Publications by authors named "Herlon Victor Rodrigues Silva"

17 Publications

  • Page 1 of 1

Effects of somatic tissue cryopreservation on puma (Puma concolor L, 1771) tissue integrity and cell preservation after in vitro culture.

Cryobiology 2021 Jun 10. Epub 2021 Jun 10.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil. Electronic address:

Somatic resource banks play a crucial role in the conservation of genetic diversity, allowing for the preservation of biological samples from different populations. Puma somatic cells can be recovered from these banks and used in assisted techniques toward enhancing their multiplication and conservation. In response to the population reduction of this ecologically importance species, we aimed to evaluate the capacity of cryopreservation of somatic tissues on the maintenance of the integrity and quality of the cells recovered after culture, with the aim of establishing a somatic tissue bank that will allow for the safeguarding of a wide genetic sampling of pumas. Cryopreservation increased the thickness of the corneum layer in the tissues, and the number of perinuclear halos and empty gaps. Nevertheless, cryopreservation was able to maintain normal fibroblast patterns, even showing an increase in the percentage of collagen fibers. Cryopreservation maintained the proliferative potential of the tissues and the parameters evaluated during in vitro culture, mainly regarding the viability, proliferative activity, and apoptosis levels. Nevertheless, cells from cryopreserved tissues showed decreased metabolism and mitochondrial membrane potential when compared to cells from non-cryopreserved tissues. In summary, we demonstrated for the first time that puma somatic tissues subjected to cryopreservation are viable and maintain tissue integrity, featuring minimal changes after warming. Although viable somatic cells are obtained from these tissues, they undergo alterations in their metabolism and mitochondrial membrane potential. Improvements in the conservation conditions of somatic samples are needed to increase the quality of somatic tissue banks in this species.
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http://dx.doi.org/10.1016/j.cryobiol.2021.06.003DOI Listing
June 2021

Characterization of seminal parameters, sperm morphometry, micromorphology, and ultrastructure in gray brocket deer (Mazama gouazoubira, Fischer, 1814).

Microsc Res Tech 2021 Feb 21;84(2):313-325. Epub 2020 Sep 21.

Faculty of Veterinary Medicine, Ceara State University, Fortaleza, Brazil.

Populations of gray brocket deer (Mazama gouazoubira) are declining; yet, knowledge on the reproductive biology of this species remains limited. Therefore, this study aimed to describe morphology, viability, membrane integrity, mitochondrial activity, morphometry, micromorphology, and ultrastructure of the gray brocket deer sperm. Three adult male gray brocket deer were used in the study. Semen collection was performed using electroejaculation. Semen were analyzed by evaluating pH, motilities, vigor, mass movement, volume, concentration, viability, membrane integrity, mitochondrial activity, morphology, and morphometry. Micromorphology and ultrastructure of sperm were analyzed using scanning and transmission electron microscopy (SEM and TEM), respectively. There was no significant difference among males regarding on pH, motilities, vigor, mass movement, volume, concentration, viability. High values for membrane integrity, mitochondrial activity, and normal sperm were observed. The most frequent defects were simple bent tail and bowed midpiece. The head length, and width, midpiece, and tail length were 8.5, 4.4, 11.5, and 41.3 μm, respectively. SEM sperm showed paddle-shaped heads, with apical ridge and serrated band on the equatorial segment. TEM revealed the nucleus, acrosome, plasma membrane, mitochondria sheath, proximal centrioles, segmented columns, axoneme, outer dense fibers, and fibrous sheath. SEM and TEM showed the presence of some abnormalities. These results are expected to provide baseline values of diverse semen parameters, contributing toward the development of reproductive biotechnologies for gray brocket deer and, other deer species at risk of extinction.
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http://dx.doi.org/10.1002/jemt.23590DOI Listing
February 2021

Influence of Microwave-Assisted Drying on Structural Integrity and Viability of Testicular Tissues from Adult and Prepubertal Domestic Cats.

Biopreserv Biobank 2020 Oct 11;18(5):415-424. Epub 2020 Aug 11.

Smithsonian Conservation Biology Institute, National Zoological Park, Washington, District of Columbia, USA.

Anhydrous preservation is a promising approach for storage of living biomaterials at nonfreezing temperatures. Using the domestic cat model, the objectives of this study were to characterize changes in histology, DNA integrity, and viability of testicular tissues from adult versus prepubertal individuals during microwave-assisted drying. Testes from each age group were cut into small pieces before reversible membrane permeabilization, exposure to trehalose, and microwave-assisted drying during different time periods. In Experiment 1, water content was monitored for up to 40 minutes of drying. Tissues from adult or prepubertal cats experienced similar decreases of water content during the first 10 minutes. Desiccation progressed slowly between 10 and 20 minutes and then remained stable. In Experiment 2, structural properties were explored at 5, 10, and 20 minutes of desiccation. Percentages of normal seminiferous tubules were lower after 20 minutes drying in adult (43%) than in prepubertal tissues (61%). At the same time point, the proportion of cell degeneration was higher in adult (53%) than prepubertal tissues (28%). Percentages of intact DNA in tissues remained above 85% regardless of the microwave time in both age groups. Lastly, adult and prepubertal tissues only lost 33% of viability in both age groups. Collective results demonstrated for the first time that normal morphology, incidence of degeneration, DNA integrity, and viability of testicular tissues remained at acceptable levels during microwave-assisted drying for 20 minutes. Overall, prepubertal testicular tissues appeared to be more resilient to microwave-assisted desiccations than adult tissues. Importantly, water loss in the presence of trehalose after 20 minutes of desiccation already is compatible with long-term storage of testicular tissues at temperatures above -20°C, which is one step closer to future storage at supra-zero temperatures.
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http://dx.doi.org/10.1089/bio.2020.0048DOI Listing
October 2020

Recovery and cryopreservation of epididymal sperm from domestic cat using powdered coconut water (ACP-117c) and TRIS extenders.

Cryobiology 2020 02 23;92:103-108. Epub 2019 Nov 23.

School of Veterinary Medicine, State University of Ceará, Fortaleza, CE, Brazil. Electronic address:

Cryopreservation of cats epididymal spermatozoa allows the conservation of the genetic material and the study of the cryogenic effect applied to the gametes of other felines. However, this biotechnique still presents variable results, being necessary the investigation of alternative extenders. Powdered coconut water (ACP-117c) has been efficient in the sperm freezing of several species and in the cat sperm refrigeration. Therefore, we aimed to evaluate the effect of the freezing stages and the quality of the cats' epididymal spermatozoa after thawing, using ACP-117c. Epididymides (n = 36) from 18 cats were processed using TRIS (n = 18) or ACP-117c (n = 18) for sperm recovery. The sperm were immediately evaluated. Then, this was cooled, glycerolized, frozen and thawed, and re-evaluated at each stage for sperm kinetics by Computer Assisted Semen Analysis, viability, functionality (HOST), mitochondrial activity (DAB) and morphology. There was a reduction in total motility and progressive motility after thawing in both groups, and TRIS was superior to ACP-117c. The curvilinear velocity reduced after thawing with ACP-117c. Viability decreased after glycerolization in TRIS. Although it also reduced after thawing in both groups, it was higher in TRIS. There was no change on HOST. Mitochondrial activity decreased during the cryopreservation steps for both extenders. Nevertheless, TRIS presented a higher percentage of spermatozoa from DAB class I and II after thawing. Morphology did not differ between extenders. Therefore, ACP-117c is an alternative for the recovery of cat epididymal spermatozoa; however, it is not efficient for freezing. Glycerolization and thawing are the most critical stages, regardless of the extender.
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http://dx.doi.org/10.1016/j.cryobiol.2019.11.042DOI Listing
February 2020

Influence of different extenders on morphological and functional parameters of frozen-thawed spermatozoa of jaguar (Panthera onca).

Cryobiology 2020 02 5;92:53-61. Epub 2019 Nov 5.

Laboratory of Carnivores Reproduction, State University of Ceará, Fortaleza, CE, Brazil. Electronic address:

Due to the global decrease in jaguar population, conservation strategies are essential and the development of effective semen cryopreservation protocols would contribute to the formation of germplasm banks. Therefore, the objectives were to (1) evaluate the use of TRIS and ACP-117c extenders for jaguar semen freezing, (2) describe the ultrastructural changes in sperm after cryopreservation, and (3) evaluate the binding capacity of the thawed sperm. Eight ejaculates from five mature individuals were collected by electroejaculation, extended in TRIS or a coconut based-extender (ACP-117c), and frozen in liquid nitrogen. Samples were evaluated for sperm motility, vigor, membrane functionality, mitochondrial activity, morphology (using light microscopy, scanning electron microscopy - SEM and transmission electron microscopy - TEM), sperm kinetic parameters (by computerized analysis - CASA), and sperm binding capability using an egg yolk perivitelline membrane assay. Samples preserved in TRIS presented better post-thaw motility (46.0 ± 7.7%) and membrane functionality (60.5 ± 4.2%) and higher mitochondrial activity (21.5 ± 3.7%) than those preserved in ACP-117c (20.9 ± 5.4% motile sperm; 47.1 ± 2.5% functional membrane; 11.8 ± 1.7% mitochondrial activity). Regarding ultrastructural evaluations, SEM showed that both extenders were able to preserve the superficial membrane of the sperm, but TEM revealed the occurrence of nuclear electron lucent points, especially in samples extended in ACP-117c. Additionally, TRIS also provided a higher number of sperm bound to the perivitelline membrane (29.5 ± 3.3%) in comparison to samples diluted in ACP-117c (18.6 ± 1.5%). Overall, we suggest the use of a TRIS-based extender for cryopreservation of jaguar semen.
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http://dx.doi.org/10.1016/j.cryobiol.2019.10.195DOI Listing
February 2020

Morphological and morphometric characterization of domestic cat epididymal sperm.

Reprod Domest Anim 2019 Dec 20;54(12):1630-1636. Epub 2019 Oct 20.

School of Veterinary Medicine, State University of Ceará (UECE), Fortaleza, Brazil.

Sperm morphometry is the tool that confers objectivity to the morphological evaluation by accurately measuring the dimensions of the gamete and its structures. Thus, the aim of the study was to perform a morphometric characterization of the domestic cat sperm. Therefore, sperm samples were collected from twenty pairs of epididymis in a TRIS extender at 37ºC. An aliquot of the sample was used to make a smear with Rose Bengal solution, and afterwards, the morphology and morphometry were analysed. In the morphology, were quantified the percentage of normal sperm cells, morphological changes of head, midpiece and tail. In morphometry, each normal sperm cell was measured for length, width, area and perimeter of head and midpiece, tail length and total length. The parameters ellipticity, elongation, regularity and rugosity were also determined. The percentage of normal sperm was 67.21%. Of the abnormalities, the curled/folded tail, followed by the curved midpiece, abnormal shaped head and detached head were the most quantified. The sperm head presented 5.56 ± 0.01 μm and 3.10 ± 0.01 μm of length and width, respectively. The head area was 16.94 ± 0.05 μm , while the perimeter was 16.16 ± 0.03 μm. In the derived parameters, the values were as follows: ellipticity of 1.81 ± 0.00; elongation of 21.39 ± 0.12; regularity of 0.81 ± 0.00; and rugosity of 0.14 ± 0.00. The midpiece presented length and width of 7.96 ± 0.01 μm and 0.76 ± 0.01 μm, respectively. The mean length of the sperm tail was 45.12 ± 0.06 μm, and the total cell size was 58.67 ± 0.06 μm. Thus, it was concluded that the cat sperm is an elongated cell, with high rugosity and regularity. The spermatic tail represents more than ¾ of the total length of the cell and the midpiece exceeds the length of the head.
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http://dx.doi.org/10.1111/rda.13572DOI Listing
December 2019

Effects of cryopreservation techniques on the preservation of ear skin - An alternative approach to conservation of jaguar, Panthera onca (Linnaeus, 1758).

Cryobiology 2019 06 25;88:15-22. Epub 2019 Apr 25.

Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil. Electronic address:

Currently, it has been observed that a considerable segment of the jaguar population is declining mainly because of hunting, and destruction and fragmentation of habitat. Given this scenario, efforts of the scientific community have been concentrated on the development of conservation strategies, such as the formation and use of somatic sample banks. We aimed to assess the effects of cryopreservation techniques of the ear skin of jaguar [slow freezing (SF) or direct vitrification in cryovials (DVC) or solid-surface vitrification (SSV)] on the morphological analysis and cell ability during the culture. All cryopreserved fragments regardless of the technique used, showed a reduction in the dermis and total thickness of the skin. Although a collagen matrix similar to the control group (fresh) has been observed only for the fragments from SF and SSV groups, all cryopreserved techniques were able to maintain normal patterns of the fibroblasts. Moreover, DVC and SSV methods maintained the proliferative activity of the tissues even after warming. After the culture, SF and SSV techniques were efficient for the recovery of the somatic cells according to most of the evaluated parameters, especially with regard to the duration of culture and cell metabolic activity. In conclusion, SSV was found to be a more efficient technique for cryopreserving jaguar skin when compared to DVC and SF. These results are relevant for the formation of somatic resource banks of this species, directed at cryopreserving adequate samplings of different individuals and generations for future applications in regenerative medicine, and assisted reproductive technologies.
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http://dx.doi.org/10.1016/j.cryobiol.2019.04.007DOI Listing
June 2019

Morphology, morphometry, ultrastructure, and mitochondrial activity of jaguar (Panthera onca) sperm.

Anim Reprod Sci 2019 Apr 26;203:84-93. Epub 2019 Feb 26.

Laboratory of Carnivores Reproduction, State University of Ceará, Fortaleza, CE, Brazil.

The jaguar is categorized as "Near Threatened". Conservation strategies, therefore, are needed which include use of reproductive biotechniques. For implementation of biotechnique use, the reproductive characteristics of the species must be understood, which is currently not the case. This study, therefore, aimed to describe the detailed morphology of jaguar sperm, and to evaluate the sperm mitochondrial activity. Five male adults were used. Slides stained with Rose Bengal were used for morphometric and morphological analyses. The length and the width of the sperm head were measured, as well as the length of the middle piece, the tail, and the total length. Scanning and transmission electron microscopy were used for ultrastructural analysis. Mitochondrial function was assessed using the marker 3,3'-diaminobenzidine (DAB). The results are expressed as means ± SEM. The most significant morphological abnormalities observed were head (9 ± 1.7%) and tail defects (12.5 ± 3.3%). The width and length of the head were 3.6 ± 0.03 μm and 4.9 ± 0.02 μm, respectively. The middle piece measured 9.7 ± 0.3 μm, the tail measured 54.5 ± 4.4 μm, and the total length of the sperm was 59.5 ± 0.1 μm. Electron-lucent regions and approximately 54 mitochondrial spirals in the middle piece were identified in the nucleus using electron microscopy. The greatest percentages of cells were classified as DAB I (46.6 ± 4.9%) and DAB II (38 ± 4.4%). The data provide detailed information on the sperm characteristics of jaguars and can support research on germplasm conservation for the species.
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http://dx.doi.org/10.1016/j.anireprosci.2019.02.011DOI Listing
April 2019

Semen Cryopreservation and Banking for the Conservation of Neotropical Carnivores.

Biopreserv Biobank 2019 Apr 27;17(2):183-188. Epub 2018 Dec 27.

3 Smithsonian Conservation Biology Institute, Washington, District of Columbia.

Neotropical carnivores include a large number of threatened and endangered species. It is critical to develop conservation efforts to ensure the sustainability of populations in situ and ex situ. The highest priorities are to protect natural habitats and better understand the biology of rare species. Conservation efforts also are directed toward the implementation of breeding programs and the development of reproductive biotechnologies in which the cryopreservation of male gametes plays a major role. It also is fundamental to create semen banks that contribute to maintaining genetic diversity in small and endangered populations. The present article aims at reviewing the state of the art in cryopreservation of semen from neotropical carnivores and discuss the development of systematic banking for the conservation of these understudied species.
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http://dx.doi.org/10.1089/bio.2018.0104DOI Listing
April 2019

Ultrastructural description of fresh and frozen/thawed sperm derived from collared peccaries (Pecari tajacu Linnaeus, 1,758).

Microsc Res Tech 2018 Nov 8;81(11):1301-1309. Epub 2018 Oct 8.

Laboratory of Animal Germplasm Conservation, Universidade Federal Rural do Semiárido - UFERSA, Rio Grande do Norte, Brazil.

The aim was to describe, through scanning electron microscopy (SEM) and transmission electron microscopy (TEM), the ultrastructure of peccaries' fresh and frozen-thawed sperm. For that, semen derived from three mature males was obtained by electroejaculation and evaluated for motility, membrane integrity, membrane functionality, chromatin integrity, and morphology through light microscopy. Samples were frozen using a Tris extender plus egg yolk (20%) and glycerol (6%). Then, fresh and frozen-thawed semen samples were mixed in different sperm pools that were processed for SEM and TEM. Sperm motility, membrane integrity, and functionality were impaired (p < .05) by freezing-thawing procedures, but sperm morphology, and chromatin integrity evaluated by light microscopy were not significantly affected. The SEM revealed that peccaries' sperm presents a flattened head in a paddle format, measuring 6.07 μm in length and 3.84 μm in width, with a vastus acrosome (4.46 μm). Normal tails measure 38.11 μm, being formed by an extensive midpiece with 15.52 μm in length. In frozen-thawed samples, both SEM and TEM provide us information about damage undetected through light microscopy as the presence of vesicles in the acrosome, loose plasma membrane, vacuolized mitochondria, dense fibers disorganized, and decondensed chromatin. In conclusion, we provide the first description of the sperm ultrasctruture in collared peccaries. Moreover, SEM and TEM help us to identify some nanometric damage provoked by freezing-thawing procedures, thus providing valuable information for the improvement of such important protocols used for biobanking formation.
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http://dx.doi.org/10.1002/jemt.23138DOI Listing
November 2018

Seminal plasma and sperm proteome of ring-tailed coatis (Nasua nasua, Linnaeus, 1766).

Theriogenology 2018 Apr 31;111:34-42. Epub 2018 Jan 31.

Laboratory of Carnivore Reproduction, State University of Ceará, Fortaleza, CE, Brazil. Electronic address:

Ring-tailed coati is listed as a species of least concern in the International Union for Conservation of Nature (IUCN) Red List, however, there has been a sharp decline in their population. The present study was conducted to evaluate the major proteins of both seminal plasma and sperm in ring-tailed coatis. Semen sample was collected from three adult coatis and evaluated for their morphological characteristics. Further, the sample was centrifuged to separate spermatozoa from seminal plasma, and then stored in liquid nitrogen. The seminal plasma and sperm proteins were subjected to one-dimensional (1-D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and identified by mass spectrometry. Gene ontology and protein networks were analyzed using bioinformatics tools. Based on sperm concentration and average protein content of the semen, the concentration of protein/spermatozoon was found to be 104.69 ± 44.43 μg. The analysis of SDS-PAGE gels showed 20.3 ± 3.1 and 17 ± 2 protein bands/lane for seminal plasma and sperm, respectively. In-gel protein digestion and peptide analysis by mass spectrometry revealed 238 and 246 proteins in the seminal plasma and sperm, respectively. The gene ontology analysis revealed that the proteins of seminal plasma mainly participated in cellular (35%) and regulatory (21%) processes. According to their cellular localization, seminal plasma proteins were categorized as structural (18%), extracellular (17%), and nuclear (14%) proteins with molecular functions, such as catalytic activity (43%) and binding (43%). The sperm proteins were also involved in cellular (38%) and regulatory (23%) processes, and mainly categorized as extracellular (17%), nuclear (13%), and cytoplasmic (10%) proteins. The major molecular functions of the sperm proteins were catalytic activity (44%) and binding (42%). These results indicated that the seminal plasma of ring-tailed coati has an array of proteins that can potentially modulate several sperm functions, from sperm protection to oocyte binding. However, further studies are necessary to interpret the roles of these major seminal plasma proteins in coatis.
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http://dx.doi.org/10.1016/j.theriogenology.2017.12.036DOI Listing
April 2018

Proteomic characterization of canine seminal plasma.

Theriogenology 2017 Jun 20;95:178-186. Epub 2017 Mar 20.

Laboratory of Carnivores Reproduction, State University of Ceara, Fortaleza, Brazil. Electronic address:

The present study was conducted to identify the major proteome of the sperm-rich fraction and prostatic fraction of canine seminal plasma. Three semen samples from four healthy dogs were obtained by digital manipulation. The pre-sperm fraction, sperm-rich fraction and prostatic fraction were separated from each ejaculate. Immediately after sperm analysis, a protease inhibitor was added to the sperm-rich fraction and prostatic fraction, and the fractions were separately centrifuged and frozen at -80 °C. The samples were thawed, re-centrifuged, and the total protein concentration was determined. Samples were subjected to 1D SDS-PAGE and Coomassie-blue stained gels, were analyzed by Quantity One 1D Analysis Software. Bands detected in the gels were excised and proteins subjected to digestion with trypsin. Proteins were identified by nano-HPLC-MS and tools of bioinformatics. Tandem mass spectrometry allowed the detection of 268 proteins in the gels of sperm-rich fraction and prostatic fraction of canine ejaculate. A total of 251 proteins were common to the sperm-rich and prostatic fractions, while 17 proteins were present in the sperm-rich fraction and absent in the prostatic fraction. The intensity of the bands detected in range 1 and 2 represented 46.5% of all of the band intensities detected in the 1D gels for proteins of the sperm-rich fraction and 53.0% of all bands in the prostatic fraction. Arginine esterase and lactotransferrin precursor were the protein with the highest intensity observed in the both fractions. Among the proteins present only in the sperm-rich fraction, the proteins UPF0764 protein C16orf89 homolog and epididymal-specific lipocalin-9 were the most abundant. In conclusion, canine sperm-rich fraction and prostatic fraction express a very diverse set of proteins, with unique biochemical properties and functions. Moreover, although most proteins are common to both sperm-rich fraction and prostatic fraction, there are some exclusive proteins in sperm-rich fraction.
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http://dx.doi.org/10.1016/j.theriogenology.2017.03.016DOI Listing
June 2017

Two-dimensional sonographic and Doppler changes in the uteri of bitches according to breed, estrus cycle phase, parity, and fertility.

Theriogenology 2017 Jun 15;95:171-177. Epub 2017 Mar 15.

Laboratory of Carnivore Reproduction, Veterinary School, State University of Ceara, Fortaleza, CE, Brazil.

This study aimed to evaluate the two-dimensional and Doppler ultrasonographic features of bitches' uteri according to breed, cycle phase, parity and fertility during the follicular and early diestrus phases. Thirty-nine pubertal bitches were divided into groups according to breed, parity, and fertility. Sonographic assessments started from the first day of vaginal bleeding and were performed weekly for a month. The diameter of the uterine body was measured longitudinally and the uterine characteristics observed sonographically were evaluated. The resistivity index (RI) and pulsatility index (PI) of the uterine artery were calculated and the spectral morphology analyzed subjectively. The uterine diameter increased from proestrus to the diestrus regardless of the breed or parity. Multiparous bitches of both breeds had higher uterine diameters than nulliparous bitches, except Fila Brasileiro bitches in estrus. In diestrus and proestrus, the uterine diameters were significantly the largest for multiparous bitches, followed by the primiparous bitches and smallest for nulliparous bitches. The uterine diameter was larger in Fila Brasileiro females than in French Bulldogs during estrus and diestrus. The RI did not differ during the different phases of the cycle for the same breed. However, the RI was higher in nulliparous and primiparous Fila Brasileiro females compared to French Bulldog females. Within Fila Brasileiro, multiparous bitches showed lower RI than nulliparous and primiparous bitches. Higher PI values were found during proestrus. All multiparous French Bulldog bitches had greater PI than nulliparous bitches, while multiparous Fila bitches had lower PI. Nulliparous and primiparous Fila bitches had larger PI larger than bitches with the same reproductive status of the breed French Bulldog. Infertile bitches had higher RI and PI than bitches considered fertile during the initial estrus and diestrus. Triphasic and types A, C, and D spectral morphologies were found in females who did not gestate; while pregnant females showed spectral morphologies of type B, C and D. Hence, it was concluded that the breed, the phase of estrus cycle and pregnancy history should be considered when studies the uterine artery flow during estrus and early diestrus using investigations such as Doppler assessment, which can be an important tool in diagnosing fertility in dogs.
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http://dx.doi.org/10.1016/j.theriogenology.2017.03.012DOI Listing
June 2017

Can maternal-fetal hemodynamics influence prenatal development in dogs?

Anim Reprod Sci 2016 Sep 3;172:83-93. Epub 2016 Aug 3.

Laboratory of Carnivore Reproduction, Veterinary School, State University of Ceara, Fortaleza, Brazil.

The goals of this study were to report embryonic and fetal ultrasound changes and compare blood flow of uteroplacental and umbilical arteries of normal and abnormal conceptus. Accordingly, from the day of mating or artificial insemination, all fetuses in 60 pregnancies were evaluated weekly. According to the ultrasound findings, the gestational age was determined and the conceptuses were divided into normal or abnormal (embryonic and fetal abnormalities). The two-dimensional ultrasound assessment consists of measuring and evaluating the echogenicity of conceptus and extra-fetal structures. Doppler velocimetry measured the resistivity index (RI) and pulsatility index (PI) of uteroplacental and umbilical arteries. Two-dimensional and Doppler measurements were expressed as mean and standard deviation. Differences between normal and abnormal groups were subject to Mann-Whitney test (P<0.05). Of 264 fetuses, 15.90% showed embryonic abnormalities (resorption) and 5.68% presented fetal abnormalities (congenital abnormalities, fetal underdevelopment and fetal death). We observed a reduced diameter and abnormalities in the contour of gestational vesicle, lack of viability, increased placental thickness, increased fluid echogenicity and increases in RI and PI of uteroplacental arteries of conceptuses with embryonic resorption between the 2nd and 4th weeks. Fetuses with abnormalities showed changes in the flow of uteroplacental and umbilical arteries prior to visualization of two-dimensional alterations and different vascular behavior according to the classification of the change. Results show that ultrasound is efficient for the detection of embryonic and fetal abnormalities. When combined with Doppler ultrasound, it allows early detection of gestational changes, as well as hemodynamic changes, in conceptuses with abnormalities, which may influence their development.
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http://dx.doi.org/10.1016/j.anireprosci.2016.07.005DOI Listing
September 2016

Semen quality, testicular B-mode and Doppler ultrasound, and serum testosterone concentrations in dogs with established infertility.

Theriogenology 2015 Sep 27;84(5):805-10. Epub 2015 May 27.

Laboratory of Carnivore Reproduction, Veterinary School, State University, Fortaleza, Ceará, Brazil.

Retrospective examination of breeding records enabled the identification of 10 dogs of normal fertility and 10 dogs with established infertility of at least 12 months of duration. Comparisons of testicular palpation, semen evaluation, testicular ultrasound examination, Doppler ultrasound measurement of testicular artery blood flow, and measurement of serum testosterone concentration were made between the two groups over weekly examinations performed on three occasions. There were no differences in testicular volume (cm(3)) between the two groups (fertile right testis = 10.77 ± 1.66; fertile left testis = 12.17 ± 2.22); (infertile right testis = 10.25 ± 3.33; infertile left testis = 11.37 ± 3.30), although the infertile dogs all had subjectively softer testes compared with the fertile dogs. Infertile dogs were either azoospermic or when they ejaculated, they had lower sperm concentration, sperm motility, and percentage of morphologically normal spermatozoa than fertile dogs. Furthermore, infertile dogs had reduced sperm membrane integrity measured via the hypoosmotic swelling test. Infertile dogs had significantly lower basal serum testosterone concentrations (1.40 ± 0.62 ng/mL) than fertile dogs (1.81 ± 0.87 ng/mL; P < 0.05). There were subjective differences in testicular echogenicity in some of the infertile dogs, and important differences in testicular artery blood flow with lower peak systolic and end-diastolic velocities measured in the distal supratesticular artery, marginal testicular artery, and intratesticular artery of infertile dogs (P < 0.05). Notably, resistance index and pulsatility index did not differ between infertile and fertile dogs. These findings report important differences between infertile and fertile dogs which may be detected within an expanded breeding soundness examination.
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http://dx.doi.org/10.1016/j.theriogenology.2015.05.015DOI Listing
September 2015

Two-dimensional and Doppler sonographic prostatic appearance of sexually intact French Bulldogs.

Theriogenology 2015 Apr 18;83(7):1140-6. Epub 2014 Dec 18.

Laboratory of Carnivore Reproduction, Veterinary School, State University of Ceará, Fortaleza, Ceará, Brazil.

The aim of this study was to determine the relationship between age and prostate size and measure and characterize the prostates of French Bulldogs through two-dimensional and Doppler ultrasound. Thirty-three healthy French Bulldogs were used in this study. The dogs were divided into three groups of 11 animals according to age: 8 to 19 months (group 1), 24 to 36 months (group 2), and 48 to 72 months (group 3). The animals were evaluated once with ultrasound to obtain the following: the two-dimensional sonographic appearance of the prostate, prostatic dimensions, and characteristics from colored Doppler (perfusion characteristics, diameter, and number of pixels of the vessel) and spectral Doppler (wave morphology, resistive index, and pulsatility). There was a high positive correlation between age and prostate volume (r = 0.906). The prostatic volume gradually increased with age (P < 0.05). The characteristics in color and spectral Doppler differed according to the location of the prostatic artery. In cranial and subcapsular location: prostatic artery diameter was 0.22 ± 0.02 and 0.15 ± 0.02; number of pixels: 15,431.09 ± 1753 and 10,095.18 ± 1079.85; resistance index: 0.86 ± 0.06 and 0.64 ± 0.05; pulsatility index: 2.45 ± 0.32 and 1.14 ± 0.11, respectively. All parameters evaluated increased between groups 1 and 3 (P < 0.05), except at the parenchymal location. The resistance and pulsatility indices were significantly lower in group 1 compared with group 3 in all locations studied. In addition, the indices were significantly reduced among the locations of the prostatic artery, with the exception of the cranial and caudal locations. Groups 1 and 3 differed in all evaluations, suggesting that there is a relationship between age and the Doppler parameters evaluated in this study. It can be concluded that two-dimensional ultrasound combined with Doppler ultrasound plays an important role in the evaluation of the canine prostate. Furthermore, the location of the prostatic artery may influence Doppler parameters, and age is an important factor that should be considered when evaluating the canine prostate.
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http://dx.doi.org/10.1016/j.theriogenology.2014.12.016DOI Listing
April 2015

Cryopreservation of canine epididymal sperm using ACP-106c and TRIS.

Cryobiology 2014 Aug 10;69(1):17-21. Epub 2014 May 10.

Laboratory of Carnivore Reproduction, School of Veterinary Medicine, State University of Ceara, Av. 1700, CEP Fortaleza, CE, Brazil. Electronic address:

The objective was to cryopreserve sperm recovered from the canine epididymal cauda immediately after an orchiectomy. The sperm was stored for 12h at 4 °C using ACP-106c and TRIS as extenders. Sixty adult male dogs were used. The testis-epididymis complex (TEC) was removed, immersed in 0.9% saline and transported to the laboratory. The 60 TEC were divided into groups according to the 4 °C cooling time (0 h or 12 h) and according to the extender used for sperm recovery (ACP-106c or TRIS), forming 4 experimental groups: G0h-ACP, G12h-ACP, G0h-TRIS and G12h-TRIS. The sperm were recovered from the epididymal cauda using the retrograde flow technique. Next, 1.0 mL of ACP-106c or 1.0 mL of TRIS (preheated to 37 °C for 5 min) was added to the sperm of each epididymis. One week later, the sperm was thawed at 37 °C for 1 min, and its morphology, functionality and total and progressive sperm motilities were analyzed. Other parameters were obtained by Computer Assisted Semen Analysis (CASA). The data were submitted to multivariate analysis of variance (MANOVA) (P<0.05). The total motility values were 52.17 ± 1.78 and 49.8 ± 1.93 for groups G0h-ACP and G12h-ACP and 50.7 ± 2.06 and 43.90 ± 2.51 for groups G0h-TRIS and G12h-TRIS, respectively. A decrease in total sperm motility was observed after 12h of cooling for both extenders (P<0.05). ACP-106c can be used as an extender for freezing canine epididymal sperm, and the freezing procedure must be performed immediately after sperm recovery.
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http://dx.doi.org/10.1016/j.cryobiol.2014.04.013DOI Listing
August 2014
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