Sperm traits and seminal plasma proteome of locally adapted hairy rams subjected to intermittent scrotal insulation.

The present study evaluated the effects of heat stress on reproductive parameters of hairy rams. Six animals were subjected to scrotal insulation during four consecutive nights (6 PM - 6 AM). Day (D) 0 was the first day of insulation. Scrotal circumference increased from 30.5 ± 0.3 cm (at pre-insulation) to 31.8 ± 0.4 cm on D4, decreased 3.9 cm on D28, returning to 30.6 ± 0.6 cm on D57. Sperm concentration decreased from 3.7 ± 0.12 ×109 sperm/mL before insulation to 2.6 ± 0.1 ×109 on D23, returning to normal on D57. Sperm motility averaged 75 ± 2.9% before insulation, was undetectable on D23, and became normal on D77. Sperm with normal morphology reached 5.9 ± 2.6% on D35 but recovered (86.8 ± 2.1%) on D91. Sperm DNA integrity decreased from 86.5 ± 4.7% before insulation to 11.1 ± 3.7% on D63, returning to pre-insulation values on D120. Sperm BSP immunostaining was reduced after scrotal insulation. Variations in seminal protein abundances coincided with changes in sperm parameters. Seminal plasma superoxide dismutase, carboxypeptidase Q-precursor and NPC intracellular cholesterol transporter 2 decreased on D18, returning to normal after D28. Albumin, inhibitor of carbonic anhydrase precursor, EGF-like repeat and discoid I-like domain-containing protein 3 and polymeric immunoglobulin receptor increased after insulation. In summary, intermittent scrotal insulation drastically altered ram sperm attributes and seminal proteins, especially those associated with oxidative stress. Knowledge of animal´s response to thermal stress is vital in the scenario of climate changes.

Mapping the major proteome of reproductive fluids and sperm membranes of rams: From the cauda epididymis to ejaculation.

The present study evaluated the major proteome of ram seminal plasma and the main secretions that contribute to its formation, such as the cauda epididymal and accessory sex gland fluids. The study also investigated sperm membrane protein profiles before and after ejaculation. First, semen was collected from six rams (using artificial vagina) to obtain seminal plasma and ejaculated sperm. Then, rams were vasectomized for collection of accessory sex gland fluid (using artificial vagina). Next, rams were slaughtered and cauda epididymal fluid (CEF), seminal vesicle fluid, bulbourethral gland fluid and cauda epididymal sperm were properly collected. Proteins from reproductive fluids and sperm membranes were analyzed by 2-D SDS-PAGE, tandem mass spectrometry and bioinformatics. There we 386 proteins and 256 isoforms identified in all samples. The most abundant seminal plasma proteins were BSP1, BSP5 and spermadhesins (bodhesin-2 and spermadhesin Z13-like). These proteins were present in similar patterns in maps of accessory sexgland fluid, with very low quantities in the CEF and absent in the bulbourethral gland secretion. Thus, practically all BSPs and spermadhesins come from seminal vesicles. Bulbourethral gland fluid brought bactericidal/permeability-increasing protein-containing Family A member 1 isoforms, superoxide dismutase [Cu-Zn] and betamicroseminoprotein to seminal plasma. CEF was the major provider of clusterin, epididymal-specific lipocalin-5-like isoform, epididymal secretory gluthathione peroxidase, epididymal secretory protein E1 and prostaglandin-H2 D-isomerase to seminal plasma. Albumin came from all reproductive fluids. BSPs and spermadhesins were present in 2-D maps of ejaculated sperm but absent in cauda epididymal sperm. These proteins come from the seminal vesicles and bind to sperm at the moment of ejaculation. Other proteins of ejaculated and epididymal sperm membranes were mostly associated to energy production, cell adhesion and proteolytic activity (ATP synthases, disintegrin, metalloproteinase domain-containing protein 32, carboxypeptidase Q and cytosol aminopeptidase). In conclusion, there is a well-orchestrated sequence of events to form the major seminal plasma proteome, with specific contributions from cauda epididymis, seminal vesicles and bulbourethral glands. The present data contribute to a better understanding of male reproductive biology and how sperm functions are affected by the noncellularmicro environment of semen.

Proteomic analysis of follicular fluid from tropically-adapted goats.

The present study was conducted to characterize the major proteome of ovarian follicular fluid from locally-adapted, "Canindé" goats in the northeast of Brazil. Eight estrous cycling goats received a hormonal treatment consisting of medroxyprogesterone acetate, D-cloprostenol and FSH. Fluid was collected by laparoscopy from small (<3mm), medium (3-4mm) and large (>4mm) follicles and then, proteins were analyzed by 2-D SDS-PAGE and tandem mass spectrometry. Thirty-six proteins were identified in the goat follicular fluid, including albumin, immunoglobulins, ceruloplasmin, complement factor B, alpha-1B-glycoprotein precursor, serotransferrin, complement C3 and serpins, among others. Albumin and immunoglobulins were the most abundant proteins. Protein concentrations were similar in the fluid from small (45.3±3.1mg/mL), medium (44.2±3.3mg/mL) and large follicles (45.1±2.3mg/mL). The intensities of spots identified in 2-D gels as serotransferrin, zinc-alpha-2-glycoprotein-like, complement factor B and complement protein C3 differed (P<0.05) among follicle categories. The amount of serotransferrin was greater in the medium than small follicles (P<0.05). Content of zinc-alpha-2-glycoprotein-like, complement factor B and complement C3 was greater (P<0.05) in the fluid of large follicles than in medium follicles. Based on gene ontology, the major molecular functions associated with goat follicular fluid proteins were binding and catalytic activity, while the main biological processes were related to regulation, cellular processing, location and the immune system. In conclusion, the major proteome of the follicular fluid from goats subjected to hormonal stimulation was elucidated in the present study. Also, molecules associated with follicle development are potential biomarkers of oocyte competence were prevalent.

Effect of increased testicular temperature on seminal plasma proteome of the ram.

The present study evaluated the effects of heat stress on the ram seminal plasma proteome. Six Morada Nova rams were scrotal insulated for 8 days. Scrotal circumference, sperm parameters, and seminal fluid proteins were evaluated before (Day 0) and twice during scrotal insulation (Days 4 and 8), and weekly until semen parameters returned to preinsulation values (normal). Seminal proteins were analyzed by two-dimensional SDS-PAGE and mass spectrometry. Scrotal circumference decreased from 30 ± 0.4 cm on Day 0 to 22.6 ± 0.6 cm on Day 36 (P < 0.05) and became equivalent to preinsulation values on Day 71. Motile sperm became nearly absent from Day 8 to Day 64 but returned to normal on Day 113. Percentage of normal sperm changed similarly and returned to normal on Day 106. Rams were azoospermic between Days 29 and 64, and sperm concentration came back to normal on Day 92. The number of spots/two-dimensional gel reduced from 256 ± 31 on Day 0 to 104 ± 14 on Day 29 (when rams were azoospermic) and then increased to 183 ± 9 on Day 113 (P < 0.05), similar to spot counts before insulation. The intensities of 24 spots, referring to 17 seminal plasma proteins, were affected by treatment (P < 0.05). After insulation, seminal plasma had greater expression of actin (two isoforms), albumin, heat shock protein 70 kDa, protein DJ-1, HRPE773-like, C-reactive protein precursor, bodhesin-2 (one isoform), spermadhesins. Most protein spots had the greatest intensity between Days 8 and 29, returning to preinsulation values on Day 113 (when many sperm criteria returned to normal). Proteins downregulated after scrotal insulation included dipeptidyl peptidase 3, isoforms of heat shock protein 90 kDa, RSVP22, MMP2 and of Bdh2. In this case, RSVP22 was reduced on Day 113 and all others, on Day 134. Expression of MMP2 and HSP90.1 was reduced throughout the study. Integrin ß5, V-type H(+)-ATPase subunit A, ZBTB 42-like protein, isoforms of Bdh2, PSP-I, and RSVP22 were upregulated after testis insulation. Intensities of these spots were maximum (P < 0.05) 8 days after insulation started or on Day 29. Expression of most of such proteins returned to normal on Day 113. In conclusion, scrotal insulation affected testis and sperm parameters of rams, indicating alterations in both spermatogenesis and sperm maturation. Changes of seminal plasma proteome were coincidental with variations in semen parameters. Proteins affected by heat challenge are potentially involved in sperm protection, maturation, and fertilization.

Perfil hematológico de cabras Saanen e mestiças (1/2 Saanen e 1/2 Anglo-nubiana) criadas em clima tropical do Ceará / Hematological profile of Saanen and mixed-breed (1/2 Saanen x 1/2 Anglo-Nubian) raised in the tropical climate of Ceará, Brazil

Objetivou-se verificar a influência das condições climáticas sobre o perfil hematológico de cabras Saanen e mestiça (1/2 Saanen x 1/2 Anglo-nubiana), bem como determinar valores de referência para esses animais criados no Ceará. Foram utilizadas 30 cabras que tiveram o sangue coletado mensalmente durante a época chuvosa (fevereiro a maio) e seca (agosto a novembro) para realização do eritrograma e leucograma. As médias foram comparadas pelo teste t-Student e Mann Whitney, quando constatada distribuição paramétrica e não paramétrica dos dados, respectivamente, com significância quando p<0,05. Procedeu-se ainda um estudo de correlações simples de Pearson dos parâmetros hematológicos com variáveis ambientais e fisiológicas. O número de hemácias foi maior em cabras Saanen e na época chuvosa, enquanto que o hematócrito foi maior na época seca (p<0,05). Os leucócitos e linfócitos foram maiores em cabras mestiças nas duas épocas (p<0,05). Nos dois genótipos, os leucócitos e linfócitos foram maiores na época seca e os neutrófilos segmentados maiores na época chuvosa (p<0,05). Os demais parâmetros não diferiram (p>0,05). As hemácias e neutrófilos segmentados apresentaram uma correlação negativa com a temperatura ambiente, porém positiva com a umidade do ar e a temperatura retal (p<0,05). O hematócrito correlacionou-se positivamente com a temperatura ambiente e frequência respiratória (p<0,05). Os leucócitos e linfócitos mostraram uma correlação negativa com a temperatura retal (p<0,05). Conclui-se, portanto, que fêmeas Saanen sofrem mais influência das variações climáticas e a época chuvosa tem maior impacto negativo sobre os parâmetros hematológicos. Os valores obtidos poderão servir como referência para esses genótipos no Ceará...

The aim of this study was to determine the influence of climatic conditions on the hematological profile of Saanen and mixed-breed (1/2 Saanen x 1/2 Anglo-Nubian) goats, as well as to define reference values for these animals bred in Ceará, Brazil. Thirty goats were utilized and blood samples were collected monthly during the rainy (February to May) and dry (August to November) periods to obtain an erythrogram a leukogram. The averange were compared by the t-Student test and Mann Whitney test, with parametric and non-parametric distribution of the data, respectively, where p<0.05 was considered significant. A study was carried out of simple Pearson correlations of the hematological parameters with environmental and physiological variables. The number of red blood cells (RBCs) was higher in the Saanen goats and in the rainy period, while the hematocrit was higher in the dry period (p<0.05). The leukocytes and lymphocytes were higher in the 1/2S1/2AN goats in the two periods (p<0.05). In the two genotypes, the leukocytes and lymphocytes were higher in the dry season and the segmented neutrophils higher in the rainy season (p<0.05). The other parameters did not differ (p>0.05). The RBCs and segmented neutrophils displayed a negative correlation with air temperature, but positive with relative humidity and rectal temperature (p<0.05). The hematocrit positively correlated with air temperature and respiratory rate (p<0.05). The leukocytes and lymphocytes showed a negative correlation with rectal temperature (p<0.05). It was therefore concluded that Saanen females are more affected by climatic variations and that the rainy season has a greater negative impact on hematological parameters. The values obtained could serve as a reference for these genotypes in Ceará...

Refrigeração do epidídimo canino a 4ºC e recuperação dos espermatozoides epididimários utilizando ACP-106c / Cooling of the canine epididymis at 4ºC and recovery of epididymal sperm using ACP-106c

Objetivou-se avaliar a qualidade dos espermatozoides recuperados da cauda do epidídimo após a refrigeração do complexo testículo-epidídimo (CTE) de cães usando o diluidor ACP-106c. Foram utilizados 60 cães machos adultos, com peso de 10-20 kg. Após a eutanásia, removeu-se o CTE que foi imerso em solução fisiológica 0,9% e transportado em caixa térmica ao laboratório a 30ºC. Para a refrigeração e recuperação dos espermatozoides epididimários, os 60 pares do CTE foram divididos em 4 grupos, de acordo com o tempo de refrigeração do CTE e posterior recuperação espermática: G0h, G6h, G12h e G18h, em que cada par do CTE permaneceu por zero, seis, doze ou dezoito horas a 4ºC, respectivamente. A recuperação dos espermatozoides da cauda do epidídimo foi realizada pela técnica de flutuação utilizando-se o diluidor ACP-106c ou Tris. Para cada epidídimo foi adicionado 1,0 mL de um dos dois diluidores, pré-aquecidos a 37ºC por 5 minutos. Em seguida foram centrifugados a 800g/5 minutos para remoção dos resíduos celulares. Avaliou-se a morfologia, funcionalidade e motilidade espermática total e progressiva, além de parâmetros obtidos pelo CASA. Os dados foram submetidos à ANOVA seguido do teste de Turkey (P < 0,05). Em todos os parâmetros avaliados, não houve diferença entre os diluidores testados (P>0,05). Os valores de motilidade total nos grupos G0h, G6h, G12h, e G18h para o ACP-106c foram 84,4±7,7; 81,6±11,6; 88,3±6,5 e 69,5±16,9, respectivamente, e para o Tris 85,2±8,7; 77,4±14,3; 79,0±17,8 e 65,4±17,9, respectivamente. Um decréscimo na qualidade espermática foi observado após 18 horas de refrigeração em ambos os diluidores. Dessa forma pode-se concluir que o ACP-106c pode ser utilizado para recuperar os espermatozoides epididimários refrigerados e podem ser viáveis por até 12h de refrigeração.

The study aimed to assess the quality of sperm recovered from the epididymal cauda after cooling the complex testis-epididymis (CTE) of dogs using ACP-106c extender. Sixty adult male dogs, weighing 10-20 kg were used. After euthanasia, CTE was removed and it was immersed in 0.9% saline and transported to the laboratory in cooler at 30ºC. For cooling and recovery of epididymal spermatozoa, the 60 pairs of CTE were divided into four groups, according to the refrigeration time of the CTE and subsequent sperm recovery: G0h, G6h, G12h and G18h, wherein each pair of CTE remained zero, six, 12 or 18 hours at 4ºC respectively. The recovery of sperm epididymal tail was conducted by flotation technique using ACP-106c or Tris extender. For each epididymis, it was added 1.0 mL of either extenders, preheated to 37ºC for 5 minutes. They were then centrifuged at 800g/5 minutes to remove the cell debris. Morphology, functionality and total and progressive motility, and parameters obtained by CASA were evaluated. Data were analyzed by ANOVA followed by Turkey test (P <0.05). In all parameters assessed, there was no difference between the extenders used (P>0.05). The values of total motility groups G0h, G6H, G12H, and G18h for ACP-106c were 84.4±7.7, 81.6±11.6, 88.3±6.5 and 69.5±16.9 respectively, and for Tris 85.2±8.7, 77.4±14.3, 79.0±17.8 and 65.4±17.9 respectively. A decrease in sperm quality was observed after 18 hours of cooling in both extenders. Thus it can be concluded that the ACP-106c may be used to recover the epididymal spermatozoa chilled and may be viable for up to 12h cooling.