Genetic dissection of photoperiod response based on GWAS of pre-anthesis phase duration in spring barley

Heading time is a complex trait, and natural variation in photoperiod responses is a major factor controlling time to heading, adaptation and grain yield. In barley, previous heading time studies have been mainly conducted under field conditions to measure total days to heading. We followed a novel approach and studied the natural variation of time to heading in a world-wide spring barley collection (218 accessions), comprising of 95 photoperiod-sensitive (Ppd-H1) and 123 accessions with reduced photoperiod sensitivity (ppd-H1) to long-day (LD) through dissecting pre-anthesis development into four major stages and sub-phases. The study was conducted under greenhouse (GH) conditions (LD; 16/8 h; ∼20/∼16°C day/night). Genotyping was performed using a genome-wide high density 9K single nucleotide polymorphisms (SNPs) chip which assayed 7842 SNPs. We used the barley physical map to identify candidate genes underlying genome-wide association scans (GWAS). GWAS for pre-anthesis stages/sub-phases in each photoperiod group provided great power for partitioning genetic effects on floral initiation and heading time. In addition to major genes known to regulate heading time under field conditions, several novel QTL with medium to high effects, including new QTL having major effects on developmental stages/sub-phases were found to be associated in this study. For example, highly associated SNPs tagged the physical regions around HvCO1 (barley CONSTANS1) and BFL (BARLEY FLORICAULA/LEAFY) genes. Based upon our GWAS analysis, we propose a new genetic network model for each photoperiod group, which includes several newly identified genes, such as several HvCO-like genes, belonging to different heading time pathways in barley

A preliminary study of the effects of organic farming on oocyte quality in ewe lambs

This study tested whether feeding Rasa Aragonesa ewes certified organic feed, from 15 days before mating until lamb weaning, improved oocyte quality and in vitro maturation (IVM) and fertilization (IVF) performances of the offspring. In a second experiment, ovaries from ewe lambs that were bred on an organic farm and were of the same breed were compared with those from conventionally bred animals. The number (± standard error of the mean) of healthy oocytes per ewe lamb did not differ significantly between organic (12.2 ± 3.3) and conventionally (13.6 ± 4.0) fed ewes. Ovaries from ewe lambs born on an organic farm had significantly (P < 0.0001) more healthy oocytes per ewe lamb (39.6 ± 5.2) than did those born on a conventional farm (25.0 ± 4.2), and higher IVM (76.5% vs. 53.1%, P < 0.0001) and IVF (97.3 vs. 91%, P < 0.05) rates. In conclusion, this preliminary approach to the study of the effect of organic procedures on the sheep oocyte quality indicates that the total integration in the complete organic system improved the oocyte quality of ewe lambs, although organic feeding alone was insufficient to improve quality

Testicular ultrasound analysis as a predictive tool of ram sperm quality

esticular ultrasound is a non-invasive technique that could be very useful for predicting ram seminal quality. Recent software developments allow macroscopic and microscopic evaluation of testicular parenchyma. Thus, the aim of this study was to evaluate the testicular echotexture using ultrasound-video analysis and investigate its possible correlation with semen quality. Nine rams were evaluated for one year using a portable ultrasound scanner and the echotexture was analyzed with ECOTEXT® software. The number of black (Ec1), white (Ec2), and grey pixels (Ec3), tubular density (TD), lumen area (LA), and lumen diameter (LD) were analyzed. Semen was collected by an artificial vagina the same day and the sperm concentration, morphology, motility, viability, phosphatidylserine (PS) translocation, reactive-oxygen-species (ROS) levels, DNA damage and capacitation state were evaluated. Ec2 and Ec3 correlated positively with “bad quality” sperm parameters (the percentage of spermatozoa with high ROS levels, with PS translocation and proximal cytoplasmic droplets), and negatively with motility. In contrast, TD and LA showed a positive correlation with “good quality” parameters (motility or normal morphology) and a negative correlation with spermatozoa with high ROS levels, with DNA fragmentation, and proximal or distal cytoplasmic droplets. Thus, echotexture analysis by ultrasound-video analysis could be a valuable tool for assessing ram fertility

Short communication. In vitro embryo production can be modified by the previous ovarian response to a superovulatory treatment in sheep

Thirty-two ewes were used to study how the ovarian response to a superovulatory treatment determines quality of oocytes recovered from ovaries after embryo collection, and their developmental capacity after in vitro maturation (IVM) and fertilization (IVF). Ewes were superovulated, and seven days after oestrus, embryos were collected and ewes divided into three groups: (+ +), n=19, ewes responding to the treatment with embryos collected after flushing; (+ –), n=8, ewes responding, but only oocytes were found; and (– –), n=5, ewes not responding to the treatment and no embryos collected. Ovaries were recovered and oocytes collected from the three groups. A significant effect of the response to the treatment was observed for oocyte quality, so that (– –) ewes presented the higher number of oocytes per ewe (p&lt;0.001). Total number of oocytes selected for IVM and IVF was significantly higher in the same group, in comparison with (+ +) and (+ –) (p&lt;0.001). Group (+ –) ewes presented the lowest maturation (p&lt;0.001), fertilization (p&lt;0.05) and cleavage rates (p&lt;0.001). In conclusion, the ovarian response to a superovulatory treatment determines the number and quality of the oocytes recovered 7 days after the oestrus induced by the hormonal treatment. In vitro techniques could be an important tool to increase embryo production by particular ewes when they are not able to produce a significant amount of in vivo embryos

Semen quality of rasa aragonesa rams carrying the fecxr allele of the BMP15 gene

The FecXR mutation is a variant of the ovine gene BMP15 in the Rasa Aragonesa breed. Information on the physiological importance of carrying the FecX polymorphism in rams is limited. The aim of this study was to compare semen quality, testicle characteristics, and fertility rate of rams that carry the FecXR allele. Rams (n = 15) were either FecXR allele carriers (n = 10) or non-carriers, wild type (++) (n = 5). FecXR rams exhibited higher mass motility (p < 0.05), proportion of rapid sperm (p < 0.05), and a lower proportion of slow sperm (p < 0.0001) than did ++ rams. The presence of the FecXR allele was not associated with mean scrotal circumference or testicular length and diameter, although season had a significant (p < 0.05) effect on these traits. Genotype (p < 0.05) and season (p < 0.01) had a significant effect on mean fertility rate, FecXR rams had a higher proportion of pregnant ewes than did ++ rams (p < 0.05). In conclusion, the FecXR allele produced high-quality semen throughout the year, and corresponded with an improvement in some sperm parameters, particularly, mass motility and the proportion of rapid sperm

Sperm behavior and response to melatonin under capacitating conditions in three sheep breeds subject to the equatorial photoperiod

In this study, we demonstrated that, in seasonal Mediterranean ovine breeds, supplement-ing the TALP medium with cAMP-elevating agents (the cocktail medium) is effective for achieving ram sperm capacitation, and that melatonin is able to regulate this phenomenon. We investigated the behavior under capacitating conditions using the TALP and cocktail mediums, and the response to melatonin, of spermatozoa from three sheep breeds (Colombian Creole, Romney Marsh, and Hampshire) subject to the equatorial photoperiod, during the dry and the rainy seasons. The cocktail medium was able to induce sperm capacitation, assayed by chlortetracycline staining and phos-photyrosine levels, to a greater extent than TALP, without a higher loss of viability (membrane in-tegrity and viable spermatozoa without phosphatidylserine (PS) translocation). The addition of mel-atonin at 100 pM or 1 µM in the cocktail medium partially prevented the decrease in viability without PS translocation and the increase in capacitated spermatozoa from all breeds, with no significant effect on phosphotyrosine levels. Differences between breeds and seasons were evidenced. This study shows that melatonin is able to exert direct effects on spermatozoa in ovine breeds under equatorial photoperiod conditions, as it does in seasonal breeds located in temperate regions

Polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and sexual activity of adult rams in spring

The aim of this study was to determine whether polymorphisms of the melatonin receptor 1A (MTNR1A) gene influence the age at first mating in autumn-born ram-lambs and influence the out-of-season sexual activity of adult rams. In experiment 1, 24 Rasa Aragonesa ram-lambs born in September were genotyped for their RsaI and MnlI allelic variants of the MTNR1A gene, and the date of their first mounting with ejaculation after a period of semen collection training was documented. In experiment 2, the reproductive behavior, testicle size, and plasma testosterone concentrations of 18 adult rams (6 rams for each RsaI genotype) were recorded at the beginning (March) and end (May) of the seasonal anestrus. The number of days of training to achieve the first mating with ejaculation in T/T (C/C: 85.17 ± 12.08 C/T: 86.60 ± 18.87; T/T; 26.50 ± 24.50 d; P < 0.05), and G/G ram-lambs (G/G: 51.57 ± 14.99; A/G: 95.58 ± 10.95 d; P < 0.05) was significantly fewer than it was in the other genotypes. Likewise, for the RsaI genotype, 55% of the vulva-sniffing (P < 0.001), 48% of the approaches (P < 0.01), 48% of the mountings (P < 0.05) and 49% total activities (P < 0.001) were performed by T/T rams in March, and 50% of the sexual events in May (P < 0.001). For the Mnll variant, G/G rams performed a significantly (P < 0.001) larger proportion of the vulva-sniffing (41%), approaches (46%) and total activities (40%) in March, and 52% of the vulva-sniffing (P < 0.001), 43%, of the approaches (P < 0.001), 46% of the mountings (P < 0.05), and 47% of the total activities (P < 0.001) in May. Scrotal circumference, testicular volume, and plasma testosterone concentrations did not differ significantly among genotypes. Results confirmed that the polymorphisms of the MTNR1A gene sequence can influence reproductive performance in young and adult rams. Autumn-born ram-lambs that carried the T/T or G/G genotype had an advanced ability to reproduce, and T/T or G/G adult rams exhibited the most intense reproductive behavior. Genotyping might be a useful procedure for identifying the correct and rational use of rams in modern sheep farming

NADPH Oxidase 5 and Melatonin: Involvement in Ram Sperm Capacitation

Reactive oxygen species (ROS) play an essential role in mammalian sperm capacitation. NADPH oxidase 5 (NOX5) has been described as the main source of ROS production in some mammalian spermatozoa, such as human and equine. On the other hand, melatonin can decrease cellular ROS levels and regulates NOX activity in somatic cells. Therefore, the objectives of this work were (1) to identify NOX5 in ram spermatozoa and analyze its possible changes during in vitro capacitation and (2) to investigate the effect of melatonin on NOX5 expression and localization and on superoxide levels in capacitated ram spermatozoa. Protein bands associated with NOX5 were detected by Western blot analysis. Likewise, indirect immunofluorescence (IIF) revealed six different immunotypes for NOX5, which varied throughout in vitro capacitation. Superoxide (O2·–), evaluated by DHE/Yo-Pro-1, rose after in vitro capacitation and in the presence of the calcium ionophore A23187 but decreased in the presence of the NOX inhibitor GKT136901. GKT also reduced the percentage of capacitated and acrosome-reacted spermatozoa that had increased during incubation in capacitating conditions. The presence of melatonin at micromolar concentrations avoided the increment in O2·– and the changes in NOX5 immunotypes provoked by capacitation. In conclusion, NOX5 is present in ram spermatozoa and the changes in its distribution, associated with sperm capacitation, can be prevented by melatonin. To this extent, it could imply that melatonin exerts its antioxidant role, at least in part, by modulating NOX5 activity during ram sperm capacitation. © Copyright © 2021 Miguel-Jiménez, Pina-Beltrán, Gimeno-Martos, Carvajal-Serna, Casao and Pérez-Pe

Underlying molecular mechanism in the modulation of the ram sperm acrosome reaction by progesterone and 17ß-estradiol

Steroid hormones progesterone (P4) and 17ß-estradiol (E2) not only have important functions in regulation of reproductive processes in mammals but also have direct effects on spermatozoa. There can be induction of the acrosome reaction in ram spermatozoa by P4 and E2 and, in the present study, there was further investigation of mechanisms underlying this effect. In a medium containing agents that increase cAMP, the presence of both P4 and E2 led to changes in the localization of proteins phosphorylated in tyrosine residues evaluated by indirect immunofluorescence. The inclusion of P4 at 1 µM in the media induced an increase in Ca2+i and mobilization in the area of the acrosome (Fluo-4 and Rhod-5 staining, respectively), an increase in ROS (H2DCFDA staining) and a substantial disruption of the acrosome (evaluated using RCA), while E2 did not have these effects. There were no effects on cAMP concentrations or PKA activity with inclusion of these hormones in the media. The inclusion of P4 at 100 pM in the media led to changes in values for sperm kinematic variables which could indicate there was an inhibition of the hyperactivation caused by agents that induce an increase in cAMP concentrations. In conclusion, results from the present study indicate that P4 and E2 promote mechanisms regulating the acrosome reaction in ram spermatozoa, however, these effects on mechanisms are different for the two hormones, and for E2, require further clarification

Influence of Non-conventional Sperm Quality Parameters on Field Fertility in Ovine

The prediction of the fertilizing ability of a seminal dose continues to be a primary aim in the field of artificial insemination (AI). To achieve this goal, in this study we have included the evaluation of some non-conventional sperm quality markers. A total of 3, 906 ewes from 52 different farms were inseminated with 357 refrigerated seminal doses obtained from 45 mature Rasa Aragonesa rams. The same samples were used for sperm quality analysis including membrane integrity, capacitation status, oxygen consumption and apoptotic-like markers such as phosphatidylserine translocation (PS), plasmalemma disorganization/mitochondrial membrane potential, caspase activation and DNA damage. Seminal doses from the breeding (B) season presented higher percentages of intact membrane (IM), non permeant (NP) membrane with high mitochondrial membrane potential (¿¿m) and IM without PS translocation spermatozoa than those from the non-breeding (NB) season. Therefore, we can conclude that there were less spermatozoa showing apoptotic-like features in the seminal doses from the B than the NB season, although these differences did not affect field fertility. Only the percentage of intact membrane, non-capacitated (IM-NC) spermatozoa showed a significant correlation with in vivo fertility (P = 0.005) and fecundity (P = 0.007) values obtained after cervical AI when all data were evaluated. When the data were sorted by season and distance to the farms where AI was performed, the correlation between the percentage of IM-NC spermatozoa and reproductive parameters increased in the NB season and progressively with remoteness from the farms. Some other sperm parameters, like NP with high ¿¿m, IM sperm without active caspases and DNA-intact spermatozoa, also showed significant correlations with the reproductive parameters in the sorted data. Moreover, the increment in both the percentage of IM-NC and DNA-intact spermatozoa would increase the probability of obtaining a fertility higher than the mean (>52%), as revealed by a multiple logistic regression analysis. In conclusion, we have identified two seminal markers—the percentage of intact membrane, non-capacitated spermatozoa, and DNA intact spermatozoa—which could be used as a test to discard males in AI programs, which is highly important from an economic point of view and can contribute to achieving satisfactory fertility rates. © Copyright © 2021 Mendoza, Casao, Domingo, Quintín, Laviña, Fantova, Cebrián-Pérez, Muiño-Blanco and Pérez-Pe