Evaluation of Sperm Kinetics and Plasma Membrane Integrity of Frozen Equine Semen in Different Storage Volumes and Freezing Conditions

In the present study, different freezing systems (Styrofoam box and Mini Digitcool ZH 400) and storage volumes (0.5- and 0.25-mL straws) were compared with regard to sperm kinetics and plasma membrane integrity of frozen and thawed semen. For that, three ejaculates from four animals were frozen in Styrofoam box and Mini Digitcool ZH 400 machine. The 0.5-mL straws were thawed at 46°C for 20 seconds, and the 0.25-mL straws were thawed at 46°C for 12 seconds. Statistical analysis was performed using program R of descriptive analysis box plot, followed by analysis of variance using PROC MIXED of SAS 9.1 package. Variances of 5% were considered as different. There was no interaction between the straw sizes and volumes; however, statistical differences were observed between the semen storage volumes. The 0.5-mL straws had higher total motility (%), progressive motility (%), average path velocity (μm/s), straight-line velocity (μm/s), curvilinear velocity (μm/s), and rapid sperm percentage (%) than the 0.25-mL straws. However, plasma membrane integrity analysis did not differ between the two straws. Thus, it is possible to conclude that equine sperm cryopreserved in 0.5-mL straws has better sperm kinetics than when stored in 0.25-mL straws. Additionally, it is possible to conclude that automated systems that enable faster freezing rates result in a seminal quality that is similar to the one obtained by the conventional system using Styrofoam boxes. © 2013 Elsevier Inc.

Effect of Storage Time and Temperature of Equine Epididymis on the Viability, Motion Parameters, and Freezability of Epididymal Sperm

The recovery of sperm from the epididymal cauda may be the last chance to obtain genetic material when sudden death or serious injuries occur in valuable stallions. However, the lack of technical knowledge regarding the storage and transportation of the epididymis often prevents the preservation of the sperm. Therefore, the aim of this study was to compare sperm parameters of sperm obtained immediately after orchiectomy with sperm recovered from epididymal cauda at different times after storage at 5°C and at room temperature (RT). For that, 48 stallions of different breeds were used. In group 1 (control group), eight stallions were used, and the harvest of the epididymal sperm was performed immediately after orchiectomy. In group 2, 40 stallions were used, which were divided into five groups according to the storage time of the epididymis after orchiectomy (6, 12, 18, 24, or 30 hours), making a total of eight stallions per group. One epididymis of each stallion was stored at 5°C, and the contralateral epididymis was stored at RT, both for the same period. The sperm parameters of total motility, progressive motility, progressive linear velocity, curvilinear velocity, percentage of rapid sperm, and plasma membrane integrity were evaluated in all the groups after sperm recovery, resuspension in a sperm freezing diluent, and thawing. In conclusion, the storage of the testis-epididymis complex at 5°C provided better preservation of epididymal sperm than the storage at RT, and regardless of the temperature, the progressive motility is the sperm parameter that is most sensitive to storage time. © 2013 Elsevier Inc.

High incidence of 'Dag-like' sperm defect in the domestic cat

The occurrence of a high incidence of sperm tail defects in a male domestic cat resembling the known 'Dag-like' defect is reported. Sperm analyses were performed in ejaculated samples collected by an artificial vagina and in testicular and epididymal sperm cells after castration. The following alterations were observed using transmission electron microscope: heavily coiled sperm tails containing several axonemal units enclosed in the same common cell membrane; aberrations in the axonemal main structure; and swollen and unevenly distributed mitochondria in the midpiece. Abnormal modifications in the mitochondrial sheath were also found in sperm cells retrieved from testes and epididymides. Considering these findings, we can conclude that this is the Dag-like defect, described previously in other domestic species and a testicular origin may be involved. © ISFM and AAFP 2012.

Slimmer or Fertile? Pharmacological Mechanisms Involved in Reduced Sperm Quality and Fertility in Rats Exposed to the Anorexigen Sibutramine

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm. © 2013 Elsevier Inc. All rights reserved.

Morphometry and expression of MyoD and Myogenin in white and red skeletal muscles of juvenile fish Colossoma macropomum (Cuvier 1818)

Colossoma macropomum, also known as tambaqui, is an economically important fish species, and interesting new studies have been published with the aim of improving the growth of this fish. In this work, we describe the morphometric characteristics, as well as the mRNA and protein expression levels of two myogenic regulatory factors (MRFs)-myod/MyoD and myogenin/Myogenin-in the white and red muscle types of tambaqui. A high proportion of white and red muscle fibers with large diameters suggest a hypertrophic growth process in the skeletal muscle during juvenile stages. Comparisons between muscle types showed that, in red muscle, myogenin transcript levels were significantly higher than those of myod. In contrast, MyoD protein levels were significantly higher than those of Myogenin in red muscle. These results suggest that in red and white muscles of juvenile tambaqui, independent post-transcriptional mechanisms for regulating MyoD and Myogenin expression may exist, which could be differentially activated during muscle growth. Furthermore, these data also suggest that specific control mechanisms may regulate distinct muscle phenotypes. © 2013 The Royal Swedish Academy of Sciences.

Cooling of ejaculated and epididymal stallion sperm

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The relationships between scrotal surface temperature, age and sperm quality in stallions

In horses, spermatogenesis normally occurs at an average intratesticular temperature of 35. °C; therefore, mechanisms for testicular thermoregulation are essential. Measuring the scrotal surface temperature by thermography is one of the methodologies used to evaluate the effectiveness of testicular thermoregulation. The objective of this study was to determine the relationship between the control of scrotal surface temperature and sperm quality in horses of different ages. In total, 24 Quarter Horse stallions were divided into three groups: YS (young stallions), AS (adult stallions) and OS (old stallions). Initially, we calculated the testicular volume (TV) and evaluated various aspects of the semen (sperm kinetics, plasma membrane integrity and sperm morphology) for all the animals. We also evaluated rectal temperature (RT), body surface temperature (BST,) and average scrotal surface temperature in the testicular region (SST) before (M0) and after sun exposure (M1). Differences were observed (p<0.05) between the RT and BST before and after sun exposure in all three groups. However, there were no differences (p>0.05) in the SST values at these two time points, thus demonstrating the efficiency of the mechanisms for testicular thermoregulation. The SST was similar (p>0.05) among all three groups. Based on these results, we conclude that fertile stallions of different age groups are able to maintain SST and measuring the heat radiating from the scrotum using a digital infrared thermographer. We can also conclude that measuring the heat radiating from the scrotum using a digital infrared thermographer is a practical and efficient tool for monitoring SST in horses. © 2013 Elsevier B.V.

Sperm motility parameters for Steindachneridion parahybae based on open-source software

Summary: The objective of this work was to evaluate the sperm motility of 13 Steindachneridion parahybae males using open-source software (ImageJ/CASA plugin). The sperm activation procedure and image capture were initiated after semen collection. Four experimental phases were defined from the videos captured of each male as follows: (i) standardization of a dialogue box generated by the CASA plugin within ImageJ; (ii) frame numbers used to perform the analysis; (iii) post-activation motility between 10 and 20 s with analysis at each 1 s; and (iv) post-activation motility between 10 and 50 s with analysis at each 10 s. The settings used in the CASA dialogue box were satisfactory, and the results were consistent. These analyses should be performed using 50 frames immediately after sperm activation because spermatozoa quickly lose their vigor. At 10 s post-activation, 89.1% motile sperm was observed with 107.2 μm s-1 curvilinear velocity, 83.6 μm s-1 average path velocity, 77.1 μm s-1 straight line velocity; 91.6% were of straightness and 77.1% of wobble. The CASA plugin within ImageJ can be applied in sperm analysis of the study species by using the established settings. © 2013 Blackwell Verlag GmbH.

Genetic parameters for scrotal circumference, breeding soundness examination and sperm defects in young Nellore bulls

A total of 51,161 records of scrotal circumference measurements at 18 mo of age (SCI 8) and 17,648 records of sperm defects and breeding soundness of Nellore bulls (mean age of 22.5 mo), raised under extensive conditions, were analyzed to estimate coefficients of heritability and genetic correlations of morphological semen traits by Bayesian inference. The observed semen traits were classified as minor (MID). major (MAD), and total sperm defects (TD). The animals were classified according to breeding soundness as satisfactory and unsatisfactory potential breeders. The (co)variance components and breeding values were estimated by Gibbs sampling using the GIBBS2F90 program under an animal model that included contemporary group as fixed effect, age of animal as linear covariate, and direct additive genetic effects as random effects. Heritabilities of 0.40 ± 0.02, 0.16 ± 0.02, 0.04 ± 0.01, 0.15 ± 0.01, and 0.10 ± 0.01 were obtained for SCI8, MID, MAD, TD, and breeding soundness, respectively. The SC18 showed a positive and moderate correlation with breeding soundness (0.56 ± 0.04) and a negative and low correlation with MID (-0.23 ± 0.03), MAD (-0.16 ± 0.02), and TD (-0.24 ± 0.02). In conclusion, scrotal circumference showed the best response to selection among the traits studied and was favorably correlated with breeding soundness and sperm morphology in young Nellore bulls. © 2013 American Society of Animal Science. All rights reserved.

Identification of phospholipase C zeta in normospermic and teratospermic domestic cat sperm

In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca2+]i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca2+]i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ~70 kDa in whole sperm lysates of domestic cat as well as in both soluble and insoluble fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males. © 2013 Elsevier Inc..

Efeito da idade do homem na avaliação do sêmen pela Motile Sperm Organelle Morphology Examination (MSOME)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Eficácia do motile sperm organelle morphology examination (MSOME) na predição de gravidez após inseminação intrauterina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Concentrações plasmáticas de testosterona e fertilidade de machos caprinos das raças Boer e Alpina durante as estações reprodutiva e não reprodutiva

Pós-graduação em Medicina Veterinária - FCAV

Sperm characteristics as additional evidence of close relationship between Lebiasina and Piabucina (Characiformes: Lebiasinidae: Lebiasininae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)