Equine frozen spermatic cell survivor after thawed and dilution using two different commercial extenders

O presente estudo teve por objetivo avaliar parâmetros de motilidade e viabilidade in vitro na diluição do crioprotetor dimetil-formamida a 5% pós-descongelação para concentrações de 2,5% e 1,25%, mediante utilização de dois diluentes comerciais adicionados junto ao sêmen. Após a descongelação, as amostras foram diluídas com a finalidade de manter as concentrações finais (2,5% e 1,25%) de crioprotetor, utilizando-se dois diluentes comerciais (FR4® e Botu-Crio®) em dois tempos: inicial (Ti) e final (Tf). Empregaram-se quinze amostras de ejaculados distintos de cinco garanhões de raças nacionais. Os parâmetros de motilidade foram observados através da análise computadorizada e os de integridade de membrana plasmática pela microscopia de epifluorescência. Verificou-se melhora nos parâmetros de motilidade total e progressiva dos espermatozóides, no tempo final (P0,05) entre os tratamentos quanto à integridade de membrana plasmática. ___________________________________________________________________________________________ ABSTRACTThe purpose of this study was to evaluate the motility and viability of stallion semen cryopreserved with 5% dimethyl-formamyde as cryoprotectant and posterior dilution to 2.5 and 1.25% as final concentration of cryoprotectant. After thawing, samples were diluted with two commercial extenders (FR4® and Botucrio® ) in two moments: initial (Ti) and final (Tf). A total of 15 distinct ejaculates from five stallions of national breeds were analysed. Motility was observed using a computer assistant system analysis and viability was analyzed using fluorescent probes. A significant (P0.05) on membrane integrity between treatments was not observed

Sensitivity evaluation of the computer-assisted sperm analysis (CASA) in the determination of frozen-thawed bull semen concentration

Tradicionalmente, a concentração espermática é avaliada por meio da contagem de células em câmara hemocitométrica de Neubauer, técnica laboriosa adotada na rotina dos laboratórios de andrologia. Uma alternativa para essa contagem é a técnica computadorizada de avaliação espermática (CASA), método que pode aumentar a eficiência e acurácia na determinação da concentração de espermatozoides em uma amostra de sêmen. O presente trabalho relata a avaliação da sensibilidade da técnica CASA para o acesso da concentração de espermatozoides bovinos em pósdescongelação. Foram selecionadas 425 doses de sêmen de reprodutores de diferentes raças, descongeladas a 37°C por 30 segundos e homogeneizadas. Alíquotas de 40 µL de sêmen foram transferidas para tubos cônicos de 1,5 mL previamente preenchidos com 960 µL de água destilada, fixando a taxa de diluição em 1:25 para contagem em câmara de Neubauer. Em contrapartida, alíquotas de 5 µL de cada dose de sêmen foram avaliadas com o emprego do sistema CASA considerando o número mínimo de cinco campos aleatórios e 2 mil espermatozoides por análise. A concentração média de células espermáticas foi de 38,96a ± 1,28 e 35,14b ± 0,82,respectivamente para amostras avaliadas em câmara de Neubauer ou sistema computadorizado, apresentando o coeficiente de correlação de 0,87 (P < 0.0001) e concordância de 0,78 (escala de 0 a 1). Conclui-se que as duas técnicas de avaliação da concentração espermática possuem eficiência similar. No entanto, em virtude da precisão, rapidez e por dispensar a diluição prévia das amostras para a contagem, a CASA é uma alternativa para a contagem de células espermáticas em câmara de Neubauer, sobretudo para grandes centrais de produção de sêmen bovino congelado.Sperm concentration is traditionally evaluated by counting cells in a hemocytometric Neubauer chamber, often a highly subjective, time-consuming, and laborious technique prevalent in andrology laboratories around the world. However, the Computer-Assisted Semen Analysis (CASA) represents a more consistent method of evaluating sperm concentration that may provide enhancing efficiencies of sperm count. The purpose of this study is to compare the results of these two methods in the analysis of post-thaw concentration of bovine semen. Four hundred and twenty five batches of semen from different bulls were selected, thawed at 37°C for 30 seconds and then homogenized. Aliquots of 40 μL of semen were diluted in 960 μL of distilled water, fixing the rate at 1:25 dilution for analysis in a Neubauer chamber. Conversely, aliquots of 5 μL for each semen dose were submitted to CASA, considered a minimum of five random fields and 2000 sperm count per analysis. The average concentration of sperm cells was 38.96a ± 1.28 in the Neubauer analysis and 35.14b ± 0.82 for the CASA, with the correlation coefficient of 0.87 (P < 0.0001) and reliability of 0.78 (scale ranging from 0 to 1) between the two methods. In conclusion, the results of two techniques for assessing sperm concentration have similar results. However the CASA methodology would yield greater benefit due to precision, consistency, and reduced disposal issues, particularly for large processing laboratories

Use of Bayesian Inference to Correlate In Vitro Embryo Production and In Vivo Fertility in Zebu Bulls

The objective of this experiment was to test in vitro embryo production (IVP) as a tool to estimate fertility performance in zebu bulls using Bayesian inference statistics. Oocytes were matured and fertilized in vitro using sperm cells from three different Zebu bulls (V, T, and G). The three bulls presented similar results with regard to pronuclear formation and blastocyst formation rates. However, the cleavage rates were different between bulls. The estimated conception rates based on combined data of cleavage and blastocyst formation were very similar to the true conception rates observed for the same bulls after a fixed-time artificial insemination program. Moreover, even when we used cleavage rate data only or blastocyst formation data only, the estimated conception rates were still close to the true conception rates. We conclude that Bayesian inference is an effective statistical procedure to estimate in vivo bull fertility using data from IVP

Novo protocolo utilizando ocitocina para induzir a ejaculação em garanhão penectomizado

Background: Several reproductive diseases can prevent ejaculation by the traditional method of collection. Neoplasias as squamous cell carcinoma is the most common tumor of the external genitalia of horses and its lesions usually prevent copulation. The pharmacological induction of ejaculation is an important alternative technique to obtain and preserve the genetic material of stallions incapable of ejaculating by traditional methods of semen collection. However, the protocols currently used have shown questionable results and new protocols are needed in order to increase the success rates. The aim of this study is to report the success of a new protocol in inducing ejaculation when oral imipramine and intravenous oxytocin and detomidine were administrated in a Crioulo stallion.Case: A 9-year-old Crioulo stallion was admitted at the Veterinary Hospital of the São Paulo State University, FMVZUNESP, Botucatu, Brazil, with a history of a mass located on the glans and body of the penis. The histopathological exam confirmed the diagnostic of Squamous cell carcinoma and penectomy was performed. After 10 days of surgery the stallion was submitted to 5 different protocols with 3 days interval between the follow protocols: Imipramine+Xylazine; Imipramine+ Xylazine+Oxytocin; Imipramine+Detomidine and Imipramine+Detomidine+Oxytocin.Discussion: The traditional protocol of pharmacologically-induced ejaculation with imipramine hydrochloride (3 mg/kg/v.o) and xylazine hydrochloride (0.66 mg/kg/iv) was not successful even when oxytocin (20 UI/iv) was added to this protocol. Administration of imipramine hydrochloride (3 mg/kg/v.o) two hours prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) also did not result in ejaculation. However, administration of imipramine hydrochloride (3 mg/kg/v.o) 2 h prior to administration of detomidine hydrochloride (0.02 mg/kg/i.v) associated with oxytocin (20 U.I/i.v) resulted in ejaculation. The stallion was submitted to three seminal collections with a three-day interval between administration of the protocol and ejaculated in all the attempts after approximately 5 min of detomidine and oxytocin injection, presenting mean values of 50 mL of total volume (TV) and concentration of 80x106 spermatozoa/mL. The VT was higher and concentration was lower when compared to ejaculates obtained by pharmacological induction in previous studies, probably due to daily stimulation with estrus mare to induce penile exposure in order to allow antisepsis of the surgical wound. Thus, it is believed that the large amount of total volume of this stallion is due to the high production of gel by the accessory sex glands, with consequent reduction of ejaculate concentration. The sperm kinetics were evaluated by the computerized method CASA (HTMA-IVOS-12) with total motility (MT) of 84% and progressive (MP) of 38%, with 70% of rapid spermatozoa (RAP), being considered normal to the equine specie and similar to those observed by other authors in pharmacolocally-induced ejaculates. Post-thaw sperm kinetics presented 42% of MT, 21% of MP and 28% of RAP probably due to an intrinsic sensitivity of the stallion to the freezing process. Thus, this report concludes that the protocol associating imipramine, detomidine and oxytocin was efficient in the pharmacological induction of ejaculation, presenting normal and characteristic sperm parameters of the specie. Fresh and refrigerated semen presented good parameters for use in conventional artificial inseminations while frozen semen is indicated for deep horn inseminations or for use in intracytoplasmic sperm injection (ICSI) programs

Estudo da técnica de coleta, congelação e descongelação de embriões de caprinos (Capra hircus), da raça Saanen, portadores da translocação 5/15

This work studied the surgical technique for collecting embryos in the goat, using heterozygous donors for the 5/15 translocation, which were bred with translocated animals. At the same, time technical characteristics related to cryopreservation, thawing and cultivation of the structures were performed. Considering the superovulatory treatment, oestrus was observer 24 or 48 hours after removing the intravaginal sponge and 71 viable structures were yielded in 43 collections. The 1 -2 Propanediol proved to be an efficient cryoprotectant agent, and allowed the cultivation of all the embryos. This result was not observed when glycerol was used as a cryoprotectant agent. Adhesions due to multiple collections constitute a great challenge for the improvement of this technique, since they are considered a limiting factor to the use of donors in the beginning of their reproductive lives.No presente trabalho foi realizado um estudo da técnica cirúrgica de coleta de embriões na espécie caprina, utilizando-se doadoras heterozigotas para a translocação 5/15, acasaladas com animal translocado. Simultaneamente foram observados aspectos técnicos inerentes à criopreservação, descongelação e cultivo das estruturas recuperadas. Frente ao tratamento superovulatório, o cio foi observado em 24 ou 48 horas após a remoção da esponja intravaginal e foram recuperadas 71 estruturas viáveis em 43 coletas. O 1 -2 Propanodiol mostrou-se eficiente criopreservador, permitindo cultivo de todos os embriões, fato não observado quando utilizou-se o Glicerol. As aderências provocadas pelas repetidas coletas constituem-se no grande desafio no sentido de se aperfeiçoar a técnica, pois são consideradas um fator limitante ao uso de doadores no início da vida reprodutiva

Morte embrionária precoce em éguas: aspectos clínicos e hormonais

The present study aimed to diagnose early embryonic death in 128 mares. Blood samples were collected at the 4th, 7th, 10th, 13th, 16th, 19th, 21st and 30th day after ovulation and the diameter of the corpus luteum was measured. The diameter and characteristics of the embryonic vesicle were evaluated by ultrasonography. Microbiology, cytology and histopathology were carried out in mares with embryonic death. Plasma oestrogen and progesterone concentration were measured by radioimmunoassay. From 128 mares, 17 (13.28%) showed early embryonic death. The embryonic losses took place at 19 days of pregnancy in 47.05% and at 21 days of pregnancy in 29.4% of the mares. The diameter of the corpus luteum in the mares that maintained pregnancy was similar to those with embryonic loss. Otherwise, the diameter of the embryonic vesicles was bigger at the 16th, 19th, 21st and 30th day of pregnancy when compared to the mares with embryonic loss. The mean plasma progesterone concentration was similar in both groups and the median plasma oestrogen concentration was higher in the pregnant mares. The cytological, microbiological and histopathological exams revealed that most of the mares had endometritis. Ultrasonography provided an early diagnosis of pregnancy (from the 12th day post-ovulation) and important information about the development of embryo and embryonic death. Endometritis was considered the main cause of embryonic losses in this study.O presente estudo objetivou diagnosticar as causas da morte embrionária precoce em 128 éguas. Amostras de soro foram coletadas nos dias 4, 7, 10, 13, 16, 19, 21 e 30 após ovulação, para dosagens hormonais e mensurações do corpo lúteo. O diâmetro e as características da vesícula embrionária foram avaliados, através da ultra-sonografia, a partir do 12º dia pós-ovulação. Das 128 éguas estudadas, 17 (13.28%) apresentaram morte embrionária. O diâmetro do corpo lúteo, bem como as concentrações plasmáticas de progesterona, foi semelhante nos grupos que apresentaram morte embrionária e nos que mantiveram a gestação. Os níveis de estrogeno plasmático foram mais elevados no grupo das fêmeas que mantiveram a gestação. Os exames citológicos, microbiológicos e histopatológicos revelaram que a maioria das éguas com diagnóstico de morte embrionária eram portadoras de endometrites

A força de centrifugação pode comprometer a integridade de membrana plasmática, acrossomal e DNA de espermatozoides caprinos?

Protocols for cooling or freezing goat semen usually recommend centrifugation for seminal plasma removal. However, little is known about the effect of this process on goat sperm viability and functionality. The present study evaluated the effects of centrifugation force on the plasma membrane, acrosomes, and DNA integrity of goat semen. Four ejaculates from each of the four different Anglo Nubian male goats were used. Semen samples were obtained using artificial vagina, and immediately after collection, ejaculates were diluted using Ringer’s sodium lactate solution and split into three groups: Control (CG, without centrifugation), G1 (centrifugation 600 x g/10 min), G2 (centrifugation 1200 x g/10 min). After centrifugation, seminal plasma was removed, the sperm pellets were resuspended using Tris-egg yolk extender (80 x 106 spermatozoa/mL) and the sperm morphology was analyzed. Samples were cooled at 5°C for 5, 24, 36, and 48 h and then sperm plasma membrane and acrosome integrity (PMAI, %) and sperm DNA fragmentation index (SDF, %) were evaluated at each time-point, using a flow cytometer. Additionally, sperm movement was determined using computer semen analysis (CASA) after 5, 24, and 48 h of refrigeration period. The semen centrifugation did not induce additional sperm morphology defect or reduction in sperm kinetics in the experimental groups. Differences were not observed (p > 0.05) in PMAI and SDF among different groups, in any of each timepointof the cooling process. In conclusion, centrifugation, even at high speeds, did not affect goat sperm integrity and functionality when submitted to refrigeration process.A maior parte dos protocolos de refrigeração e criopreservação do sêmen caprino recomenda o uso de centrifugação para remoção do plasma seminal. No entanto, não existe consenso sobre o risco que esse tipo de processamento pode ocasionar à viabilidade espermática. Nesse contexto, o presente trabalho investigou os possíveis efeitos deletérios da centrifugação sobre a integridade estrutural e DNA de espermatozoides caprinos. Para a pesquisa foram selecionados quatro reprodutores para colheita de sêmen (n = 4 ejaculados/bode). Cada ejaculado foi fracionado em três alíquotas iguais, diluídas em ringer e divididas em três grupos: Controle (GC, não centrifugado), G1 (centrifugação a 600 g/10 minutos) e G2 (centrifugação a 1200 g/10 minutos). As amostras seminais por grupo foram diluídas em meio Tris gema respeitando-se a concentração final de 80 milhões de espermatozoides/mL e foram submetidas à avaliação de morfologia espermática. Todas as amostras foram acondicionadas a 5°C, sendo analisadas nos momentos 5, 24, 36 e 48 horas do processo de refrigeração por meio da avaliação da integridade de membrana plasmática e acrossomal (MPAI, %) e índice de fragmentação de DNA (IDF, %). Adicionalmente, a cinética espermática foi avaliada com o emprego de um sistema computadorizado de análise (CASA) nos momentos 5, 24 e 48 horas da refrigeração. A centrifugação não induziu a manifestação de defeitos morfológicos ou redução significativa da cinética de espermatozoides caprinos. Não foram observadas diferenças para a integridade de membrana plasmática e para o índice de fragmentação de DNA quando comparados, respectivamente, GC, G1 e G2 em cada um dos quatro momentos experimentais. Conclui-se que mesmo quando empregadas altas forças de rotação não ocorre lesão à ultraestrutura dos espermatozoides caprinos submetidos ao processo de refrigeração

How to Perform and Interpret Testicular Fine Needle Aspiration in Stallions

Although several methods of testicular biopsy have been proposed previously, testicular fine needle aspiration (FNA) has proved to be the simplest, the most rapid, inexpensive, and overall the least invasive technique for obtaining testicular biopsies Testicular FNA is indicated for fertility investigations in stallions with oligozoospermia or azoospermia It is also used for differential diagnosis of testicular enlargement After sedation the stallion's testis is punctured to obtain testicular parenchyma samples containing cells mainly from the seminiferous epithelium the material obtained is used to perform smears which are analyzed for identification and quantification of term cells and Sertoli cells The results are based on the presence of the cell types found in the smears and the proportions of Sertoli cells per germ cells In addition to being a very useful diagnostic tool, testicular FNA is also used for follow-up examinations, as it is minimally invasiv

Cytological identification and quantification of testicular cell types using fine needle aspiration in horses

Fifteen stallions of different breeds, age 3-11 years, had their right testicles evaluated by fine needle aspiration cytology (FNAC). Cytological analysis showed the following spermatogenic cell types: spermatogonia (1.6% +/- 1.1); spermatocyte I (3.4% +/- 2.2); spermatocyte II (0.8% +/- 0.7); early spermatids (25.5% +/- 9.5); late spermatids (37.0% +/- 9.3). Spermatozoal numbers were expressed as the spermatic index (SI = 31.5% +/- 8.5) and Sertoli cells mere expressed as the Sertoli cell index (SEI = 20.9% +/- 17.0) (means +/- s.d). Identification of cell types was relatively easy and no immediate adverse effects of aspiration were noted. The results suggest that FNAC of testis may assist clinical diagnosis in the study of male equine infertility

Efeitos do hipotireoidismo no sistema reprodutor masculino

Thyroid hormones are essential for growth, development and metabolism in many tissues and organs. Currently, it is known that these hormones play an important activity in testicular function in several species through specific receptors located, mainly, in Sertoli cells. The thyroid dysfunction, particularly hypothyroidism, affects testicular steroidogenesis and spermatogenesis and may lead to infertility. Although several reproductive changes have been related in animals with hypothyroidism, the results of some studies are controversial and little is known about the direct effects of thyroid hormones in the reproductive system. Through this article, we report a revision in which we will describe the effects of hypothyroidism in the testicular function and accessory glands.Las hormonas tiroideas son esenciales para el crecimiento, el desarrollo y el metabolismo en varios tejidos y órganos. En la actualidad, se sabe que estas hormonas desempeñan una actividad importante en la función testicular en varias especies a través de receptores específicos situados principalmente en las células de Sertoli. Las disfunciones de la tiroides, en especial el hipotiroidismo, afecta a la espermatogénesis y la esteroidogénesis testicular pudiendo llevar a la infertilidad. A pesar de haber sido descritas diversas alteraciones reproductivas relacionadas con el hipotiroidismo en animales, los resultados de las investigaciones son contradictorios y poco se sabe sobre los efectos directos de las hormonas tiroideas en el sistema reproductor. En esta revisión se describen los efectos del hipotiroidismo sobre la función testicular y glándulas accesorias.Os hormônios tireoidianos são essenciais para o crescimento, o desenvolvimento e o metabolismo de diversos tecidos e órgãos. Atualmente, sabe-se que estes hormônios desempenham uma importante atividade na função testicular em diversas espécies por meio de receptores específicos situados, principalmente, nas células de Sertoli. As disfunções tireoidianas, em especial o hipotireoidismo, afeta a espermatogênese e a esteroidogênese testicular podendo levar à infertilidade. Embora sejam relacionadas diversas alterações reprodutivas em animais que apresentam hipotireoidismo, os resultados das pesquisas são controversos e pouco se conhece sobre a ação direta dos hormônios tireoidianos no sistema reprodutor. Por meio deste artigo, objetiva-se apresentar uma revisão na qual serão descritos os efeitos do hipotireoidismo na função testicular e glândulas acessórias