Influence of Genotype on Endometrial Angiogenesis during Early Pregnancy in Piau and Commercial Line Gilts.

This study aimed to evaluate the endometrial angiogenesis of pregnant commercial line and Piau gilts during early pregnancy. We used 27 gilts, divided into three groups according to the type of mating: Commercial (n = 9), commercial line females mated with commercial line males; Cross-mated (n = 9), Piau females mated with commercial line males; and Piau (n = 9), Piau females mated with Piau males. Each group was divided into three subgroups based on gestational age at the time of slaughter (7, 15, and 30 days of pregnancy). Immediately after slaughter, endometrial samples were obtained for histological evaluation and for analysis of the relative transcript abundance (RTA) of angiogenesis-related genes (HIF1α, FGF9, ANG1, TEK, VEGFA, ANGPT1, and ANGPT2). The number of endometrial glands was similar among groups but decreased with gestational age (p < 0.05). Piau females showed a higher number of blood vessels (p < 0.05) at 7 and 15 days of pregnancy, but no differences were observed among groups at 30 days, suggesting an influence of the male genotype on the pattern of uterine vascularization. There were no differences among groups for RTA of the FGF9, HIF1α, TEK, VEGFA, ANGPT1, and ANGPT2 genes. The HIF1α-gene RTA was higher at 7 and 15 days of pregnancy; for TEK and ANGPT1, the RTA was higher at 15 days of pregnancy; and the RTA of VEGFA and ANGPT2 genes were higher at 30 days of pregnancy. The ANG1 RTA was similar for pregnancies in the commercial and Piau groups but was higher (p < 0.05) at 15 days in the Cross-mated group, suggesting an interaction between genotypes. Overall, the pattern found for the RTA of angiogenesis-related genes was similar among the groups in this study, although some phenotypic differences could be noted, such as the highest number of blood vessels being found during early pregnancy of Piau gilts. The results of the gene RTA when crossed with phenotypic data led to conclusions that are conflicting with those reported in the literature. However, noteworthy is that angiogenesis is a complex process in which the balance between stimulatory and inhibitory factors may be related to time.

Correction: Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

[This corrects the article DOI: 10.1371/journal.pone.0218963.].

Bovine herpesvirus 1 can cross the intact zona pellucida of bovine oocytes after artificial infection.

Bovine herpesvirus 1 (BHV1) is an important bovine pathogen, responsible for respiratory diseases and reproductive problems. This study investigated the penetration capacity of BHV1 into oocytes after co-incubation for either 1 h or 24 h. Immunofluorescence assays in cumulus-oocyte complexes (COCs) and denuded oocytes (without the presence of cumulus cells) were performed and evaluated using confocal laser scanning microscopy. Blood samples and ovaries from BHV1 seronegative cows were used. The oocytes recovered were divided into two groups. Group I comprised COCs (n = 312) and denuded oocytes (n = 296), which were experimentally infected with BHV1 and incubated for 1 h at 38.5°C and 5% CO2. Group II comprised COCs (n = 425) and denuded oocytes (n = 405), which were co-incubated with BHV1 under the same conditions for 24 h. The negative control of these two groups was respectively subjected to the same protocol, except for exposure to BHV1. To our knowledge, this study provides the first evidence of BHV1 detection within COCs and denuded oocytes exhibiting intact zona pellucida when co-incubated with the virus for 24 h. Immunolocalization also confirmed the presence of BHV1 in the cytoplasm of the cumulus cells of all COCs exposed to the virus after both incubation periods. In conclusion, detection of BHV1 inside oocytes has a great meaning for the field of animal reproduction. The detection of BHV1 in different layers of cumulus cells also demonstrates that these cells are sources of viral infection.

Use of nonlinear mixed models for describing testicular volume growth curve in Nellore bulls.

This study aimed to describe longitudinal testicular volume (TV) data of Nellore bulls by using nonlinear mixed models. Dataset consisted of 2,294 TV measurements from 505 bulls with ages ranging from 563 to 4,307 days. Nine nonlinear models were evaluated: Brody, Gompertz, Hill, Logistic I and II, Meloun, Michaelis-Menten, Mitscherlich and von Bertalanffy. Goodness of fit was evaluated by Akaike's information criterion (AIC), Bayesian information criterion (BIC), adjusted R2, percentage of convergence, error sum of squares (ESS), mean absolute deviation (MAD), and average prediction error (APE). These criteria were used to select the best model, then the absolute growth rate (AGR) for TV was estimated by the first derivate of the adjusted model related to time (∂Y/∂t). The values of adjusted R2, ESS, MAD and APE were similar among models. Percentage of convergence was higher for the Logistic I (76.8%), Logistic II (75.5%) and Mitscherlich (78.6%) models, but Logistic I and II showed the lowest values of AIC and BIC, indicating a better fit, so the Logistic I model was chosen for subsequent analyses. The TV growth occurred at a high rate until the inflection point, which was estimated at approximately 22 months of age; it stabilized and reached a plateau at approximately 2,500 days of age. This may suggest that TV is more related to sexual maturity than precocity. Additionally, the Logistic I model was used to estimate the growth curve and the AGR of the testicular length and width. As a result, testicular length increased at a higher rate than testicular width until approximately 1,600 days of age, indicating that the testes become longer with increasing age.

Detection of bovine herpesvirus 1 in genital organs of naturally infected cows.

Bovine herpesvirus 1 (BoHV-1) is a causative agent of respiratory diseases in cattle, and infection with BoHV-1 can cause reproductive failure. There are few studies regarding infections in natural conditions in the reproductive organs of bovine animals. In this context, this study investigated the presence of BoHV-1 in the uterus, oviducts, and ovarian tissues of naturally infected cows. The three genital structures were evaluated for the presence or absence of BoHV-1 by immunofluorescence assay using confocal scanning laser microscopy. Blood and genital organ samples of 75 cows unvaccinated against BoHV-1 were used. Fragments of uterus, oviduct, and ovarian tissue were processed and analyzed by confocal scanning laser microscopy. Neutralization by antibodies was observed in 54.7% (41/75) of the serum samples tested. BoHV-1 were detected in the uterus of all the seropositive cows. The oviducts contained BoHV-1 in 73.2% of the samples and the ovaries contained BoHV-1 in 58.5% of the samples from seropositive animals. The presence of the virus was not observed in any of the genital organs of seronegative animals. There was no correlation between the antibody titer and the detection of BoHV-1 in positive tissue in the different genital organs or with the number of infected structures per animal. The detection of BoHV-1 in 100% of the uterus samples from seropositive cows suggests that this organ may be a source of infection for the fetus, resulting in abortion. Further studies on the mechanism by which BoHV-1 infects the fetus via the uterine route should be performed.

Use of two new formulations as bovine embryo manipulation solution.

This study aimed to evaluate the effect of two Embryo Manipulation Solutions (EMS and EMS supplemented) in maintenance of the viability of embryos, initially using structures derived from mice (first phase). Next, the efficiency of these solutions in routines of bovine embryo transfer was evaluated (second stage). Mice embryos were used in the stages of early blastocyst, and compact morula grades I and II. These embryos were initially randomly distributed and maintained for four hours in three solutions: Modified phosphate buffered saline (PBS; Control); EMS (treatment 1), and EMS supplemented (treatment 2). Subsequently, they were cultured in TCM 199 medium and evaluated in terms of total number of cells, morphometric characteristics, ultra structural aspects, detection of cell apoptosis, and quantification of Hsp70.3 gene expression. In the second phase, these same solutions were tested in the transfer of quality I and II bovine embryos (excellent and good). These embryos were transferred fresh to 58 recipients. The results showed that the total number of cells in embryos expanded blastocyst (ExB), the number of apoptotic cells, the cell, nuclear, nucleolar diameter and the nucleus/nucleolus ratio was similar among the treatments. The pregnancy rate shown on second phase was also similar. However, the EMS supplemented expressed more Hsp70.3 than EMS. The expression of Hsp70.3 was also greater for embryos in EMS than that of EMS supplemented. The McII embryos, EMS and EMS supplemented samples also expressed more Hsp70.3 compared to control embryos. In conclusion, the tested solutions can be used in routine embryo transfer techniques, replacing modified PBS solution as an effective media in maintaining embryo viability.

Latent bovine herpesvirus 1 and 5 in milk from naturally infected dairy cattle.

Bovine herpesvirus 1 and 5 (BoHV-1 and -5) are antigenically and genetically related and can establish latent infection. We aimed to analyze the applicability of the milk sample to detect latently BoHV-infected cattle. BoHV-1 non-vaccinated clinically healthy cows from five dairy cattle herds (herd 1, n=24; herd 2, n=39; herd 3, n=39; herd 4, n=36; herd 5, n=70) were studied. We confirmed the presence of BoHV-1, and for the first time, BoHV-5 in the milk of naturally infected dairy cattle.

Relationship of testicular biometry with semen variables in breeding soundness evaluation of Nellore bulls.

This study aimed to evaluate the correlation between testicular biometry and semen variables, as well as, to relate testicular variables to the probability of selecting Nellore bulls with desirable sperm morphology when conducting breeding soundness evaluations (BSE). A total of 2055 BSEs from 506 bulls comprised the dataset. Biometric variables evaluated were: scrotal circumference, testicular volume, width, length, ratio and eccentricity; and semen variables were sperm motility, major sperm defects, minor sperm defects and normal sperm. Data of testicular biometry were correlated with data for semen variables using the Pearson's correlation assessment. Effects of testicular variables in selecting for sperm morphology of bulls in the BSE were evaluated by logistic regression. Scrotal circumference, testicular volume, length and width were positively correlated to sperm motility (0.18 to 0.19) and normal sperm (0.24 to 0.27) and negatively correlated with values for major defects (-0.24 to -0.27), but for testicular ratio and eccentricity there were coefficients near zero for all semen traits. Testicular ratio and eccentricity were not suitable for predicting the probability of selecting a bull based on semen variables using the BSE, but scrotal circumference, testicular volume, length and width were highly significant (P < 0.0001) with moderate values of area under ROC (Receiver Operating Characteristics) curve (0.608 to 0.620).

Sêmen sexado através de citometria de fluxo e centrifugação por gradiente de concentração / Semen sexed using flow cytometry and concentration gradient centrifugation / Semen sexado a través de citometría de flujo y centrifugación por gradiente de concentración

Resumo O objetivo deste trabalho foi apresentar uma revisão completa e atualizada sobre a origem do sêmen sexuado, os requisitos e as técnicas de sexagem espermáticas. Nas últimas décadas, desenvolveram-se várias tecnologias na área da reprodução animal. O sêmen sexado é uma biotecnologia recente que ainda se encontra em fase de estudo e aperfeiçoamento em diversas etapas, devido a que o processo de sexagem causa estresse ao espermatozoide, e o deixa mais sensível ao processo de armazenamento; isto interfere diretamente na fertilidade. Esta tecnologia, que chegou ao Brasil em 2004, vem ganhando adeptos entre produtores que desejam produzir animais mais homogêneos para o sacrifício ou para a produção de leite. O uso do sêmen sexado tem sido difundido em muitos rebanhos do mundo, já que se apresenta como uma técnica com um direcionamento maior na seleção do sexo. Muitas técnicas têm sido utilizadas com a finalidade de buscar uma maior separação de espermatozoides portadores de cromossomas X e Y, com mínima agressão espermática. Desta forma, a utilização da sexagem espermática tem obtido resultados na aplicação de biotecnologias da reprodução assistida, e com isso tem elevado o lucro no melhoramento genético e tem otimizado a produtividade no sexo escolhido. Entretanto, ainda existem muitas limitações, principalmente referentes al tempo de exposição, o índice de aproveitamento e os resultados de índices de gestação em condições de campo. Por outro lado, a dose de sêmen sexado pode custar de duas a oito vezes o valor de uma convencional; da mesma forma, o seu uso avança no Brasil.

Abstract This study aimed to present a complete and updated review on the origin of sexed semen, as well as on the requirements and techniques of sperm sexing. In the last decades, several technologies were developed in the field of animal reproduction. Sexed semen is a recent biotechnology that is still under study and refinement in various stages, because the sexing process causes stress to the sperm, leaving it more sensitive to the storage process; this directly interferes with fertility. This technology, which arrived in Brazil in 2004, has been gaining support among producers who want to produce more homogeneous animals for slaughter or for dairy production. The use of sexed semen has been disseminated in many herds of the world, since it is a technique with better results in the selection of sex. Many techniques have been used in order to seek a greater separation of sperms carrying X and Y chromosomes, with minimal sperm aggression. In this way, the use of sperm sexing has obtained results in the application of assisted reproductive biotechnologies, which has increased the gain in genetic improvement and has optimized productivity in the chosen sex. In the meantime, there are still many limitations, mainly concerning exposure time, exploitation rate, and the results of pregnancy rates in field conditions. On the other hand, the dose of sexed semen can cost two to eight times the value of conventional semen; nevertheless, its use progresses in Brazil.

Resumen El objetivo de este trabajo fue presentar una revisión completa y actualizada sobre el origen del semen sexado, los requisitos y las técnicas de sexaje espermáticas. En las últimas décadas, se desarrollaron varias tecnologías en el área de la reproducción animal. El semen sexado es una biotecnología reciente que aún se encuentra en fase de estudio y perfeccionamiento en diversas etapas, debido a que el proceso de sexaje causa estrés al espermatozoide, y lo deja más sensible al proceso de almacenamiento; esto interfiere directamente en la fertilidad. Esta tecnología, que llegó a Brasil en 2004, viene ganando adeptos entre productores que desean producir animales más homogéneos para el sacrificio o para la producción lechera. El uso del semen sexado ha sido difundido en muchos rebaños del mundo, ya que se presenta como una técnica con un direccionamiento mayor en la selección del sexo. Muchas técnicas han sido utilizadas con el fin de buscar una mayor separación de espermatozoides portadores de cromosomas X y Y, con mínima agresión espermática. De esta forma, la utilización del sexaje espermático ha obtenido resultados en la aplicación de biotecnologías de la reproducción asistida, y con eso ha elevado la ganancia en el mejoramiento genético y ha optimizado la productividad en el sexo escogido. Entretanto, aún existen muchas limitaciones, principalmente referentes al tiempo de exposición, la taza de aprovechamiento y los resultados de tazas de gesta ción en condiciones de campo. Por otro lado, la dosis de semen sexado puede costar de dos a ocho veces el valor de una convencional; así mismo, su uso avanza en Brasil.

Cell population indexes of spermatogenic yield and testicular sperm reserves in adult jaguars (Panthera onca).

The intrinsic yield of spermatogenesis and supporting capacity of Sertoli cells are the desirable indicators of sperm production in a species. The objective of the present study was to quantify intrinsic yield and the Sertoli cell index in the spermatogenic process and estimate testicular sperm reserves by histological assessment of fragments obtained by testicular biopsy of five adult jaguars in captivity. The testicular fragments were fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 microm thickness. In the seminiferous epithelium of the jaguar, 9.2 primary spermatocytes in pre-leptotene were produced by "A" spermatogonia. During the meiotic divisions only 3.2 spermatids were produced by a primary spermatocyte. The general spermatogenic yield of the jaguar was about 23.4 cells and each Sertoli cell was able to maintain about 19.2 germ cells, 11 of them were round spermatids. In each seminiferous epithelium cycle about 166 million spermatozoa were produced by each gram of testicular tissue. In adult jaguars, the general spermatogenic yield was similar to the yield observed in pumas, greater than that observed for the domestic cat, but less compared to most domestic animals.

Organization and quantification of the elements in the intertubular space in the adult jaguar testis (Panthera onca, LINNAEUS, 1758).

The endocrine portion of mammal testicle is represented by Leydig cells which, together with connective cells, leukocytes, blood and lymphatic vessels, form the intertubular space. The arrangement and proportion of these components vary in the different species of mammals and form mechanisms that keep the testosterone level--the main product of the Leydig cell--two to three times higher in the interstitial fluid than in the testicular blood vessels and 40-250 times higher in these than in the peripheral blood. Marked differences are observed among animal species regarding the abundance of Leydig cells, loose connective tissue, development degree and location of the lymphatic vessels and their topographical relationship with seminiferous tubules. In the jaguar about 13% of the testicular parenchyma is occupied by Leydig cells, 8.3% by connective tissue and 0.3% by lymphatic vessels. Although included in standard II, as described in the literature, concerning the arrangement of the intertubular space, the jaguar has grouped lymphatic vessels in the intertubular space instead of isolated ones. In the jaguar the average volume of the Leydig cell was 2386 microm3 and its average nuclear diameter was 7.7 microm. A great quantity of 2.3 microm diameter lipidic drops was observed in the Leydig cell cytoplasm of the jaguar. The Leydig cells in the jaguar occupy an average 0.0036% of the body weight and the average number per gram of testicle was within the range for most mammals: between 20 and 40 million.

Sertoli cell index and spermatic reserves in adult captive African lions (Panthera leo, Linnaeus, 1758).

The intrinsic yield of spermatogenesis and the supporting indexes of the Sertoli cells are the best indicators for the spermatic production capacity in a species. The aim of the present study was to quantify the intrinsic yield of the spermatogenetic process, as well as the Sertoli cell index and spermatic reserves. Testicular fragments of five adult African lions was fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 microm thickness. In the seminiferous epithelium of the African lions, 10.3 primary spermatocytes at pre-leptotene phase are produced by the type-A spermatogonia. During meiotic divisions, only 2.7 spermatids were produced from the primary spermatocytes. The general spermatogenesis production in the African lions was approximately 22.1 cells, and each Sertoli cell was able to sustain and maintain approximately 14.9 cells of the germinative line, from which 7.9 are round spermatids. A total of 103x10(6) spermatozoa are produced by each testis gram at each cycle of the seminiferous epithelium. The spermatic reserve of lion is below the amplitude observed in mammals.