Abstract The identification of putative prognostic factors in canine mammary neoplasms (CMNs) has been focused on tissue-specific biomarkers, but the serum biomarkers, including cancer antigen 15-3 (CA 15-3), c-reactive protein (CRP), and lactate dehydrogenase (LDH) have been demonstrated to display clinical application in cases of CMNs. The aim of the study was to evaluate the levels of these serum biomarkers and their association with well-established prognostic factors in CMNs. Samples from 15 female canines with CMNs and 15 clinically healthy ones were collected. The results were evaluated using the Tukey’s, Pearson, or Spearman tests. The cut-off point, sensitivity, specificity, and area under curve (AUC) were evaluated using the receiver operating characteristic (ROC) curve analysis in a logistic regression model (P<0.05). The levels of CA 15-3, CRP and LDH were significantly higher in the serum of female dogs with CMNs compared to the healthy ones. Moreover, these factors were positively correlated with ulceration, tumor size, histopathological grade, metastatic lymph node, and clinical staging. Female dogs with CMNs were found to exhibit highest serum levels of CA 15-3, CRP, and LDH. Therefore, they can be applied to improve the efficacy of the diagnosis and prognostic evaluation in casas of CMNs.

Abstract Heterologous in vitro fertilization (IVF) is an important tool for assessing fertility of endangered mammals such as the jaguar, considering difficult access to females for artificial insemination and to obtain homologous oocytes. We aimed to evaluate the fertility of jaguar sperm cryopreserved with different extenders, using domestic cat oocytes to assess the development of hybrid embryos. Semen from four captive jaguars was obtained by electroejaculation. Samples were cryopreserved in powdered coconut water (ACP-117c) or Tris extender containing 20% egg yolk and 6% glycerol. Thawed spermatozoa were resuspended (2.0 × 106 spermatozoa/mL) in IVF medium and co-incubated with cat oocytes matured in vitro for 18 h. Presumptive zygotes were cultured for 7 days. After 48 h, cleavage rate was evaluated, and non-cleaved structures were stained for IVF evaluation. On days 5 and 7, the rate of morula and blastocyst formation was assessed. Data were analyzed using the Fisher exact test (p < 0.05). No difference was observed between ACP-117c and Tris extenders, respectively, for oocytes with 2nd polar body (2/51, 3.9 ± 2.9% vs. 2/56, 3.6 ± 3.1%), pronuclear structures (5/51, 9.8 ± 4.7% vs. 8/56, 14.3 ± 8.0%), and total IVF rates (7/36, 19.4 ± 5.0% vs. 10/37, 27.0 ± 13.8%). All the samples fertilized the oocytes, with 22.9 ± 3.2% (16/70) and 16.7 ± 3.6% (12/72) cleavage of mature oocytes for ACP-117c and Tris extenders, respectively. Morula rates of 4.3 ± 2.3% (3/70) and 5.6 ± 2.2% (4/72) were observed for ACP-117c and Tris, respectively. Only the Tris extender demonstrated blastocyst production (2/12, 16.7 ± 1.5% blastocyst/cleavage). We demonstrated that jaguar ejaculates cryopreserved using ACP-117c and Tris were suitable for IVF techniques, with blastocyst production by ejaculates cryopreserved in Tris. This is a first report of embryos produced in vitro using jaguar sperm and domestic cat oocytes through IVF.

Abstract Morphological sperm evaluation supported by the morphometry can be used in the determination of the seminal quality and in the investigation of potential extenders. Although there are studies comparing TRIS and ACP extenders, there are no comparative studies between them for the computerized assisted semen analysis (CASA), sperm viability, membrane functionality and sperm morphometry parameters of cryopreserved canine semen. Hence, we aimed to evaluate the effects of ACP-106c and TRIS on post-freezing canine sperm quality. Five dogs were submitted to semen collection twice with one-week interval. The semen was evaluated within the parameters: total motility, vigor, concentration, viability, plasma membrane functionality, morphology and morphometry. In the morphometric evaluation, the morphologically normal sperm was measured as: length, width, area and perimeter of the head and the midpiece, tail length and total length. The parameters of ellipticity, elongation, regularity and roughness were determined. Then, the semen was divided into two aliquots that were diluted in TRIS or ACP-106c, with the addition of egg yolk and glycerol. The diluted semen was refrigerated and frozen. The thawed samples were evaluated. Total motility, viability, sperm membrane functionality and normal morphology reduced after thawing in both extenders (morphology reduced from 89.60 ± 1.3% to 84.40 ± 1.8 and 84.60 ± 1.1% in TRIS and ACP-106c, respectively). However, it did not differ between TRIS and ACP-106c. In the ACP-106c the sperm head defects in cryopreserved semen were higher compared to fresh semen (P < 0.05). For all the morphometric parameters evaluated, there were no differences between fresh and cryopreserved samples (3.70 ± 0.4% vs. 2.30 ± 0.5%). In kinetics, with an interval of one week statistical differences between the extenders were found only in the parameters ALH and LIN (P < 0.05). Regardless of the extender, there were no changes in the morphometric parameters of sperm after thawing.

Resumo: Este estudo foi realizado para determinar o efeito de hidroxitolueno butilado (BHT) sobre a qualidade do sêmen canino congelado e descongelado, utilizando o diluidor à base de água de coco em pó (ACP-106c). Para tanto, foram realizadas quinze coletas de sêmen provenientes de cinco cães. O sêmen obtido foi diluído em ACP-106c acrescido de glicerol e gema de ovo. As amostras foram então transferidas para tubos contendo diferentes concentrações de BHT (0; 0,5; 1,0 e 2,0 mM). Em seguida, as amostras foram envasadas, congeladas e armazenadas em nitrogênio líquido. O sêmen coletado foi avaliado in natura quanto aos seguintes parâmetros: coloração, volume da fração espermática, motilidade total, vigor, concentração, morfologia e funcionalidade de membrana espermática. Após uma semana, as amostras foram descongeladas e avaliadas por meio de análise computadorizada, como também foram realizadas análises da funcionalidade de membrana e da morfologia espermática. A motilidade progressiva no grupo BHT 2,0 mM foi significativamente superior (P < 0,05) do que a do grupo BHT 0 mM (27,6 ± 11,7% vs. 19,0 ± 9,5%, respectivamente). Em todos os demais parâmetros avaliados, não houve diferença entre os grupos testados. Portanto, conclui-se que a adição do BHT ao diluidor ACP-106c não afetou a qualidade do sêmen canino pós-descongelação.

Abstract: This study was conducted to determine the effect of butylated hydroxytoluene (BHT) on the quality of canine sperm frozen and thawed using the powdered coconut water based (ACP-106c) extender. Therefore, fifteen ejaculates were collected from five dogs. Semen obtained was diluted in ACP-106c added of glycerol and egg yolk. The samples were then transferred to tubes containing different concentrations of BHT (0, 0.5, 1.0 and 2.0 mM). After that, the samples were filled into straws, frozen and stored in liquid nitrogen. Fresh semen was evaluated for the following parameters: color, sperm fraction volume, total motility, vigor, concentration, morphology, and HOST test. After one week, the samples were thawed and evaluated by computer analysis, as well as for membrane functionality and sperm morphology. Progressive motility in the 2.0 mM BHT group was significantly higher (P <0.05) than that of the 0 mM BHT group (27.6 ± 11.7% vs. 19.0 ± 9.5%, respectively). Regarding all other parameters evaluated, there was no difference between the groups tested. Therefore, the addition of BHT to the ACP-106c extender did not affect the quality of canine semen after thawing.

RESUMO: A criopreservação do tecido testicular possibilita a manutenção da capacidade reprodutiva em diferentes espécies animais e contribui para a formação de bancos de germoplasma nas espécies ameaçadas de extinção. As espermatogônias presentes nos testículos podem ser cultivadas in vitro e os espermatozoides obtidos utilizados em programas de reprodução artificial. Assim, esta revisão teve como objetivo descrever as principais técnicas de criopreservação testicular, os principais crioprotetores utilizados e os avanços obtidos até o presente momento nas diferentes espécies animais. Na última década, os avanços obtidos com a utilização do tecido testicular para obtenção de células germinativas viáveis e funcionais foram significativos. Todavia, o estabelecimento de protocolos que possam ser utilizados na rotina clínica em diferentes espécies ainda necessitam de maiores esclarecimentos.

ABSTRACT: Cryopreservation of testicular tissue enables the maintenance of reproductive capacity in different animal species, and contributes to the formation of gene banks for endangered species. The spermatogonia present in the testes can be grown in vitro and the sperm obtained can be used in artificial breeding programs. This review aimed to describe the main techniques of testicular cryopreservation, the main cryoprotectants used, as well as the progress made in different animal species thus far. In the last decade, significant progress has been made in obtaining viable and functional germ cells from testicular tissue. However, more research is needed to better establish protocols that can be used in clinical practice with various species.

RESUMO: Pesquisas têm sido realizadas para identificar as proteínas presentes no plasma seminal canino, com o intuito de relacioná-las com a qualidade espermática, bem como buscar por marcadores moleculares de patologias do trato reprodutivo. Há evidências de que as proteínas ligadoras de heparina, ligadoras de zinco, a lactoferrina, bem como as enzimas matrix metalloproteinase, superoxide dismutase, catalase e a glutationa peroxidase estão relacionadas com a qualidade seminal canina. Outras pesquisas indicam que a prolactina, e as enzimas arginina esterase, fosfatase ácida e fosfatase alcalina poderiam ser utilizadas com sucesso como biomarcadores de doenças reprodutivas. Assim, esta revisão de literatura objetiva abordar aspectos relacionados às proteínas do plasma seminal canino, suas influências sobre a fertilidade, e sua importância como biomarcadores de doenças reprodutivas.

ABSTRACT: Studies have been performed to identify the proteins present in canine seminal plasma (SP) and relate them to sperm quality as well as to discover molecular markers of reproductive tract diseases. There is evidence that heparin-binding proteins, zinc-binding proteins, and lactoferrin as well as the matrix metalloproteinase, superoxide dismutase, catalase, and glutathione peroxidase enzymes are associated with canine sperm quality. Other studies indicate that prolactin and enzymes like arginine esterase, acid phosphatase, and alkaline phosphatase could be successfully used as biomarkers of reproductive disorders. Thus, the present literature review aims to address aspects related to proteins of the canine SP, their influence on fertility, and their importance as biomarkers of reproductive disorders.

A estocagem do sêmen por um longo período, permitindo o seu posterior uso representa uma importante ferramenta para criadores que desejam resguardar o potencial genético de seus reprodutores. O objetivo do presente trabalho foi avaliar o efeito da água de coco, gema de ovo e glicerol sobre o resfriamento e a criopreservação de sêmen canino. A fração espermática do ejaculado de 12 cães foi avaliada macro e microscopicamente e, em seguida, dividida em quatro alíquotas, submetidas à congelação em quatro diluidores, sendo todos à base água de coco e diferindo quanto à presença ou não da gema de ovo e glicerol. Durante o resfriamento, não se observou diferença entre os grupos, entretanto, após o congelamento e descongelamento, o diluidor adicionado de gema de ovo e glicerol (ACGG) foi superior aos demais quanto à motilidade, vigor e morfologia espermática. Nesse grupo, os valores de motilidade (%), vigor (0-5) e alterações morfológicas totais (%) após a descongelação foram 56,7 ± 16,1, 3,4 ± 0,5 e 23,8 ± 8,4, respectivamente. Diante dos resultados, concluiu-se que a adição de gema de ovo e glicerol ao diluidor foi necessária para a preservação da qualidade espermática após criopreservação de sêmen canino.

The long-term storage of frozen semen represents a tool for breeders that want to preserve the genetic potential of their sires. The aim of this experiment was to evaluate the effect of coconut water, egg yolk and glycerol on canine semen cooling and freezing. The sperm rich fraction of 12 dogs was submitted to macroscopic and microscopic evaluation. This fraction was divided into four aliquots and frozen with coconut water extender regarding the presence of egg yolk and glycerol. After cooling, there was no difference among groups. However, after thawing, coconut water plus egg yolk and glycerol (ACGG) was superior than others concerning to sperm motility, vigor and morphological abnormalities. In this group, motility (%), vigor (0-5) and abnormalities (%) were 56.7 ± 16.1, 3.4 ± 0.5 e 23.8 ± 8.4, respectivelly. In conclusion, it is necessary to add egg yolk and glycerol to the coconut water extender to improve the cryopreservation of canine semen.

O presente trabalho objetivou avaliar a eficiência do diluidor à base de água de coco acrescido de ácido 3-indol acético (AIA) e gema de ovo na conservação do sêmen canino a 37ºC. Coletou-se o sêmen de seis cães adultos por manipulação digital, fracionando-o em três alíquotas. Procedeu-se a diluição do sêmen com o diluidor à base de água de coco (grupo A), com este acrescido de 4% de AIA (grupo B) e, com o mesmo acrescido de 20% de gema de ovo e 4%AIA (grupo C). Avaliou-se a motilidade, o vigor e a percentagem de alterações morfológicas espermáticas imediatamente após a coleta e periodicamente durante 180 minutos (T0, T5, T15, T30, T60, T90, T120, T180), avaliando-se também a taxa de degradação média de motilidade espermática (TDM). Os diluidores foram comparados pelo teste de Whitney-Mann (P<0,05). Não foram verificadas diferenças estatísticas entre os grupos até T60. A motilidade no uso do grupo A foi superior à dos demais grupos a partir de T90, ficando em torno de 95%.A TDM foi significativamente menor no diluente A (7,5%) em relação aos demais diluentes. Dessa forma, o diluente à base de água de coco mostrou-se adequado para a preservação do sêmen canino a 37ºC por 180 minutos.

The aim of this study was to evaluate the canine semen diluted with coconut water extender, added of 3-indol acetic acid (IAA) and egg yolk at 37ºC. Sperm of six stud dogs was collected by digital manipulation and divided in three equal aliquots. Dilution was performed by the addiction of coconut water (extender 1), coconut water plus 4% of IAA (extender 2) and coconut water plus 4% of IAA and 20% of egg yolk (extender 3). Sperm motility, vigor and morphology immediately after dilution and periodically until 180 minutes, as well as media degradation motility rate (DMR), were evaluated. Extenders were compared by Whitney-Mann test (P<0.05). Statistical differences were not shown between the extenders up to 60 minutes. After 90 minutes, sperm motility on the extender A was superior to the other extenders (95%). The DMR on the extender A (7.5%) was significantly inferior to the other extenders. Thus, coconut water extender is adequate for the preservation of canine semen at 37ºC for 180 minutes.

O objetivo do presente trabalho foi comparar os diluidores à base de água de coco e à base de Tris na congelação do sêmen canino, utilizando-se diferentes concentrações de gema de ovo e glicerol. Cães Pastores Alemães (n=5) tiveram seu sêmen coletado por manipulação digital. Sua fração espermática foi avaliada quanto aos parâmetros macro e microscópicos e misturada para a formação de um pool de esperma que foi diluído em água de coco ou Tris, acrescidos de gema de ovo (10 ou 20%) e glicerol (4, 6 ou 8%). Em seguida, congelado pelo método de NUNES et al. (1997), sendo descongelado a 37ºC após uma semana. Observou-se uma superioridade do diluidor à base de Tris na conservação dos parâmetros vigor (2,3 ± 0,8), alterações espermáticas totais (14,8 ± 5,1%) e secundárias (14,4 ± 5,5%) no par onde os diluidores foram acrescidos de 20% de gema de ovo e 6% de glicerol. Nos demais protocolos, não se evidenciaram diferenças significativas entre os dois diluidores, em nenhum dos parâmetros observados. Esses resultados sugerem uma superioridade do diluidor à base de Tris em relação ao diluidor à base de água de coco na congelação do sêmen canino, no uso dos mesmos acrescidos de 20% de gema de ovo e 6% de glicerol.

The aim of the present research was to compare the efficiency of coconut water and Tris extenders on canine semen freezing. The semen from 5 German Shepherd dogs was collected by digital manipulation. The spermatic fraction of different dogs was evaluated about its macro and microscopic parameters and mixed in a pool. It was extended in coconut water or Tris, added of egg yolk (10 or 20%) and glycerol (4, 6 or 8%). Then, it was frozen by the method of NUNES et al. (1997) and thawed at 37ºC after one week. A Tris extender superiority was shown at the conservation of vigor (2.3 ± 0.8), total (14.8 ± 5.1%) and secondary (14.4 ± 5.5%) spermatic defects in the protocols with 20% of egg yolk and 6% of glycerol. In all other protocols, there were no significant differences for the observed parameters. These results suggest a superiority of Tris extender over the coconut water extender on canine semen freeze, in the use of them added of 20% of egg yolk and 6% of glycerol.