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.

Phenotypic and genetic relationships among anogenital distance, anti-Müllerian hormone, and in vitro embryo production in Gyr dairy cattle.

Anti-Müllerian hormone (AMH) concentration and number of recovered oocytes (ROOC) are phenotypic parameters associated with in vitro embryo production (IVEP). More recently, anogenital distance (AGD) has been proposed as a proxy for fertility in dairy cattle that is easy to collect at a low cost. The aim of this study was to characterize the AGD and its phenotypic and genetic associations with AMH and IVEP in Bos indicus Gyr dairy cattle. The hypothesis was that the number of ROOC, in vitro-produced embryos, and AMH concentration would increase as the AGD decreases. From July to December 2021, a single morphometrical measurement of AGD was collected in 552 donors from 6 herds in Brazil. A subset of donors had AMH assayed on the same day. Only ovum pick-up events that occurred up to 12 mo preceding and 7 mo succeeding the AGD measurement were used to assess the association between AGD, AMH, and IVEP. Thus, 472 donors (1,551 ovum pick-up events and 140 donors with AMH) were considered in the analysis. A raw average was calculated for each individual donor's ROOC, viable oocytes, total produced embryos, viability rate, and embryo rate (defined as total produced embryos/viable oocytes). Comparisons were conducted within the age categories of 3 to <6 yr or 6 to <10 yr. Phenotypic associations were performed in SAS software (SAS Institute Inc., Cary, NC). Genetic correlations were estimated using the BLUPF90 family of programs. The AGD (128.7 mm ± 14; mean ± standard deviation) had a normal distribution and was highly variable (83 to 172 mm) among the Gyr population. Our experimental hypothesis was partially supported by a phenotypic association of a greater number of total produced embryos (R2 = 0.023) as AGD decreased. Our results failed to support an increase in AMH concentration along with a decrease in AGD. In addition, positive and low genetic correlations were observed between AGD and viable oocytes (r = 0.08), and embryo rate (r = 0.20). A greater number of viable oocytes and embryos were observed in donors in the high compared with intermediate and low ROOC categories within both age categories. The age interval of 3 to <6 yr showed a greater number of recovered and viable oocytes for the high AMH compared with the low category, but no differences were observed among the AGD categories. In summary, for the Gyr breed, AGD was phenotypically inversely associated with a quantity-related parameter, such as the total number of produced embryos. In contrast, AGD showed a low genetic correlation with qualitative-related outcomes such as viable oocytes and embryo rate. Further studies should be performed to validate these retrospective analyses and to better understand the association between AGD and IVEP.

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.

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.

Calving date as a potential predictor for the probability of approval in the first breeding soundness evaluation of Nellore bulls.

Beef production systems primarily use natural service (NS) for breeding. However, a significant number of bulls used for NS are subfertile, limiting the profitability of the cow-calf operations. Therefore, producers should select bulls based on breeding soundness evolutions (BSE) to ensure higher pregnancy rates. Several factors can affect the bull ability to pass a BSE. We hypothesize that calving date would be a factor that affects the bull probability of approval at the first BSE. For this purpose, a multivariate logistic regression in a dataset of 14,737 BSEs from young Nellore bulls was used. Correlations between calving date, biometrics, and semen traits were evaluated using Pearson's correlation coefficient. Our results demonstrated that the calving date affected the probability of approval at the first BSE (p < .05). Indeed, the variable that added more information to our model was the calving date, far more than the age group of the bulls according to Akaike's information criterion. Hence, bulls born on day 0 of the calving season have 1.26 more chances to be approved at the first BSE than bulls born 21 days later. This result highlights the importance of getting the dams of future bulls pregnant as soon as possible in the breeding season. In addition, the calving season should be no longer than 47 days to achieve 80% BSE approval in 20-22 months old Nellore bulls. The strongest correlation was with SC, which decreased as the calving date increased. Therefore, the calving date may be used as a predictor of the outcome of the first BSE in young bulls. In that way, the calving date can help seedstock producers to maximize efficiency in making crucial management decisions during the breeding and calving season including nutrition, reproductive, and culling.

Climatic seasons and time of the day influence thermoregulation and testicular hemodynamics in Santa Inês rams raised under humid tropical conditions.

This study evaluated the possible association between the diurnal variations of climatic factors during the rainy (RS) or less rainy (LS) seasons on the testicular hemodynamics and thermoregulatory responses of hair sheep rams raised in a humid tropical climate. Santa Inês rams (n = 6) underwent evaluation of general and testicular physiological parameters (heart and respiratory rates, internal and scrotal temperatures, internal-scrotal temperature gradient, scrotal distention, and color Doppler ultrasound evaluation of the spermatic cords and spectral analyses of testicular arteries) over six consecutive weeks per season at three separate times daily (morning = 8:00 a.m., noon = 12:00 p.m., and afternoon = 5:00 p.m.) during the RS and LS. Climatic air temperature and relative humidity data were recorded, and the temperature and humidity index (THI) was calculated. Higher thermal challenge was observed in LS relative to RS (air temperature = 28.0 vs. 30.9 °C; relative humidity = 84.1 vs. 69.9%; THI = 80.0 vs. 82.5; P < 0.05). In both seasons, respiratory rate and internal temperature were normal, demonstrating the animals' adaptability. In RS, however, a higher scrotal temperature was recorded in relation to LS (35.0 vs. 34.7 °C; P < 0.05), with a gradual increase from morning to afternoon. Lower resistivity (0.40 vs. 0.64; P < 0.05) and pulsatility (0.55 vs. 1.14; P < 0.05) indices, and a higher rate of high-velocity blood flow of testicular arteries (71.1 vs. 60.6%; P < 0.05) were observed in RS compared to LS. The lowest correlations between testicular hemodynamic, physiological variables, and environmental parameters (P < 0.05) were observed in the morning. In conclusion, testicular thermoregulation and testicular hemodynamics were influenced by the climatic seasons and time of the day, being more efficient in the LS season and with less interference from environmental factors in the morning.

Luteal function in cyclic goats treated with human chorionic gonadotropin administered by intramuscular or intravaginal routes at the time of artificial insemination.

Human chorionic gonadotropin (hCG) has been used to improve goats reproductive efficiency. This study aimed to (i) evaluate if hCG administered by the intramuscular (i.m.) or intravaginal (i.vag.) route can be detected by a rapid ß-hCG test in blood plasma samples and (ii) document ovarian effects of hCG administered by both routes at the time of artificial insemination (AI) performed 60 h after oestrus synchronization in goats. Twenty-two Alpine goats received two i.m. injections of 30 µg of d-cloprostenol (Prolise®, Tecnopec, São Paulo, Brazil) 7.5 days apart. One day after the onset of oestrus (at the time of AI), the goats were randomly allocated to one of the three groups that received: control (n = 7): 0.3 ml of saline solution intravaginally; hCGi.m. (n = 7): 300 IU of hCG (Vetecor®; Hertape-Calier, São Paulo, Brazil) i.m. and hCGi.vag. (n = 8): 300 IU of hCG deposited intravaginally. Blood samples were drawn at -1, 3, 6, 9 and 24 h after as well as on days 3, 7, 10, 13, 17 and 21 after hCG treatment/AI. All animals tested negative for hCG (ECO Diagnóstica, Corinto, Brazil) at -1 h, and all control animals tested negative throughout the entire blood collection period. All hCGi.m. animals tested positive from 3 h until D3 post-AI but only 50% of hCGi.vag. goats tested positive during the present study. In all animals studied, mean circulating P4 concentrations increased (p < .05) from D3 to D7 after AI and then declined (p < .05) from D10 to D17 in control and hCGi.m. groups and from D17 to D21 in the hCGi.vag. group. Total cross-sectional luteal area (CLA), mean colour Doppler area (DA), DA/CLA, mean high-velocity Doppler area and HVDA/CLA all declined (p < .05) by D17-D21 in all animals studied. In summary: (i) human chorionic gonadotropin could consistently be detected in blood samples using the rapid ß-hCG test only in the hCGi.m. group; and (ii) there were no significant differences in the mean pregnancy rate, circulating P4 concentrations and various luteal parameters studied among Control, hCGi.m. and hCGi.vag. dose.

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.

Human chorionic gonadotropin affects original (ovulatory) and induced (accessory) corpora lutea, progesterone concentrations, and pregnancy rates in anestrous dairy goats.

Two experiments were conducted in acyclic Alpine (A) and Saanen (S) goats that received intravaginal sponges containing 60 mg of medroxyprogesterone acetate for 6 days, as well as 200 IU of eCG and 30 µg d-cloprostenol i.m. 24 h before sponge removal. On day 7 (day 0 = onset of synchronized estrus), all goats were randomly divided into two groups: animals treated with 300 IU of hCG i.m. (hCG; Exp.1: n = 8A; Exp.2: n = 75A + S) and untreated controls (Control; Exp.1: n = 8A; Exp. 2: n = 70A + S). In Exp.2, all goats were artificially inseminated. Transrectal ovarian ultrasonography and blood collection were done on days 7, 10, 13, 17, and 21 (Exp.1), and pregnancy detection on day 60 (Exp.2). Estrus and ovulations occurred in five hCG and seven Control animals. Accessory CL (aCL) were detected in all hCG does. The total luteal area of ovulatory corpora lutea (oCL) increased (P < 0.05) on day 10 in hCG does and remained greater (P < 0.05) than in Control until day 21. Total and high-velocity color Doppler area were greater (P < 0.05) for oCL of hCG does on days 13 and 17. Progesterone concentrations were greater (P < 0.05) in hCG does from days 13 to 21 and related directly to the total luteal and oCL area for the duration of the study in all does. The pregnancy rate was higher (P < 0.05) in hCG than in Control by 22.5 %. Human chorionic gonadotropin given on day 7 of the synchronized estrous cycle positively affected CL function and pregnancy rates in seasonally anovular dairy goats.

Bicarbonate-Stimulated Membrane Reorganization in Stallion Spermatozoa.

Classical in vitro fertilization (IVF) is still poorly successful in horses. This lack of success is thought to be due primarily to inadequate capacitation of stallion spermatozoa under in vitro conditions. In species in which IVF is successful, bicarbonate, calcium, and albumin are considered the key components that enable a gradual reorganization of the sperm plasma membrane that allows the spermatozoa to undergo an acrosome reaction and fertilize the oocyte. The aim of this work was to comprehensively examine contributors to stallion sperm capacitation by investigating bicarbonate-induced membrane remodelling steps, and elucidating the contribution of cAMP signalling to these events. In the presence of capacitating media containing bicarbonate, a significant increase in plasma membrane fluidity was readily detected using merocyanine 540 staining in the majority of viable spermatozoa within 15 min of bicarbonate exposure. Specific inhibition of soluble adenylyl cyclase (sAC) in the presence of bicarbonate by LRE1 significantly reduced the number of viable sperm with high membrane fluidity. This suggests a vital role for sAC-mediated cAMP production in the regulation of membrane fluidity. Cryo-electron tomography of viable cells with high membrane fluidity revealed a range of membrane remodelling intermediates, including destabilized membranes and zones with close apposition of the plasma membrane and the outer acrosomal membrane. However, lipidomic analysis of equivalent viable spermatozoa with high membrane fluidity demonstrated that this phenomenon was neither accompanied by a gross change in the phospholipid composition of stallion sperm membranes nor detectable sterol efflux (p > 0.05). After an early increase in membrane fluidity, a significant and cAMP-dependent increase in viable sperm with phosphatidylserine (PS), but not phosphatidylethanolamine (PE) exposure was noted. While the events observed partly resemble findings from the in vitro capacitation of sperm from other mammalian species, the lack of cholesterol removal appears to be an equine-specific phenomenon. This research will assist in the development of a defined medium for the capacitation of stallion sperm and will facilitate progress toward a functional IVF protocol for horse gametes.

Nonlinear Models to Describe the Testicular Size Growth Curve of Mangalarga Marchador Stallions.

This study aimed to describe the testicular growth curve in Mangalarga Marchador stallions fitting nonlinear models to data of total scrotal width (TSW) and testicular volume (TV). A cross-sectional study was conducted with 120 stallions, aging from 1.75 to 21.93 years. The parameters of nonlinear models (Brody, Gompertz, Hill, Logistic I and II, Meloun I and II, Michaelis-Menten, Mitscherlich, and von-Bertalanffy) were estimated by Gauss-Newton iterative process. Goodness of fit was evaluated by the corrected Akaike (AICc) and Bayesian (BIC) information criteria, adjusted R2, error sum of square, mean absolute deviation and average prediction error. Based on the goodness of fit, the Logistic I model fitted better for TSW while Logistic II fitted better for TV growth curve. The inflection point (IP) of TSW growth was estimated in 5.23 cm at 0.76 years; the IP of TV growth was estimated in 139.36 cm3 at 2.57 years. The TV growth curve reached a plateau later than TSW growth curve, mainly because testicular height maintains growth for a longer period. In addition, there were moderate and positive correlations between whiter height and TSW and TV (r = 0.51 and r = 0.53, respectively). Findings suggest that TV is more associated with sexual maturity than sexual precocity. In addition, the moderate and positive correlations between whiter height and TSW and TV indicate that testicular traits can be included in selection programs of Mangalarga Marchador stallions with the same relevance as the morphometric traits.

Effect of protein supplement level on the productive and reproductive parameters of replacement heifers managed in intensive grazing systems.

Evaluations of replacement heifers in intensively managed grazing systems in tropical conditions are warranted. Thus, we aimed to evaluate performance, muscle and mammary gland development, oocyte quality, and in vitro production of embryos of crossbred heifers grazing an intensively managed pasture and supplemented with high or low protein concentrates. Eighteen pubertal crossbred heifers (Holstein x Gyr) with an initial weight of 350 ± 8.0 kg were used in a 60-day trial. Two supplement types, 12% crude protein (CP) (S12CP) or 24% CP (S24CP), and a control treatment (mineral mixture, CON) were randomly distributed to the heifers. Throughout the experiment, four digestibility trials were performed over four consecutive days. Four ovarium pick-ups were performed to evaluate oocyte quality and in vitro embryo production. Lastly, ultrasounds of carcasses and mammary glands were performed. The intakes of dry matter (DM), digestible energy (DE), and CP were greater for supplemented (SUP) compared with CON heifers. The SUP heifers had a greater average daily gain (ADG) (645 versus 390 g/d) and rib eye area (58.78 versus 53.32 cm2) than the CON heifers. Oocyte recovery, quality, and follicle features were not affected by supplementation strategy. However, the cleavage rate (47.17% versus 30.31%) and blastocyst rate (27.91% versus 10.12%) were negatively affected by supplementation. The S12CP presented a blastocyst rate much lower than the S24CP (3.02% versus 17.23%). Carcass ultrasonography indicated a trend for greater rib eye area for S24CP and mammary ultrasonography indicated no effects of supplementation on mammary gland development. In summary, supplementation seems to be an appropriate strategy for satisfactory performance, with greater muscle deposition and no negative impacts on mammary gland development. However, in vitro embryo production was impaired when the animals received the supplementation with 12% CP.

Expression of apoptosis and myogenesis related genes during prenatal life in two divergent breeds of pigs.

We aimed to evaluate apoptosis and myogenesis related genes expression in embryos and fetuses from two divergent genetic groups of pigs: Piau breed and a commercial line. Thirty females (15 Piau and 15 commercial line) were selected at 120 days of age. Estrous cycle was observed and on the third estrus females were considered sexually mature. Gilts were inseminated with semen from males of the respective breed. Three females from each breed were slaughtered at five different gestational ages: 15, 30, 45, 60 and 90 days. Whole embryos (15 and 30 d) and samples of longissimus dorsi muscle from fetuses (45, 60 and 90 d) were collected for RNA extraction. Expression of apoptosis and myogenesis related genes (BAX, BCL2, FGF4, IHH, HHIP, SHH, SOX2, WNT1 and WNT4) were evaluated by quantitative real time PCR. There was significant effect of interaction between breeds and gestational ages for all genes evaluated (P < 0.05). The BCL2 gene expression differed throughout pregnancy in Piau group with lower expression on day 15. The IHH gene expression throughout pregnancy was lower on days 15 and 60 in Piau group and lower on day 90 in commercial line group; and the SHH gene expression throughout pregnancy was higher on day 30 and lower on day 60 in Piau group and lower on day 45 in commercial group. The WNT1 gene expression along pregnancy was lower on day 15 and higher on days 30 and 45 in Piau group, and it was higher on day 30 than days 15 and 60 in commercial group. WNT4 gene expression throughout pregnancy was lower on day 15 in Piau group, and it was lower on day 30 in commercial pigs. Piau group presented higher expression of the FGF4 gene on days 45, 60 and 90, and commercial group showed higher expression on day 15 and 90. SOX2 gene expression was lower on day 15 in Piau pigs and it was constant throughout pregnancy in commercial group. Overall, the expression of IHH, SHH, WNT1, WNT4 and FGF4 genes were higher in commercial than Piau pigs on day 15, besides expression of BCL2 gene in Piau embryos was lower on day 15; these results might indicate that the muscle precursor cells are allowed to proliferate for a longer time in commercial than in Piau embryos by the balance of proliferative and apoptotic genes. Therefore, the expression differential between breeds can stimulate proliferation and differentiation of cells in different ways, explaining the postnatal differences in the muscularity between pigs from Piau breed and a commercial line.

Genetic parameters for fertility traits in Nellore bulls.

Nellore is the main cattle breed used in Brazil, being the largest commercial herd in the world. Beyond the importance of male reproductive efficiency for farm profit, the use of reproductive techniques, mainly artificial insemination, turns the evaluation of male reproductive traits even more important. Estimation of genetic parameters increases the knowledge on traits variances and allows envisaging the possibility of the inclusion of new traits as selection criterion. Genetic parameters for fifteen traits that can be classified as testicular biometry or physical and morphological semen traits were estimated for a Nellore bull population ranging from 18 to 36 months. Single-trait and bi-trait animal models were used for (co)variance components estimation. The contemporary group was considered as fixed effect and age at measurement as covariable. Scrotal circumference presented heritability of 0.47 ± 0.12. This value is similar to the heritabilities found for all testicular biometry traits (0.34-0.48). Sperm progressive motility, which has a direct effect on bull fertility, presented low heritability (0.07 ± 0.08). Major and total sperm defects presented moderate to high heritabilities (0.49 ± 0.18 and 0.39 ± 0.15, respectively), indicating that great genetic gain can be obtained through selection against sperm defects. High and positive genetic correlations were observed among testicular biometry traits, which also presented favourable genetic correlations with physical and morphological traits of the semen with magnitude ranging from high to low. Scrotal circumference presented moderate to high and favourable genetic correlations with sperm progressive motility, sperm turbulence, major sperm defects and total sperm defects. Thus, the selection for scrotal circumference results in favourable correlated genetic response for semen quality. The results show that the use of scrotal circumference as reference trait for bull fertility is appropriate, since it presents high heritability and favourable genetic correlation with semen quality.

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.].

Dietary L-arginine supplementation during early gestation of gilts affects conceptuses development.

l-arginine supplementation of sows has led to improvement of reproductive performance, but the mechanisms responsible for the positive effects of arginine during gestation on conceptuses survival and development are still poorly understood. Thus, we aimed to evaluate effects of 1.0% l-arginine supplementation (ARG) on phenotypic traits of commercial gilts, embryos and fetuses, concentration of gilts' blood metabolites, expression of developmental and cellular apoptosis genes in conceptuses of 25 and 35 days. At 25 days, IGF1 gene was more expressed in embryos from ARG than in embryos from control gilts (CON) (P = 0.05). At this same gestational age, ARG embryos tended to be heavier compared to CON (P = 0.07) and ARG gilts showed a trend to have a greater arginine concentration in blood plasma (P = 0.06). However, at 35 days of gestation, arginine concentration in blood plasma of ARG gilts tended to be lower compared to CON (P = 0.06) and ARG fetuses showed smaller cephalic-caudal length (P = 0.05). These results indicate that duration of supplementation is determinant for arginine effects, not only on the females performance but also on the conceptuses, since supplementation upregulated IGF1 expression at 25 days, in addition to the reduction of cephalic-caudal length of 35-day fetuses.

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.

Comparison of the intravenous and intravaginal route of oxytocin administration for cervical dilation protocol and non-surgical embryo recovery in oestrous-induced Santa Inês ewes.

This study compared the effects of intravaginal and intravenous routes of oxytocin (OT) administration in 46 oestrous-induced Santa Inês ewes (6-day treatment with progestin-releasing intravaginal sponges and a single injection of 200 IU of eCG at the time of sponge removal) that underwent transcervical embryo recovery 6-7 days after oestrous onset and mating. All ewes received 37.5 µg of d-cloprostenol via latero-vulvar route, and 1 mg of oestradiol benzoate i.m. 16 hr before and 50 IU of OT 20 min before non-surgical embryo recovery (NSER), with OT being administered intravenously (n = 21) or intravaginally (n = 21). An overall oestrous response was 95.6% (44/46), and adequate cervical retraction could be accomplished in 78.6% (33/42) of ewes. The percentage of successful NSER procedures was 57% (24/42) or 72.7% (24/33) of animals with sufficient cervical retraction. The duration of NSER procedure averaged 28 min (range: 17-40 min) and ~96% of flushing fluid could be recovered (range: 85%-100%). Out of 18 ewes that could not undergo NSER, 12 (66.6%) presented various anatomical barriers, whilst the other 33.4% did not present these barriers and still could not be traversed. Excluding the ewes with those anatomical features, the overall success rate of NSER was 80% (24/30). The route of OT administration had no effect on NSER efficiency or the ease with which transcervical embryo flushing was performed. Both routes of OT administration can be used for cervical dilation protocol. Discarding ewes with anatomical features precluding cervical penetration is highly recommended to increase the efficacy of NSER in sheep.