhões produzem ejaculados de baixa qualidade e alta sensibilidade à refrigeração, necessitando de técnicas de processamento especializadas para otimizar a qualidade do sémen. Recentemente, foi disponibilizada uma técnica de centrifugação com monocamada (SLC) de Androcoll-E®, que tem demonstrado selecionar os espermatozoides (SPZ) de boa qualidade. No presente estudo pretendeu-se avaliar o efeito do tratamento com SLC na qualidade do sémen refrigerado, durante 72 horas. As amostras tratadas mostraram valores numéricos superiores para os parâmetros de motilidade, mas apenas se obtiveram valores estatisticamente significativos (p<0,05) para a velocidade curvilínea (VCL) e retilínea (VSL), medidas com um sistema computorizado de análise de sémen, nas amostras tratadas. O reduzido número de amostras e a elevada variabilidade das características seminais entre ejaculados, podem ser responsáveis pela ausência de diferenças estatisticamente significativas, pelo que seria de interesse alargar este estudo a um maior número de garanhões para consolidar as conclusões; ABSTRACT: A significant number of stallions produce ejaculates of low quality and high sensitivity to cooling, requiring specialized processing techniques to optimize the quality of sperm. Recently, a new protocol has been developed - the single layer centrifugation (SLC) with Androcoll-E®, which in some studies has been able to select good quality spermatozoa (SPZ). The objective of this study was to investigate the effect of SLC treatment on the quality of semen refrigerated for 72h. In our study, numerical improvements of motility parameters were seen in the treated samples, but only the curvilinear (VCL) and linear velocities (VSL) measured by a computer analysis system were significantly (p<0.05) higher in the treated samples. Lack of significance may have been due to the small sample size and high variability among ejaculates. Expanding this data set with the use of further ejaculates from different stallions is warranted to draw more definitive conclusions.

As biotécnicas da reprodução na espécie equina avançaram na última década e os criadores de equinos começaram a questionar as possibilidades de interferência na determinação do sexo dos potros. A determinação do sexo é importante, pois o sexo do potro possui grande influência no valor comercial do mesmo. O objetivo do presente estudo foi avaliar a influência do momento da cobertura em relação ao momento da ovulação no sexo dos potros e outros fatores como: idade da égua e garanhão; indutor da ovulação; ovário em que ocorreu a ovulação e o diâmetro do folículo pré-ovulatório também foram avaliados. O estudo foi realizado em Haras na região de Bagé e Aceguá (RS) – Brasil (31°24'06.1''S 54°07'47.5''W) e (31°30'16.0''S 54°07'45.5''W) nas estações de monta de 2011 a 2015. Utilizou-se 259 ciclos reprodutivos de 160 éguas e 22 garanhões da raça Puro Sangue de Corrida. Informações como agente indutor de ovulação utilizado (Deslorelina; n = 187 ou hCG; n = 72); data da cobertura (n = 259); momento da ovulação em relação a cobertura (+ 24 horas; n = 69 e - 24 horas; n = 190); idade da égua (G1: até 8 anos; n = 123, G2: 9 até 14 anos n = 110 e G3: >14 anos; n = 26); idade do garanhão (Até 14 anos; n = 11 e >15 anos; n = 11) ; ovário em que a ovulação ocorreu (Direito; n = 122 e Esquerdo; n = 137); foram registrados e avaliados No total de potros nascidos, 136 foram machos (52,51%) e 123 foram fêmeas (47,49%). O tempo decorrido após a cobertura não influenciou o sexo dos produtos, nas éguas que ovularam com – 24h após a indução da ovulação: 104 potros (54,74%) eram machos e 86 (45,26%) eram fêmeas, enquanto que nas éguas que ovularam com + 24h, 32 potros (46,38%) eram machos e 37 (53,62%) eram fêmeas. A porcentagem de fêmeas nascidas em relação a idade da égua foi de 46,34% (n=57), 47,27% (n=52) e 46,15% (n=12) nos grupos G1, G2 e G3 respectivamente. Garanhões com idade até 15 anos tiveram 44,14% (n=49) de fêmeas e com mais de 15 anos 49,66% (n=73) eram fêmeas. Não houve diferença no sexo dos produtos quanto ao agente indutor da ovulação (Deslorelina x hCG) e ovário em que ocorreu a ovulação. O presente estudo concluiu que nenhum dos fatores estudados alterou a proporção macho:fêmea dos potros nascidos.
The biotechnologies of reproduction in equine species have been improved in the last decade and the horse breeders started to inquire about the possibility of intervention regarding to the sex determination in foals. Sex determination is important because the sex of the foal has a great influence on the commercial value of the foal. The aim of the present study is was to evaluate the influence of the time of the breeding in relation to time of ovulation in the sex of the foals, besides analyzing other factors such as: the age of the mare and the stallion; ovulation inductor, ovary and the diameter of the preovulatory follicle. The study was accomplished in studs located Bagé/RS and Aceguá/RS – Brazil (31°24'06.1''S 54°07'47.5''W) and (31°30'16.0''S 54°07'45.5''W) during the breeding seasons from 2011 to 2015, using 259 reproductive cycles of 160 mares and 22 stallions of Thoroughbred breed. Information like the induction of ovulation agent that was used (Deslorelin; n = 187 or hCG; n = 72); date of breeding (n = 259); time of ovulation in relation to the breeding (+24 hours; n = 69 and -24 hours; n = 190); age of the mare (G1: up to the age of 8; n = 123 G2: from the age of 9 to the age of 14; n = 110 and G3: >14 years old; n = 26); age of the stallion (up to the age of 14; n = 11 and >15 years old; n = 11); ovary in which the ovulation occurred (Right; n = 122 and Left; n = 137) were catalogued and evaluated As result, 136 (52,51%) were born colts and 123 (47,49%) were born fillies. The elapsed time from breeding to ovulation did not influence on the sex of the product, on mares that ovulated in less than 24 hours after the ovulation induction: 104 foals (54,74%) were male and 86 (45,26%) were female, whereas in the mares that ovulated in more than 24 hours, 32 foals (46,38%) were male and 37 (53,62%) were female. The percentage of born females regarding to the age of the mare was 46,34% (n = 57), 47,27% (n = 52) and 46,15% (n = 12) in the groups G1, G2 and G3, respectively. From stallions up to the age of 15 years, 44,14% (n = 49) were females and from those which were older than 15 years old, 49,66% (n = 73) were females. There was no difference regarding the sex of the product in relation to the ovulation inducer agent (Deslorelin x hCG) and ovary in which the ovulation occurred. None of the factors studied modified the male:female proportion of the born foals.

Beurteilung von nativen und aufgetauten Spermatozoen fertiler und subfertiler Hengste mit Hilfe der Phasenkontrast- und Transmissionselektronenmikroskopie. Institut für Veterinär-Pathologie der Veterinärmedizinischen Fakultät, Universität Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In dieser Arbeit wurde die Ultrastruktur von nativen und tiefgefrorenen Spermien mittels Phasenkontrast- und Transmissionselektronenmikroskopie (TEM) untersucht. Für die Beurteilung der Spermienmotilität und der Morphologie von in Formolzitrat fixierten Spermien standen jeweils drei Ejakulate von 50 Hannoveraner Hengsten des Niedersächsischen Landgestüts Celle zur Verfügung. Aus dieser Gruppe wurden drei fertile, drei subfertile Hengste und 6 Hengste mittlerer Fertilität ausgewählt, von denen sowohl die Nativ-Proben als auch eine Tiefgefrierprobe (TG-Probe) für die TEM im Institut für Veterinär-Pathologie der Universität Leipzig gemäß des Standardprotokolls des Institutes aufbereitet wurden. Die Spermien wurden gewaschen und das Seminalplasma der nativen Proben oder der Verdünner der TG-Proben abpipettiert und durch eine 5%-ige Glutaraldehydlösung in einem 0,1 M Kakodylatpuffer (pH 7,2) ersetzt. Die Fixierungslösung wurde anschließend entfernt und das Pellet gewaschen und danach mit Gelatine gemischt. Die spermienreichen Stellen wurden aus der Gelatine herausgeschnitten und in Glutaraldehyd aufbewahrt. Nach einer Nachfixierung in OsO4 und einer Entwässerung in Ethanollösungen erfolgte eine Einbettung in einer Eponmischung. Nach einer Polymerisation von 5 Tagen wurden die eingebetteten Eponblöckchen angetrimmt und die Semi- und Ultradünnschnitte angefertigt. Die Ultradünnschnitte wurden auf ein Kupfergrid gelegt, mit Uranylazetat und Bleizitrat kontrastiert und mit dem Transmissionselektronenmikroskop (Zeiss EM 900, Oberkochem) bei 80 kV analysiert. In den nativen Proben wurden insgesamt 360 Spermien pro Hengst beurteilt, in den TG-Proben 120 Spermien pro Hengst. Die Qualität der elektronenmikroskopischen Aufnahmen war sehr gut, doch die Plasmamembran zeigte fixierungsbedingte Artefakte. Nach dem Auftauen waren die Bilder heller und der Kontrast etwas geringer. Es gab eine Zunahme an Akrosomdefekten, akrosomreagierten Spermien und Beschädigungen der Plasmamembran, der Mitochondrien, sowie der Mantel- und Ringfasern. Durch die Membranbeschädigungen trat auch eine Verringerung der Anzahl proximaler und distaler Zytoplasmatropfen auf. Sowohl geschwollene Akrosome mit einer niedrigeren Dichte der akrosomalen Matrix als auch Mitochondrien mit einer zu hellen mitochondrialen Matrix waren typische Befunde in den TG-Proben. Die Studie der Ultrastruktur und die wahrgenommenen Defekte führten zur Erstellung eines Standardprotokolls für die transmissionselektronenmikroskopische Beurteilung von Hengstspermien. Die Beurteilung mittels TEM sollte aber nicht zu einer quantitativen, sondern zu einer qualitativen Aussage führen. Sie ermöglicht die Diagnose von Kern- (Kerndeformationen und Taschenbildung im Kern) und Akrosomabweichungen (deformierte Akrosome mit oder ohne Vakuolenbildung, abgehobene Akrosome und akrosomreagierte Spermien), Anomalien der Mitochondrien (Unterbrechung der Mitochondrienscheide, zu viele Mitochondrien, anormale Dichte der mitochondrialen Matrix), Defekten des Axonemas (Ordnung oder Anzahl der Mikrotubuli, Mantel- und Ringfasern) und der Anwesenheit immaturer Spermienvorstufen. Diese Methode eignet sich für die Diagnostik subfertiler Hengste mit normalen Spermienparametern bei der routinemäßige Spermienbeurteilung und kann sowohl in nativen als auch in TG-Proben angewendet werden. Im Vergleich zur Phasenkontrastmikroskopie waren die elektronenmikroskopischen Bilder wegen ihrer stärkeren Vergrößerung und der Darstellung innerer Spermienstrukturen viel aussagekräftiger. Für die Beurteilung von Halsansatzdefekten, abweichende Geißelformen und Mehrfachmißbildungen ist die Phasenkontrastmikroskopie die am besten geeignete Methode
Evaluation of fresh and frozen-thawed semen samples of fertile and subfertile stallions by light microscopy and transmission electron microscopy. Institut of Pathology of the Faculty of Veterinary Medicine, University of Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In this study the ultrastructure of fresh and frozen-thawed semen samples of 50 stallions from the National Stud of Lower Saxony (Celle, Germany) were evaluated by light microscopy and transmission electron microscopy (TEM). Three ejaculates of each stallion were available for the motility analysis and the morphological analysis by lightmicroscopy after fixation in formol citrate. Based on the fertility data, the ejaculates of 12 stallions (3 fertile stallions, 3 subfertile stallions and 6 stallions of average fertility) were selected for the morphological analysis by TEM. The native samples and one frozen-thawed sample from these stallions were prepared for the TEM at the Institute of Pathology of the Faculty of Veterinary Medicine, Uni-versity of Leipzig. The sperm cells were washed and the seminal plasma from the native samples and the diluents of the frozen-thawed samples were replaced by a 5%-glutaraldehyde solution in a 0,1 M cacodylate buffer pH 7,2. The fixative was removed, the pellet was washed again and mixed with gelatin. The sperm rich fraction in the gelatin mass was excised and stored in glutaraldehyde. A second fixation in OsO4 was followed by a dehydratation in ethanol and a polymerization phase in epon. After 5 days of polymerization the starred samples were used for semi- and ultratight cuts. The latter were placed on a copper grid, contrasted with uranyl acetate and lead citrate and analyzed with the transmission electron micro-scope (EM 900) by 80 kV. In the fresh samples, 360 sperm cells were examined per stallion, whereas in the frozen-thawed samples only 120 sperm cells per stallion were evaluated. The microscopic pictures were of a high quality. However, the sperm plasma membrane showed some fixation artifacts. In the thawed samples a lower contrast was noticed than in the fresh samples. The sperm cells in the frozen-thawed samples showed an increase in acrosome defects, acrosome reactions, damage of the cell plasma membrane, mitochondria, fibrous sheet and outer dense fibers. The latter defect was associated with a decrease in proximal and distal cytoplasmatic droplets. Swollen acrosomes with a lower matrix density and a bright mitochondrial matrix were typically present in the cryopreserved samples. The ultrastructural defects in these samples, examined by TEM, have led to the development of a standard evaluation protocol with the most common sperm defects in stallion semen. TEM is an expensive and time consuming technique, which cannot be used to obtain quantitative results, but is considered as an accurate method for the qualitative examination of semen samples in cases of unexplained subfertility. TEM can especially be recommended for the diagnosis of nuclear (nuclear malformations and pouches) and acrosomal defects (acrosome deformations, acrosome vacuoles, detached acrosomes and acrosome reactions), mitochondrial (mitochondrial sheet defects, mitochondrial proliferation, decrease in mitochondrial matrix density) and axonema malformations (anormal position or quantity of microtubules and fibrous sheet or outer dense fibers defects) and the detection of immature sperm cells in ejaculates. The results of this study state that TEM can be useful for the evaluation of both fresh and frozen-thawed semen samples. Compared to the light microscopic evaluation of stallion sperm, the TEM images give more precise information because of their higher magnification rate and the ability to reveal internal sperm structures. However, light microscopy remains the best method to detect sperm neck defects, deformed tailes and sperm cells with multiple heads or tails

Incrementar a eficiência do processo de refrigeração do sêmen é de grande interesse na reprodução da espécie equina, à medida que a população de equinos cresce, também aumenta a demanda para a comercialização e transporte de sêmen refrigerado. Esse trabalho foi realizado para verificar os efeitos de diferentes diluidores para refrigeração de sêmen equino a 5°C sobre a cinética, membranas, morfologia e cromatina espermáticas durante 12 horas de armazenagem. Foram utilizados quatro ejaculados de quatro garanhões de diferentes raças, colhidos em intervalos semanais. Imediatamente após a colheita, o sêmen in natura foi avaliado quanto ao movimento espermático utilizando-se o sistema computadorizado de análise espermática (CASA), integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial, utilizando-se sondas fluorescentes (PI, H342, FITCPSA e JC-1, por microscopia de epifluorescência), morfologia espermática por microscopia de contraste de interferência diferencial (DIC) e desnaturação da cromatina pela coloração de Azul de Toluidina. Logo após as análises, o sêmen foi diluído usando-se três diluidores diferentes: SMT, à base de leite desnatado e meio de Tyrode (adaptado de: PADILLA; FOOTE, 1991), BSAG, diluidor contendo BSA (adaptado de: GIBB et al., 2011) e SMK, à base de leite desnatado (KENNEY et al., 1975; controle), em seguida foi envasado em bisnagas na concentração de 50 x 106 sptz/mL e refrigerado a 5°C em caixas BotuFLEX® (Botupharma, Botucatu-SP), durante um período de 12 horas. O sêmen foi analisado 5 minutos após a diluição (T0), 4 (T4), 8 (T8) e 12 horas (T12) após a refrigeração quanto ao movimento espermático, integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial, morfologia espermática e desnaturação da cromatina. A análise estatística foi realizada empregando-se o programa computacional Statistical Analysis System (SAS inst. Inc.). Para todas as variáveis foi utilizada a análise de variância (ANOVA). Para comparação das médias, foi utilizado o método de Tukey sendo considerado o nível de significância de 5%. Foram observadas interações entre tempo X tratamento para a maioria das características de cinética espermática. Dentre os efeitos encontrados dos diluidores, destaca-se que SMT e SMK foram superiores na preservação das motilidades total e progressiva e porcentagem deespermatozoides rápidos em detrimento ao diluidor BSAG. Quanto à preservação das membranas o diluidor SMT (56,6±18,7%) foi significativamente superior ao SMK (49,6±18,6%), que por sua vez foi superior ao BSAG (25,8±14,8%) quanto à porcentagem de espermatozoides com membranas plasmática e acrossomal íntegras e alto potencial mitocondrial (PIAIA). Comportamento semelhante foi observado para o percentual de membrana plasmática e de potencial de membrana mitocondrial. Contrariamente, a membrana acrossomal não foi afetada pela refrigeração do sêmen a 5ºC por até 12 horas independente do diluidor. Foi encontrado maior percentual de defeitos totais quando o sêmen foi refrigerado utilizando o diluidor BSAG (50±12,4%) do que SMT (42,6±11,2%) e SMK (41,8±12,4%). Quando se avaliou a cromatina espermática não foram notados efeitos do tempo de refrigeração nem dos diluidores; todavia, ao se comparar com o sêmen in natura notou-se um aumento no percentual de espermatozoides com desnaturação intermediária da cromatina após 12 horas de refrigeração a 5ºC com o diluidor BSAG. Conclui-se que a refrigeração do sêmen equino a 5ºC altera a cinética espermática de forma oscilante entre os diluidores no decorrer do tempo até 12 horas. Os diluidores SMT e o SMK preservam melhor a cinética, integridade de membranas e morfologia espermática do que o diluidor BSAG.
To develop the efficiency of cooling semen is the great advantage in the equine reproduction, as according to the equine population to grow up, too increase the demand to market and transport of cooling semen. This experiment was performed to verify the effects of different extenders to equine cooling semen at 5°C on the sperm kinetic, membranes, morphology and chromatin during 12 hours of storage. Were utilized four ejaculates from four stallions of different breeds, collected a week. Immediately after the collection, the in natura semen was evaluated as the sperm movement using the Computer-Assisted Semen Analysis (CASA), integrity of plasma and acrossomal membranes and mitochondrial membrane potential, using the fluorescent probes (PI, H342, FITC-PSA and JC-1, by epifluorescência microscopy), sperm morphology by microscopy of differential interference contrast (DIC) and chromatin denaturation by Toluidine blue. As soon as finished the analyses, the semen was diluted using three different extenders: SMT, skim milk based and Tyrode medium (adapted from PADILLA; FOOTE, 1991), BSAG, extender containing BSA (adapted from GIBB et al., 2011) and SMK, skim milk based (KENNEY et al., 1975; control), following was packed in flasks at 50 x 106sperm/mL concentration and cooling at 5°C in BotuFLEX® (Botupharma, Botucatu-SP) box, during 12 hours. The semen was analyzed 5 minutes after dilution (T0), 4 (T4), 8 (T8) and 12 hours (T12) after cooling about sperm movement, plasma and acrossomal membranes integrity and mitochondrial membrane potential sperm morphology and chromatin denaturation. The statistical analysis was performed using Statistical Analysis System (SAS inst. Inc.) software. To all variables was utilized the analysis of variance (ANOVA). To media comparison was utilized the Tukey method, considering the significant level at 5%. Were observed interactions between times X treatment to majority of sperm kinetic characteristic. Among the found effects of the extenders, it was detached that SMT and SMK were superior to preserve total and progressive motility and percentage of rapid sperm in detriment to BSAG extender. It about the membranes preservation the SMT extender (56.6±18.7%) was significantly superior to SMK (49.6±18.6%), which was superior to BSAG (25.8±14.8%) as percentage of sperm with plasma and acrossomal membranes integrity and high mitochondrial potential (PIAIA). Behavior similar was observed to the plasma membrane integrity and potential mitochondrial membrane percentage. Contrary, The acrossomal membrane was not affected by semen cooling at 5ºC until 12 hours independently of the extender. It was found major percentage of defect total to cooling semen using BSAG (50±12.4%) extender compared to SMT (42.6±11.2%) and SMK (41.8±12.4%). When evaluated the sperm chromatin were not found effect of the cooling time neither of the extenders; however, when compared to in natura semen notice an increase on the percentage of spermatozoa with moderate chromatin denaturation after 12 hours of cooling at 5ºC with BSAG extender. It was concluded that the equine semen cooling at 5ºC change the sperm kinetic oscillating way among extenders during the time until 12 hours. The SMT and SMK extenders are better to preserve the sperm kinetic, membranes integrity and morphology than BSAG extender.

Em equinos é descrita uma variabilidade na qualidade do sêmen congelado, relacionada principalmente a variações consideráveis na composição da membrana plasmática do espermatozoide. Neste contexto, estudos investigam alternativas para aumentar a fertilidade ao se usar sêmen congelado, e a adição de ácidos graxos polinsaturados ao diluente de congelamento é sugerida. O objetivo deste estudo foi avaliar o efeito da adição de diferentes níveis de ácidos graxos poli-insaturados: ácido eicosanóico e ácido docosahexaenóico, em meio diluente específico para a espécie sobre as características do espermatozoide pós-descongelamento. Foi utilizado sêmen de quatro garanhões (quatro ejaculados por garanhão) da raça Crioula. O sêmen foi diluído em diluente de congelamento comercial a base de gema de ovo, glicerol e dimetilformamida (Botu-Crio©) ajustando a concentração para 200 x 10⁶ espermatozoides viáveis/mL (grupo controle) e, na sequência, os demais tratamentos: adição de ácido docosahexaenóico nas doses 25μm e 50μm /mL e ácido eicosanóico nas doses 25μm e 50μm /mL. Após o descongelamento foram realizadas análises de cinética espermática no sistema de Análise Computadorizada para Avaliação Espermática, das seguintes variáveis: motilidade total, motilidade progressiva, motilidade rápida, motilidade lenta, motilidade local, velocidade de trajeto, velocidade progressiva, velocidade curvilinear, amplitude do deslocamento lateral da cabeça, frequência de batimentos, retilinearidade e linearidade; e avaliação da integridade física através do uso de sondas fluorescentes, e funcionalidade de membrana pelo teste hiposmótico. Não ouve diferença nas variáveis avaliadas. Este é o primeiro estudo que descreve a adição de ácido eicosanóico ao sêmen equino. Concluí-se a adição de ácido docosahexaenóico e eicosanóico nas concentrações testadas não alterou as variáveis avaliadas no sêmen de garanhões da raça Crioula.
In equines, a variability in the quality of frozen semen is described, mainly related to the considerable variations in the composition of the sperm plasma membrane. In this context, studies investigate alternatives to increase fertility when using frozen semen, and the addition of polyunsaturated fatty acids to the freezing diluent is suggested. The objective of this study was to evaluate the effect of the addition of different levels of polyunsaturated fatty acids: eicosanoic acid and docosahexaenoic acid, in specific diluent medium for the species on the characteristics of the post-thawing spermatozoa. Semen was used of four stallions (four ejaculates per stallion) of the Crioula breed. The semen was diluted in commercial freezing diluent based on egg yolk, glycerol and dimethylformamide (Botu-Crio ©) by adjusting the concentration to 200 x 10 vi viable spermatozoa / mL (control group) and, in sequence, the other treatments: addition of docosahexaenoic acid at 25μm and 50μm / mL and eicosanoic acid at 25μm and 50μm / mL. After thawing, sperm kinetics analyzes were performed in the Computerized Analysis System for Sperm Evaluation, of the following variables: total motility, progressive motility, rapid motility, slow motility, local motility, path velocity, progressive velocity, curvilinear velocity, lateral head displacement amplitude, beating frequency, linearity and linearity; and evaluation of physical integrity through the use of fluorescent probes, and membrane functionality by the hyposmotic test. No difference in the variables evaluated. This is the first study to describe the addition of eicosanoic acid to equine semen. We concluded that with the addition of docosahexaenoic and eicosanoic acid at the concentrations tested did not alter the variables evaluated in the semen of Crioula stallions.

Orientador: Frederico Ozanam Papa
Resumo: O presente estudo propõe avaliar o efeito do tratamento oral com pentoxifilina sobre a qualidade seminal, morfometria, histologia e expressão gênica testicular de garanhões submetidos ao estresse térmico escrotal. Além disso, objetiva testar a eficiência de novo método de aquecimento escrotal na espécie equina. Para isso 14 garanhões foram divididos em três grupos: Controle (CT, n=4), Degenerado (DG, n=5) e Degenerado Tratado (PTX, n=5). O insulto térmico escrotal foi realizada utilizando uma bolsa térmica acoplada a uma fonte de ar aquecido a 50º C, durante uma hora no início da manhã e uma hora no final da tarde, no D-1 e D0. Um dia após o insulto (D1), o tratamento com 17 mg/kg pentoxifilina oral, a cada 12 h, foi iniciado e conduzido por 30 dias. Os animais foram coletados duas vezes por semana do D-24 ao D60 e o sêmen avaliado quanto a cinética, morfologia espermática, integridade de membrana plasmática, geração do ânion superóxido intracelular e mitocondrial, índice de peroxidação lipídica e índice de caspases ativadas 3 e 7. No D30 e D60 biópsias testiculares foram realizadas e as amostras destinadas a histopatologia, e ao RT-qPCR, quanto a resposta a apoptose, choque térmico e estresse oxidativo. As medidas testiculares de comprimento, altura e largura foram mensuradas utilizando paquímetro, e o volume testicular relativo foi calculado, uma vez por semana do D-5 ao D60. Dentre os resultados obtidos, o método de estresse térmico escrotal utilizado se mostrou eficiente ... (Resumo completo, clicar acesso eletrônico abaixo)
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Orientador: Frederico Ozanam Papa
Resumo: Durante o processo de criopreservação os espermatozoides de garanhões sofrem danos osmóticos que são irreversíveis. Mesmo utilizando-se as melhores técnicas de criopreservação, este processo ainda é prejudicial às células espermáticas sendo em média a taxa de sobrevivência espermática em torno de 50%. Além disso, ainda há uma porcentagem de espermatozoides sobreviventes portadores de danos subletais, fazendo com que seja limitada a capacidade de sobrevivência das células espermáticas após a descongelação e como consequência existe uma queda na fertilidade. Está bem estabelecido que os espermatozoides de diferentes garanhões domésticos variam significativamente em criosensibilidade, este fato pode estar relacionado com o efeito do crioprotetor, assim como pode ter relação com o tamanho molecular do crioprotetor penetrante e curva de refrigeração utilizada. Enquanto o glicerol continua a ser o crioprotetor mais comum para espermatozoides, existem fatores limitantes pelo seu uso, incluindo danos na membrana através de um efeito osmótico, sendo assim, as amidas têm sido estudas com o objetivo de minimizar os efeitos causados pelo glicerol durante o processo de criopreservação, por apresentarem melhor crioproteção da membrana das células espermáticas. Sendo assim, a proposta desse trabalho foi avaliar diferentes crioprotetores em diferentes curvas de refrigeração sobre a qualidade do sêmen equino. Para tanto, o estudo foi realizado para avaliar o efeito dos crioprotetores glicerol... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: During the cryopreservation process, sperm from stallions suffer osmotic damage that is irreversible. Even using the best cryopreservation techniques, this process is still harmful to sperm cells, with an average sperm survival rate of around 50%. In addition, there is still a percentage of surviving sperm with sublethal damage, causing the sperm cells' ability to survive after thawing to be limited and as a consequence there is a fall in fertility. It is well established that sperm from different domestic stallions vary significantly in cryosensitivity, this fact may be related to the effect of the cryoprotectant, as well as it may be related to the molecular size of the penetrating cryoprotectant and the cooling curve used. While glycerol remains the most common cryoprotectant for sperm, there are limiting factors for its use, including damage to the membrane through an osmotic effect, so amides have been studied with the aim of minimizing the effects caused by glycerol during cryopreservation process, as they present better cryoprotection of the sperm cell membrane. Thus, the purpose of this work was to evaluate different cryoprotectants in different refrigeration curves on the quality of equine semen. To this end, the study was carried out to evaluate the effect of cryoprotectants glycerol, methylformamide, dimethylformamide and dimethylacetamide in concentration at 5% in four refrigeration curves: 1°C/min-1, 0.25°C/min-1, 4°C/min-1 with stabilization at 5°C for 15min and... (Complete abstract click electronic access below)
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A motilidade espermática depende do consumo de energia. Em algumas espécies, a mitocôndria espermática tem um importante papel na produção de energia para o batimento flagelar. A irradiação com Laser de baixa intensidade aumenta a produção de energia funcionando como uma ferramenta de biomodulação. O objetivo deste estudo foi analisar o efeito da irradiação contínua de 650 nm de comprimento de onda de Laser Diodo, com dose de 6 J/cm2 por 120s, na motilidade, integridade das membranas plasmática e acrossomal e no potencial de membrana mitocondrial em espermatozóides in natura e criopreservados. Cinco ejaculados foram obtidos de cinco garanhões (n=25). O sêmen foi acondicionado em palhetas de 0,5mL com 200x106 cells/mL em diluidor Botu-CrioTM (Biotech-Botucatu-Ltda/ME, Botucatu, Brasil) e congelado utilizando o sistema automático programável (TK3000®, TK Tecnologia em Congelação_Ltda, Uberaba, Brasil). As amostras in natura foram divididas em dois grupos: espermatozóides tratados COM LASER e espermatozóides não tratados - SEM LASER. As amostras congeladas foram divididas em três grupos: espermatozóides tratados COM LASER antes da congelação; espermatozóides tratados COM LASER depois da congelação e espermatozóides criopreservados não tratados SEM LASER. As amostras criopreservadas foram analisadas imediatamente após a descongelação (tempo zero) e duas horas após a descongelação (tempo 2). A motilidade foi avaliada pelo sistema de análise espermática computadorizada (CASA, Ivos-Ultimate of Hamilton Thorne Biosciences), a integridade das membranas plasmática e acrossomal e o potencial de membrana mitocondrial foram avaliados pela técnica de citometria de fluxo (FACSaria-Beckton-Dickeson, San Jose, USA). Os dados foram analisados pelo programa SAS, com nível de significância de 5%. A freqüência de batimentos flagelar (BCF) foi alta (P<0,05) para o grupo de espermatozóides in natura tratados COM LASER (34,8 ± 0,7%) quando comparado com o grupo de espermatozóides in natura não tratados SEM LASER (33,4 ± 0,8%). No tempo zero, o potencial de membrana mitocondrial foi baixo no grupo de espermatozóides tratados COM LASER antes da congelação (40,7 ± 1,5%) quando comparados com o grupo de espermatozóides congelados não tratados - SEM LASER (47,4 ± 2,4%) (P<0,05). Duas horas após a descongelação, as membranas plasmática e acrossomal apresentavam maior porcentagem de integridade no grupo de espermatozóides tratados COM LASER antes da congelação (8,3 ± 0,7%) do que no grupo congelado não tratados - SEM LASER (6,2 ± 0,6%) (P<0,05). O grupo de espermatozóides tratados COM LASER depois da congelação não diferiu (P>0,05) dos outros grupos do tempo 2, porém no tempo zero a porcentagem de espermatozóides com motilidade progressiva foi baixa (2,0 ± 0,3%) e diferente (P<0,05) dos outros grupos (6,5 ± 1,3 nos espermatozóides tratados COM LASER antes da congelação e 5,5 ± 1,1% nos espermatozóides congelados não tratados - SEM LASER). Estes resultados indicam que a irradiação de 650 nm de comprimento de onda aumenta a freqüência de batimentos flagelar no sêmen fresco e confere melhor proteção à membrana plasmática e acrossomal ao longo do tempo (após 2h de incubação). Os resultados com sêmen congelado permitem concluir que o melhor momento para a aplicação do laser é antes da criopreservação. Novos estudos com diferentes comprimentos de onda, potência do raio e dosagem devem ser conduzidos para se obter um melhor protocolo visando aprimorar a criopreservação do sêmen eqüino.
Sperm motility depends on energy consumption. In some species sperm mitochondria play an important role in the production of energy for tail activity. Low-level laser irradiation increases this production as a modulation tool. The objective of this study was to analyze the effect of a continuous 650 nm wavelength diode laser irradiation, with dose 6 J/cm2 for 120s, in the motility, plasma and acrosomal membrane integrity and mitochondrial membrane potential in fresh and frozen equine spermatozoa. Five ejaculates were obtained from five stallions (n=25). Semen was packaged into 0.5mL straws with 200x106 cells/mL in a Botu-CrioTM (Biotech-Botucatu-Ltda/ME, Botucatu, Brazil) and frozen by automated technique using a programmed machine (TK3000®, TK Tecnologia em Congelação-Ltda, Uberaba, Brazil). Fresh samples were divided in two groups: spermatozoa treated with laser and without laser (non treated spermatozoa), and frozen samples in three groups: spermatozoa treated with laser before freezing; spermatozoa treated with laser after thawing and without laser (cryopreserved spermatozoa). Cryopreserved samples were analyzed immediately after thawing (time 0) and two hours after thawing (time 2). Motility was evaluated by computer assisted sperm analysis (CASA, Ivos-Ultimate of Hamilton Thorne Biosciences), plasma and acrosomal membrane integrity and mitochondrial membrane potential were evaluated by flow cytometry (FACSaria-Beckton-Dickeson, San Jose, USA). The data were analyzed by the SAS program, at a 5% level. Beat cross frequency (BCF) test was higher (p<0.05) for fresh semen group treated with laser (34.8 ± 0.7%) as compared to non treated group (without Laser - 33.4 ± 0.8%). At time zero, mitochondrial membrane potential was lower in laser treatment before freezing group (40.7 ± 1.5%); compared to without laser treatment group (cryopreserved spermatozoa - 47.4 ± 2.4%) (P<0.05). Two hours after thawing, plasma and acrosomal integrity was higher (P<0.05) in the group were spermatozoa were treated with laser before freezing (8.3 ± 0.7%) compared with the group without laser treatment (cryopreserved spermatozoa) (6.2 ± 0.6%). The group treated with laser after thawing didn´t show any difference (P>0.05) compared to the others groups at this period. However, at time 0 percentage of progressive motility was lower (2.0 ± 0.3%) (P<0.05) than groups treated with laser before freezing (6.5 ± 1.3%) and cryopreserved without laser (5.5 ± 1.1%). These results indicated that the irradiation of 650 nm wavelength diode laser improves beat cross frequency in the fresh semen and support a long term (2 hours) protection to plasma and acrosomal membrane of equine spermatozoa. Based on the results of frozen semen, the best moment for laser application is before cryopreservation protocol. New studies with different diode laser wavelength, different power and energy doses should be driven in order to improve stallion semen cryopreservation.

As biotécnicas da reprodução na espécie equina avançaram na última década, tanto em conhecimento agregado por pesquisas como também pela demanda do mercado. No entanto, na espécie equina machos com subfertilidade são diagnosticados frequentemente com elevado número de espermatozoides com alterações morfológica e/ou imóveis. A utilização apenas de células viáveis para realizar o processo de resfriamento busca evitar perda de material (diluente) e produção de metabólitos tóxicos aos espermatozoides viáveis. O objetivo deste estudo foi utilizar a separação espermática (lã de vidro e centrifugação com Androcoll®) pré-resfriamento para incrementar a viabilidade do sêmen de garanhões pôneis refrigerados a 5ºC durante 48h. Os parâmetros motilidade, funcionalidade de membrana (HOST), viabilidade espermática (CFDA/PI), atividade mitocondrial e morfologia espermática foram avaliados no sêmen fresco e refrigerado (24 e 48h). A utilização da filtração por lã de vidro ou Androcoll® pré-resfriamento do sêmen equino selecionou espermatozoides com maior motilidade, funcionalidade de membrana, viabilidade espermática e atividade mitocondrial. Adicionalmente, a filtração por lã de vidro proporcionou refrigerar sêmen com elevado número de células morfologicamente normais sem perdas significativas de espermatozoides pelo processo de filtração. Tanto a técnica de lã de vidro como a centrifugação com Androcoll® mostraram-se eficientes em separar ejaculados com maior viabilidade para o resfriamento. Já a técnica de lã de vidro apresenta-se como uma técnica de baixo custo e de fácil execução para ser aplicada tanto para pequenos, ou grandes volumes de sêmen.
The reproduction biotechnologies in equine species have advanced in the last decade both in aggregate knowledge by research as well as the market demand. However, it in the equine species often with male subfertility where the ejaculate has a high number of sperm with morphological and / or property changes. The use of only viable cells to perform the cooling process seeks to avoid loss of material (diluent) and production of toxic metabolites to viable sperm. The aim of this study was to use the sperm separation (glass and spin wool with Androcoll®) pre-cooling to increase the viability of semen chilled ponies stallions at 5 ° C for 48 hours. We evaluated the motility parameters, membrane functionality (HOST), sperm viability (CFDA / PI), mitochondrial activity and morphology in fresh and chilled semen (24 and 48h). The use of filtration glass wool or Androcoll® pre-cooling of equine semen selected sperm with higher motility, functionality membrane, sperm viability and mitochondrial activity. In addition to filtration through glass wool afforded cooling semen with a high number of morphologically normal cells without significant losses of spermatozoids the filtration process. Both glass wool technique as centrifugation with Androcoll® were efficient in separating ejaculated more viability for cooling. Already glass wool technique presents itself as a low cost and simple technique to be applied to both small or large volumes of semen.

After ejaculation, mammalian spermatozoa must undergo a series of complex and poorly understood cellular events known as “capacitation” in order to be able to fertilize an oocyte. Among these, biochemical changes such as an increase in tyrosine phosphorylation of some sperm proteins have been correlated with the sperm capacity to fertilize an egg and found to be regulated by a cAMP dependent pathway. The influx of ions such as Ca2+ and HCO3- induce the activation of a soluble adenylyl cyclase (SACY) increasing the cAMP levels within the cell that leads to the activation of a protein kinase A (PKA), and a subsequent increase in protein tyrosine phosphorylation. This modification in sperm proteins seems to be essential for induction of a change in the motility pattern known as hyperactivation that enables the sperm to penetrate the zona pellucida of the oocyte and initiate fertilization. Since PKA is a serine/threonine kinase, it is not clear how it mediates protein tyrosine phosphorylation during sperm capacitation. Based on the finding that in somatic cells PKA activates c-Src, it has been proposed that the Src family of protein kinases (SFK) are the intermediate players involved in tyrosine phosphorylation induced by PKA activity. In order to better understand the molecular mechanisms involved in stallion sperm capacitation, the objectives of our study were: (1) To analyze PKA activity during stallion sperm capacitation (2) To evaluate the involvement of the Src family of protein kinases (SFK) on stallion sperm phosphorylation events associated with capacitation. Standard In Vitro Fertilization (IVF) has not been reproducibly successful in the horse. Recent data indicate that good fertilization rates may be achieved after treatment of sperm with procaine to induce hyperactivation. Our objectives were also to determine if drugs used in other species as well as procaine induce hyperactivation in stallion sperm and to evaluate biochemical changes such as protein tyrosine phophorylation.

Some stallions are expected to perform athletically and breed contemporarily. Athletic activity has the potential, especially during the summer months, to induce thermal stress to the testes, resulting in reduced reproductive capability due to decreased sperm quality and libido. There is concern in the horse industry about what level of exercise, if any, affects the reproductive capability of a stallion. Thermal stress associated with training and exercise may impact sperm quality and the future reproductive capability of the stallion. The goal of this study was to determine the effect of strenuous exercise on stallion sperm quality. The objectives were to measure changes in body and scrotal temperatures following strenuous exercise and sperm quality following strenuous exercise. Miniature Horse stallions (n = 7), implanted with subdermal thermosensory devices in the subcutaneous neck and scrotal tissue, were assigned to treatment group based on age and semen quality. Exercising stallions (EX; n = 3) were exercised 4 d/wk for 90 min for 12 wk, while non-exercising stallions (CN) were tied in the shade. Semen was collected from stallions for 5 consecutive days every 4 wk to evaluate semen quality (raw, 24 h and 48 h cooled). Subcutaneous scrotal (SQST), rectal (RT) and neck (NT) temperatures were recorded along with heart rate. Spermatozoa data were normally distributed; therefore, they were subjected to parametric analysis by repeated measures (wk) using the PROC MIXED procedure (SAS v 9.1; SAS Inst. Inc., Cary, NC). Model included treatment (CN or EX), time (wk 0, 4, 8, or 12), and stallion as the subject of the repeated measures. Compared to the CN group, EX stallions had elevated temperatures (avg RT 39.27 vs 37.07 degrees, NT 39.77 vs 37.44 degrees C, and SQST 34.90 vs 33.40 degrees C; P < 0.0001). There was no difference in sperm quality between treatment groups (P > 0.05). In this study, strenuous exercise in Miniature Horse stallions, did not affect sperm quality. This suggests that anecdotal reports of reduced sperm quality in stallions in training may have other causes other than elevated scrotal and body temperature. While previous studies have illustrated that prolonged insulation of the testes reduces semen quality, strenuously exercising stallions for up to 90 min under hot and humid ambient conditions may not be harmful to spermatogenesis.

Artificial insemination using cooled, transported semen has become a popular practice in the equine industry. However, equine sperm are assumed to show a decline in their fertilizing ability after 24 to 48 hours of cooled storage. Two measures that are commonly used to estimate the fertility of an ejaculate are sperm motility and sperm membrane integrity (SMI). Recently, it has been suggested that SMI may have a better correlation with fertility of an inseminate than sperm motility. The effect of cooled-storage on sperm quality over an extended time period was evaluated to illustrate changes in sperm characteristics that might be related to an ejaculate’s fertility. Semen was stored at 4°C in INRA 96 extender containing 10% seminal plasma for a period of 10 days. Data were collected daily on sperm motion characteristics, SMI, mitochondrial membrane potential, and DNA quality. To measure daily changes in SMI in stallion sperm, two fluorescent vital-staining protocols used in flow cytometric analysis were compared – a combination of SNARF-1, Yo-Pro-1, and Ethidium Homodimer 1 (SYE) and a combination of lectin from Pisum sativum and propidium iodide (PSA/PI). We hypothesized that the SYE protocol adapted for use with stallion sperm could detect more subtle, and perhaps earlier, damage to the sperm plasma membrane than the PSA/PI protocol. A combination of SYBR 14, propidium iodide, and JC-1 (SYPIJC) was used to measure mitochondrial membrane potential, as well as SMI. A computer-assisted sperm motion analysis (CASA) instrument was used to evaluate sperm motion characteristics; the sperm chromatin structure assay (SCSA) was used to measure the degree of DNA fragmentation. In this study, with the exception of sperm motility, the measures of sperm quality retained values consistent with “viability” after 10 days of cooled-storage. This suggests that the fertility of some stallions may last considerably longer than previously assumed, which could ultimately alter the time-table used for artificial insemination using cooled, transported semen.

Density gradient centrifugation of sperm is a common assisted-reproduction procedure in humans used to improve semen quality. The technique allows sperm separation based on their isopycnic points. Sperm with morphologic abnormalities are often more buoyant, leading to their retention above centrifuged density gradients, with structurally normal sperm passing through the gradient. Three experiments were conducted to evaluate the effects of tube size, sperm number following centrifugation, and density gradient volume (height) on stallion sperm quality and recovery rate in sperm pellets following centrifugation. In all three experiments, equine semen was initially centrifuged to increase sperm concentration. In Experiment 1, one-mL aliquots were layered over EquiPure? Bottom Layer (1-Layer) or over-tiered EquiPure? Top and Bottom Layers (2-Layer). For Experiment 2, one-mL aliquots were layered over three different heights of EquiPure? Bottom Layer in 15-mL or 50-mL conical-bottom tubes. For Experiment 3, four different aliquots containing a sperm load of 1-4x were layered over a constant volume of EquiPure? Bottom Layer in 15-mL or 50-mL conical bottom tubes. The tubes were then centrifuged. Resulting sperm pellets were evaluated for morphologic quality, DNA integrity, motility and recovery rate. Sperm-EquiPure? centrifugation yielded improvements in motility, morphology and DNA integrity parameters (P<0.05), as compared to controls. The 1-Layer method resulted in a higher recovery rate than the 2-Layer method (P<0.05). Sperm processed in the 15-mL tubes yielded higher velocity and higher recovery rates than sperm processed in the 50-mL tubes (P<0.05). Within tube type, gradient volume did not impact parameters of semen quality or recovery rate. An increase in sperm number for density gradient centrifugation resulted in a decreased recovery rate (P<0.05) when 15-mL tubes were used.

In the stallion, inconsistent sexual behavior and variable semen quality are common. This reproductive variability has been attributed to differences in circulating hormone concentrations. In order to further examine this relationship, 7 miniature stallions were observed for sexual behavior and semen characteristics. Blood was also drawn from each stallion 15 min before mating (time -15), immediately following ejaculation (time 0) and at times following ejaculation (times +15, +30, and +60). Plasma was later analyzed for concentrations of testosterone (T), estrone sulphate (ES) and cortisol. Semen was evaluated for volume, sperm concentration and progressive motility. Sexual behavior was quantified by assigning a libido score to each stallion, recording reaction time and the number of jumps required for ejaculation. Upon statistical analysis, data revealed both ES and cortisol increased at the time of semen collection (P < 0.05), while T did not. Regression analysis revealed that ES and the ratio of ES to T at times -15, +30, and +60 were negatively correlated to libido scores. Additionally, a positive relationship was found between ES at times -15 and +60 and reaction time, as well as between cortisol at times -15, 0, and +15 and libido scores. No relationship was observed between T and sexual behavior. However, T at time -15 was positively correlated to progressive motility, and the ratio of ES/T at time -15 was negatively correlated to progressive motility. No other association was detected between ejaculate parameters and hormone concentrations. These results not only serve to enhance understanding of stallion hormone profiles, but also provide further insight into the hormonal control of sexual behavior and sperm production. This knowledge can be used to generate improved management techniques for stallions that are inconsistent in sexual behavior and sperm output.

Beurteilung von nativen und aufgetauten Spermatozoen fertiler und subfertiler Hengste mit Hilfe der Phasenkontrast- und Transmissionselektronenmikroskopie. Institut für Veterinär-Pathologie der Veterinärmedizinischen Fakultät, Universität Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In dieser Arbeit wurde die Ultrastruktur von nativen und tiefgefrorenen Spermien mittels Phasenkontrast- und Transmissionselektronenmikroskopie (TEM) untersucht. Für die Beurteilung der Spermienmotilität und der Morphologie von in Formolzitrat fixierten Spermien standen jeweils drei Ejakulate von 50 Hannoveraner Hengsten des Niedersächsischen Landgestüts Celle zur Verfügung. Aus dieser Gruppe wurden drei fertile, drei subfertile Hengste und 6 Hengste mittlerer Fertilität ausgewählt, von denen sowohl die Nativ-Proben als auch eine Tiefgefrierprobe (TG-Probe) für die TEM im Institut für Veterinär-Pathologie der Universität Leipzig gemäß des Standardprotokolls des Institutes aufbereitet wurden. Die Spermien wurden gewaschen und das Seminalplasma der nativen Proben oder der Verdünner der TG-Proben abpipettiert und durch eine 5%-ige Glutaraldehydlösung in einem 0,1 M Kakodylatpuffer (pH 7,2) ersetzt. Die Fixierungslösung wurde anschließend entfernt und das Pellet gewaschen und danach mit Gelatine gemischt. Die spermienreichen Stellen wurden aus der Gelatine herausgeschnitten und in Glutaraldehyd aufbewahrt. Nach einer Nachfixierung in OsO4 und einer Entwässerung in Ethanollösungen erfolgte eine Einbettung in einer Eponmischung. Nach einer Polymerisation von 5 Tagen wurden die eingebetteten Eponblöckchen angetrimmt und die Semi- und Ultradünnschnitte angefertigt. Die Ultradünnschnitte wurden auf ein Kupfergrid gelegt, mit Uranylazetat und Bleizitrat kontrastiert und mit dem Transmissionselektronenmikroskop (Zeiss EM 900, Oberkochem) bei 80 kV analysiert. In den nativen Proben wurden insgesamt 360 Spermien pro Hengst beurteilt, in den TG-Proben 120 Spermien pro Hengst. Die Qualität der elektronenmikroskopischen Aufnahmen war sehr gut, doch die Plasmamembran zeigte fixierungsbedingte Artefakte. Nach dem Auftauen waren die Bilder heller und der Kontrast etwas geringer. Es gab eine Zunahme an Akrosomdefekten, akrosomreagierten Spermien und Beschädigungen der Plasmamembran, der Mitochondrien, sowie der Mantel- und Ringfasern. Durch die Membranbeschädigungen trat auch eine Verringerung der Anzahl proximaler und distaler Zytoplasmatropfen auf. Sowohl geschwollene Akrosome mit einer niedrigeren Dichte der akrosomalen Matrix als auch Mitochondrien mit einer zu hellen mitochondrialen Matrix waren typische Befunde in den TG-Proben. Die Studie der Ultrastruktur und die wahrgenommenen Defekte führten zur Erstellung eines Standardprotokolls für die transmissionselektronenmikroskopische Beurteilung von Hengstspermien. Die Beurteilung mittels TEM sollte aber nicht zu einer quantitativen, sondern zu einer qualitativen Aussage führen. Sie ermöglicht die Diagnose von Kern- (Kerndeformationen und Taschenbildung im Kern) und Akrosomabweichungen (deformierte Akrosome mit oder ohne Vakuolenbildung, abgehobene Akrosome und akrosomreagierte Spermien), Anomalien der Mitochondrien (Unterbrechung der Mitochondrienscheide, zu viele Mitochondrien, anormale Dichte der mitochondrialen Matrix), Defekten des Axonemas (Ordnung oder Anzahl der Mikrotubuli, Mantel- und Ringfasern) und der Anwesenheit immaturer Spermienvorstufen. Diese Methode eignet sich für die Diagnostik subfertiler Hengste mit normalen Spermienparametern bei der routinemäßige Spermienbeurteilung und kann sowohl in nativen als auch in TG-Proben angewendet werden. Im Vergleich zur Phasenkontrastmikroskopie waren die elektronenmikroskopischen Bilder wegen ihrer stärkeren Vergrößerung und der Darstellung innerer Spermienstrukturen viel aussagekräftiger. Für die Beurteilung von Halsansatzdefekten, abweichende Geißelformen und Mehrfachmißbildungen ist die Phasenkontrastmikroskopie die am besten geeignete Methode.
Evaluation of fresh and frozen-thawed semen samples of fertile and subfertile stallions by light microscopy and transmission electron microscopy. Institut of Pathology of the Faculty of Veterinary Medicine, University of Leipzig Reproduktionsmedizinische Einheit der Kliniken der Tierärztlichen Hochschule Hannover In this study the ultrastructure of fresh and frozen-thawed semen samples of 50 stallions from the National Stud of Lower Saxony (Celle, Germany) were evaluated by light microscopy and transmission electron microscopy (TEM). Three ejaculates of each stallion were available for the motility analysis and the morphological analysis by lightmicroscopy after fixation in formol citrate. Based on the fertility data, the ejaculates of 12 stallions (3 fertile stallions, 3 subfertile stallions and 6 stallions of average fertility) were selected for the morphological analysis by TEM. The native samples and one frozen-thawed sample from these stallions were prepared for the TEM at the Institute of Pathology of the Faculty of Veterinary Medicine, Uni-versity of Leipzig. The sperm cells were washed and the seminal plasma from the native samples and the diluents of the frozen-thawed samples were replaced by a 5%-glutaraldehyde solution in a 0,1 M cacodylate buffer pH 7,2. The fixative was removed, the pellet was washed again and mixed with gelatin. The sperm rich fraction in the gelatin mass was excised and stored in glutaraldehyde. A second fixation in OsO4 was followed by a dehydratation in ethanol and a polymerization phase in epon. After 5 days of polymerization the starred samples were used for semi- and ultratight cuts. The latter were placed on a copper grid, contrasted with uranyl acetate and lead citrate and analyzed with the transmission electron micro-scope (EM 900) by 80 kV. In the fresh samples, 360 sperm cells were examined per stallion, whereas in the frozen-thawed samples only 120 sperm cells per stallion were evaluated. The microscopic pictures were of a high quality. However, the sperm plasma membrane showed some fixation artifacts. In the thawed samples a lower contrast was noticed than in the fresh samples. The sperm cells in the frozen-thawed samples showed an increase in acrosome defects, acrosome reactions, damage of the cell plasma membrane, mitochondria, fibrous sheet and outer dense fibers. The latter defect was associated with a decrease in proximal and distal cytoplasmatic droplets. Swollen acrosomes with a lower matrix density and a bright mitochondrial matrix were typically present in the cryopreserved samples. The ultrastructural defects in these samples, examined by TEM, have led to the development of a standard evaluation protocol with the most common sperm defects in stallion semen. TEM is an expensive and time consuming technique, which cannot be used to obtain quantitative results, but is considered as an accurate method for the qualitative examination of semen samples in cases of unexplained subfertility. TEM can especially be recommended for the diagnosis of nuclear (nuclear malformations and pouches) and acrosomal defects (acrosome deformations, acrosome vacuoles, detached acrosomes and acrosome reactions), mitochondrial (mitochondrial sheet defects, mitochondrial proliferation, decrease in mitochondrial matrix density) and axonema malformations (anormal position or quantity of microtubules and fibrous sheet or outer dense fibers defects) and the detection of immature sperm cells in ejaculates. The results of this study state that TEM can be useful for the evaluation of both fresh and frozen-thawed semen samples. Compared to the light microscopic evaluation of stallion sperm, the TEM images give more precise information because of their higher magnification rate and the ability to reveal internal sperm structures. However, light microscopy remains the best method to detect sperm neck defects, deformed tailes and sperm cells with multiple heads or tails.