Oxidative stress associated with proteomic and fatty acid profiles of sperm from Nellore bulls at rest†.

Sexual rest is a transient condition, which compromises conception rates, characterized by large volumes of ejaculate with high percentages of dead sperm observed in bulls. The biochemical mechanisms leading to this ejaculate pattern are not fully understood. Six adult resting Nellore bulls were submitted to Breeding Soundness Evaluation by four consecutive semen collections through the electroejaculation method during a 30 min period. Each ejaculate had its semen phenotypic parameters; morphology and physical aspects were evaluated. To assess enzymatic activity (superoxide dismutase, catalase, and glutathione S-transferase), lipid peroxidation (concentrations of malondialdehyde and nitric oxide), fatty acid, and proteomic profile aliquots of spermatozoa from the first and fourth ejaculates were used. All sperm parameters differed between the first and fourth ejaculates. Spermatozoa from the first ejaculate showed lower enzymatic activity and a higher concentration of lipid peroxidation markers. Among the 19 identified fatty acids, 52.7% are polyunsaturated. Relative abundance analysis showed that C12:0 and C18:0 fatty acids differed between the first and fourth ejaculates, being the fourth ejaculate richer in spermatozoa. The proteomics analysis identified a total of 974 proteins in both sample groups (first and fourth ejaculates). The majority of identified proteins are related to cellular processes and signaling. Quantitative proteomics showed 36 differentially abundant proteins, 6 up-regulated proteins in the first ejaculate, and 30 up-regulated proteins in the fourth ejaculate. Spermatozoa from bulls at sexual rest have less antioxidant capacity, causing changes in their fatty acid composition and protein profile, which generates the observed sperm pattern and lower fertilization capacity.

Human milk proteins differentiate over the sex of newborns and across stages of lactation.

BACKGROUND & AIMS: Human milk (HM) is a complete food that meets the nutritional and energy demands of the newborns. It contains numerous bioactive components, including functional proteins. Variations in HM energy and lipid content have already been reported related to the newborn's sex, but differences between protein profiles are still scarce. This work aimed to identify differences between HM proteins produced by mothers of female and male newborns, in the lactation stages of colostrum and mature milk, and the metabolic pathways involved. METHODS: A total of 98 HM samples were collected from 39 lactating women and classified according to the newborn's sex, stages of lactation, and three mothers' age groups, and evaluated about protein concentration and one-dimensional electrophoretic profile. Next, to assess samples with the greatest differences, the HM proteins regarding the newborn's sex and the stages of lactation were compared using nano-LC-MS/MS, in 24 HM samples randomly rearranged into four groups: female and male infants, and colostrum and mature milk. Functional classification, metabolic pathways, and protein interaction networks were analyzed by Gene Ontology, KEGG, and STRING, respectively. RESULTS: The soluble protein content of HM decreased throughout lactation, with differences regarding isolated factors, such as mothers' age group, child's sex and stages of lactation, and also in terms of their interactions. A total of 146 proteins were identified, 42 of which showed different abundances over the sexes of newborns and 53 between the stages of lactation. In general, proteins related to metabolic processes were up-regulated for mothers of male infants and in the mature stage of lactation, while proteins related to defense were up-regulated in mothers of female infants and in the colostrum phase. CONCLUSION: This study indicated that there are differentiated and specific nutritional and defense needs of newborns, by sex and by lactation phase, which is highly relevant for a more appropriate supply of food to infants receiving HM from donor mothers.

Dataset of seminal plasma proteome of Nellore bulls with high and low percentage of abnormalities sperm.

OBJECTIVES: Bovine seminal plasma proteins perform several functions related to sperm function. Changes in the expression pattern or abundance of seminal proteins are related to changes in the fertilizing capacity of bulls. Considering the role of seminal plasma proteins in sperm function and animal reproduction, we investigated changes in the protein abundance profile in response to sperm morphological changes using a proteomic approach. DATADESCRIPTION: In our present investigation, we employed liquid chromatography coupled with mass spectrometry to elucidate the proteomic composition of seminal plasma obtained from Nellore bulls exhibiting varying percentages of sperm abnormalities. Following semen collection, seminal plasma was promptly isolated from sperm, and proteins were subsequently precipitated, enzymatically digested using porcine trypsin, and subjected to analysis utilizing the Acquity nano UHPLC System in conjunction with a mass spectrometer. This dataset encompasses a total of 297 proteins, marking the inaugural instance in which a comparative profile of seminal plasma proteins in young Nellore bulls, categorized by their sperm abnormality percentages, has been delineated using LC-MS/MS. The comprehensive nature of this dataset contributes pivotal proteomic insights, representing a noteworthy advancement in our understanding of the reproductive biology of the Nellore breed.

Proteomic profile of seminal plasma from Pêga donkeys (Equus asinus) with high sperm motility and vigor: Implications for assisted reproduction.

Donkeys of the Pêga breed (Equus asinus) have been used for two centuries to produce breeding stock and create hybrids for labor and transport in southeast Brazil, and for exporting meat and milk to other countries. Furthermore, they are used in competitions, as they are docile and easy to handle. However, assisted reproduction success rates for frozen donkey semen are remarkably low, with no standardized method for cryopreserving sperm after removal of seminal plasma. This work aims to reveal the biological involvement of seminal plasma proteins from Pêga donkeys in aiding the development of assisted reproduction. This study was carried out with 14 ejaculates collected every eight days, throughout the breeding season, from three healthy fertile Pêga donkeys, with an average age of four years. After confirming the high freezability of fresh semen by evaluating quality parameters, the seminal plasma was separated by centrifugation and an aliquot from each collection was microfiltered and frozen. A label-free technique followed by LC-MS/MS analysis applied to pools of seminal plasma samples from each animal revealed 522 proteins in the proteomic profile, of which 49.8 % (260 proteins) are related to cellular energy transformation, and many proteins involved in reproduction (76), spermatogenesis (38), fertilization (29), among other biological process. By comparison with literature, Pêga donkeys share many proteins with donkeys of Dezhou breed that present great potential as fertility biomarkers. Our results showed proteins positively related to fertilization for different breeds of donkeys around the world, helping to enhance the assisted reproduction of Pêga donkeys.

Relative Abundance of Spermadhesin-1 in the Seminal Plasma of Young Nellore Bulls Is in Agreement with Reproductive Parameters.

This study aimed to evaluate the proteomic profile of seminal plasma from young Nellore bulls. We used 20 bulls aged between 19.8 and 22.7 months, divided into two groups according to the results of the Breeding Soundness Evaluation (BSE): approved (FIT n = 10) and not approved (UNFIT n = 10). The scrotal perimeter was measured and a semen collection was performed through electroejaculation. The percentage of sperm motility, mass motility, and sperm vigor were calculated using conventional microscopy, and the percentage of sperm abnormalities was calculated using phase-contrast microscopy of all ejaculates. Seminal plasma was separated from spermatozoa using centrifugation and processed for proteomic analysis by LC-MS/MS. Seminal plasma proteins were identified using MASCOT Daemon software v.2.4.0 and label-free quantification analysis was carried out by SCAFFOLD Q+ software v.4.0 using the Exponentially Modified Protein Abundance Index (emPAI) method. Functional classification of proteins was performed based on their genetic ontology terms using KOG. Functional cluster analysis was performed on DAVID. There were no differences in scrotal perimeter and physical semen characteristics between FIT and UNFIT groups of bulls. The percentage of sperm abnormalities was higher (p < 0.05) in the UNFIT group of bulls. A total of 297 proteins were identified for the two groups. There were a total of 11 differentially abundant proteins (p < 0.05), two of them more abundant in FIT bulls (Spermadhesin-1 and Ig gamma-1 chain C region) and nine in UNFIT bulls (Vasoactive intestinal peptide, Metalloproteinase inhibitor 2, Ig lambda-1 chain C regions, Protein FAM3C, Hemoglobin beta, Seminal ribonuclease, Spermadhesin 2, Seminal plasma protein BSP-30kDa, and Spermadhesin Z13). Spermadhesin-1 was the protein with the highest relative abundance (36.7%) in the seminal plasma among all bulls, corresponding to 47.7% for the FIT bulls and 25,7% for the UNFIT bulls. Posttranslational modification, protein turnover, and chaperones were the functional categories with the highest number of classified proteins. Protein functional annotation clusters were related to Phospholipid efflux, ATP binding, and chaperonin-containing T-complex. The differentially abundant proteins in the group of FIT bulls were related to sperm capacitation and protection against reactive species of oxygen. In contrast, differentially expressed proteins in the group of UNFIT bulls were related to motility inhibition, intramembrane cholesterol removal and oxidative stress. In conclusion, the proteomic profile of the seminal plasma of FIT bulls presents proteins with participation in several biological processes favorable to fertilization, while the proteins of the seminal plasma of UNFIT bulls indicate a series of alterations that can compromise the fertilizing capacity of the spermatozoa. In addition, the relative abundance of spermadhesin-1 found in the seminal plasma of young Nellore bulls could be studied as a reproductive parameter for selection.

Kallikrein proteoforms and reproductive parameters in stallion are conditioned by climate.

Horses are seasonal polyoestrous animals, and the photoperiod is the main factor modulating their reproductive activity. There is no consensus on the andrological and biochemical factors that influence breeding seasonality. To assess the involvement of climate in reproduction, Mangalarga Marchador stallions were monitored over 1 year regarding semen quality and seminal plasma proteome. Here, we show that kallikrein (KLKs) proteoforms in seminal plasma are involved in climate conditioning of reproduction. During the breeding season, greater abundance and different types of KLKs occurred simultaneously to lower sperm motility, greater semen volumes and higher concentrations of glucose and cholesterol. Considering that vasodilation due to activation of the kallikrein-kinin system and the consequent inhibition of the renin-angiotensin system may be associated with lower sperm motility, unravelling the involvement of KLK proteoforms in reproductive seasonality is a priority in horse breeding.

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.

Estadio de madurez sexual en toros de la raza nelore / State of Sexual Maturity of Nelore Bulls / Estádio de maturidade sexual em toros da raça nelore

La presente investigación se planteó con el objetivo de determinar el estado de madurez sexual de toros jóvenes de la raza nelore y su relación con el perímetro escrotal y las características seminales. Se estudiaron 1985 animales con edades comprendidas entre los 19 y los 23 meses de edad, alimentados con pasturas tropicales (Brachiaria brizantha, Brachiaria decumbes y Panicum maximun) y evaluados a través de examen andrológico. Se valoraron las características físicas del eyaculado, la morfología espermática y el perímetro escrotal (PE). Tras el examen andrológico, los animales se clasificaron como aptos para la reproducción (clase andrológica 1); aptos para la reproducción en régimen de monta natural (clase 2); no aptos para la reproducción temporalmente (clase 3) y descartados (clase 5). Para comparar las medias encontradas entre las clases andrológicas se utilizaron dos pruebas: la de Tukey, con probabilidad del 5 % de error, y la de correlaciones simples de Pearson, para verificar las relaciones entre las características estudiadas. Se encontró que el 84,5 % de los toros son sexualmente maduros. El 39,75 % de los animales aptos para la reproducción presentó un PE mayor a 34 cm, y solo el 0,71 % de la población estudiada exhibió una circunferencia escrotal inferior a 28 cm. Se registraron correlaciones altas favorables entre PE y las características físicas del semen. Se concluye que el PE es una excelente característica para evaluar y seleccionar toros jóvenes de la raza nelore.

This research aimed to determine the state of sexual maturity of young Nelore bulls and its relation to scrotal circumference and seminal characteristics. 1985 animals (aged between 19 and 23 months), fed with tropical pastures (Brachiaria brizantha, Brachiaria decumbes, and Panicum maximum), were evaluated through andrological examination. Physical characteristics of the ejaculate, sperm morphology and scrotal circumference (SC) were examined. After the andrological examination, animals were classified as suitable for reproduction (andrological class 1); suitable for reproduction in natural mating system (class 2); temporarily unsuitable for reproduction (class 3); and discarded (class 5). To compare the measurements found in different andrological classes, two tests were used: the Tukey test with 5% probability of error, and simple Pearson correlations to verify the relationships between the studied characteristics. 84.5% of the bulls were found to be sexually mature. 39.75% of the animals suitable for reproduction presented a SC greater than 34 cm, and only 0.71% of the study population showed a scrotal circumference less than 28 cm. Favorable high correlations between SC and the physical characteristics of semen were evidenced. The article concludes that scrotal circumference is an excellent characteristics for evaluating and selecting young Nelore bulls.

Esta pesquisa foi proposta com o objetivo de determinar o estado de maturidade sexual de touros jovens da raça nelore e sua relação com o perímetro escrotal e as características seminais. Realizou-se um estudo com 1985 animais com idades compreendidas entre os 19 e os 23 meses de idade, alimentados com pastagens tropicais (Brachiaria brizantha, Brachiaria decumbes e Panicum maximun) e avaliados através de exame andrológico. Avaliaram-se as características físicas do ejaculado, a morfologia espermática e o perímetro escrotal (PE). Depois do exame andrológico, os animais se classificaram como aptos para a reprodução (tipo andrológica 1); aptos para a reprodução em regime de monta natural (tipo 2); não aptos para a reprodução temporariamente (tipo 3) e descartados (tipo 5). Para comparar as médias encontradas entre os tipos andrológicos se utilizaram dois testes: o de Tukey, com probabilidade de 5 % de erro, e a de correlações simples de Pearson, para verificar as relações entre as características estudadas. Encontrou-se que o 84,5 % dos touros são sexualmente maduros. O 39,75 % dos animais aptos para a reprodução apresentou um PE superior a 34 cm, e somente o 0,71 % da população estudada exibiu uma circunferência escrotal inferior a 28 cm. Registraram-se correlações altas favoráveis entre PE e as características físicas do sêmen. Conclui-se que o PE é uma excelente característica para avaliar e selecionar touros jovens da raça nelore.