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

La actividad sexual de corderos a los ocho meses de edad como predictor de su actividad sexual adulta

Predecir la actividad sexual de un morueco lo antes posible puede ser una herramienta de gran utilidad, especialmente en centros de selección y recogida de semen. El objetivo de este trabajo fue estudiar la concordancia de la actividad sexual —medida a través de test de comportamiento— de moruecos a los 8 y a los 32 meses de edad, para predecir el comportamiento sexual adulto a una edad temprana. Para ello, se sometió a sendos test de monta en el mes de junio (20 minutos de exposición a tres ovejas en celo) a 18 moruecos nacidos en octubre, a los 8 y 32 meses de edad. Se midieron el número de aproximaciones a las hembras y los intentos de monta con y sin eyaculado de cada animal. Cada macho fue clasificado en un ranking del 1 al 18 (1: mejor macho; 18: peor macho) tras calcularse su calificación (número total de eventos observados). Se aplicó un modelo de regresión lineal para predecir el ranking y la calificación adulta a partir de la juvenil. Ambos parámetros adultos se relacionaron en gran medida con los tempranos (r2 = 0, 594 y 0, 365, respectivamente; p < 0, 0001). La predicción fue especialmente buena para los peores machos, de modo que los ocho últimos corderos del ranking fueron igualmente los ocho últimos moruecos a los 32 meses. En conclusión, la actividad sexual adulta de un morueco podría predecirse con una alta determinación a partir de su medición a edades tempranas, siendo una herramienta especialmente útil para eliminar precozmente los peores machos. The possibility of predicting sexual activity of rams during their adult life at the earliest opportunity could be a very useful tool, especially in ram selection centers. With the aim of predicting serving capacity of adult rams by their performance at 8 months of age, 18 October-born rams were used. At 8 and 32 months of age (June) they were individually exposed for 20 min to three estrous ewes, and the number of approaches, attempted mountings, and mountings were recorded. Rams were ranked (1 to 18) after the calculation of their scores (total number of observed events). A regression model was applied to predict sexual rank at 32 months of age based on the 8-month ranking. Adult ram ranking and score were largely the result of the rank at 8 months of age (r(2) = 0.594 and 0.365, resp.; p < 0.0001). The prediction of the rank at 30 months of age was outstanding for the worst rams, so that the worst 8 rams ranked at 8 months were the same than at 32 months of age. Serving capacity tests carried out with ram-lambs at early ages may be a tool to estimate sexual activity of adult rams, especially to eliminate the worst ram-lambs before they reach adult life

The continuous presence of ewes in estrus in spring influences testicular volume, testicular echogenicity and testosterone concentration, but not LH pulsatility in rams

The continuous presence of active male small ruminants prevents seasonal anestrus in females, but evidence of the same mechanism operating from the females to the males is scarce. This study assessed the effects of the continuous presence of ewes in estrus in spring on ram sexual activity, testicular size and echogenicity, and LH and testosterone concentrations. On 1 March, 20 rams were assigned to two groups (n = 10 each): isolated (ISO) from other sheep, or stimulated (STI) by 12 ewes, which were separated from the rams by an openwork metal barrier, allowing contact between sexes. Each week, four ewes were induced into estrus by intravaginal sponges. Live weight, scrotal circumference, testicular width (TW) and length (TL) were recorded at the beginning and at the end of the experiment, and testicular volume (TV) was calculated; at the same time, testicular ultrasonography and color Doppler scanning were performed. Blood samples (March to May) were collected once per week for testosterone determinations, and at the end of the experiment, blood samples were collected for 6 h at 20-min intervals for LH analysis. Rams were exposed to four estrous ewes in a serving-capacity test. Scrotal circumference, TW and TL were higher in the STI than in the ISO rams (P < 0.05) in May, and TV was higher (P < 0.05) in the STI (391 ± 17 cm3) than in the ISO rams (354 ± 24 cm3). In ISO rams, the number of white pixels was higher (P < 0.01) in May (348 ± 74) than in March (94 ± 21) and differed significantly (P < 0.01) from that of the STI rams in May (160 ± 33). In ISO rams, the number of grey pixels was higher (P < 0.05) in May (107 ± 3) than it was in March (99 ± 1). Stimulated and ISO rams did not differ significantly in mean LH plasma concentrations (0.8 ± 0.5 v. 0.9 ± 0.4 ng/ml), LH pulses (2.1 ± 0.5 v. 2.2 ± 0.2) and amplitude (2.0 ± 0.4 v. 3.2 ± 0.7 ng/ml, respectively). Stimulated rams had significantly higher testosterone concentrations than ISO rams from April to the end of the experiment. Stimulated rams performed more (P < 0.05) mountings with intromission (3.0 ± 0.4) than did ISO rams (1.5 ± 0.5). In conclusion, after 3 months in the continuous presence of ewes in estrus in spring, rams had higher TV and some testicular echogenic parameters were modified than isolated rams. Although exposed rams also had higher levels of testosterone after 2 months in the presence of estrous ewes, their LH pulsatility at the end of the study was not modified

Effect of body condition score, treatment period and month of the previous lambing on the reproductive resumption of melatonin-treated sarda breed sheep during spring

Stakeholders place great emphasis upon rationalizing the management and rearing techniques which are utilized within sheep farms. The present study aimed to investigate factors which may improve the reproductive performance of melatonin-treated Sardinian sheep via a series of three trials. The first trial (n = 100) investigated the effect of melatonin treatment alongside body condition score (BCS), the second trial (n = 150) investigated the effect of treatment alongside the date of treatment (treatment period) and the third trial (n = 150) investigated the effect of treatment alongside the previous lambing of the ewes. The findings indicated that melatonin is an effective tool for anticipating and improving the reproductive activity of in Sarda breed sheep during the springtime. Furthermore, to obtain optional results, melatonin implantation should be conducted in April, in ewes that have a BCS of >2.5 and that have passed their third month of lactation. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

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

Changes in melatonin concentrations in seminal plasma are not correlated with testosterone or antioxidant enzyme activity when rams are located in areas with an equatorial photoperiod

In temperate climates, photoperiod and melatonin regulate ram reproduction, modulating hormonal secretions, sperm quality, and seminal plasma composition. Information on the effect of an equatorial photoperiod (12L:12D) on ram reproduction, however, is scarce, and no data on hormonal concentrations and antioxidant enzyme activity in seminal plasma have been reported. Thus, the variation was investigated of melatonin and its relationship with testosterone and antioxidant enzyme activity in the seminal plasma of three sheep breeds in Colombia, when there was a consistent photoperiod during two dry and two rainy seasons per year. Semen was collected once a week from 12 mature rams (four of each breed: Colombian Creole, Hampshire, and Romney Marsh). Seminal plasma was obtained by centrifugation. The concentration of melatonin and testosterone were quantified along with the enzymatic activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase (CAT). Correlation analyses between melatonin and testosterone concentrations or enzymatic activity were also performed. Melatonin concentration was affected by season (P < 0.05) but not breed, with lesser concentrations in the first rainy season. Testosterone concentration, however, was affected by breed and season, with greater concentrations (P < 0.01) in the Hampshire and Romney Marsh rams during the second dry season. Regarding antioxidant enzyme activity, there was only seasonal variation in GPx activity (P < 0.05). When correlation analyses were used for data assessments, there was a negative correlation between melatonin and testosterone concentrations in Hampshire rams. In conclusion, melatonin concentrations in seminal plasma of rams that were located in an area with an equatorial photoperiod was affected by the climatological season but there was no positive correlation with testosterone concentration or antioxidant enzyme activity

Vasectomy and photoperiodic regimen modify the protein profile, hormonal content and antioxidant enzymes activity of ram seminal plasma

This work aimed to determine the contribution of the testis and epididymis and the effect of the photoperiodic regimen on ram seminal plasma (SP). Semen was collected from 15 mature rams located in an equatorial (Colombian Creole and Romney Marsh, eight intact and two vasectomized) or a temperate climate (Rasa Aragonesa, three intact and two vasectomized). SP proteins were analyzed by Bradford, SDS-PAGE and difference gel electrophoresis (DIGE). Melatonin and testosterone concentrations were quantified by ELISA, and activity of glutathione peroxidase (GPx), glutathione reductase (GRD), and catalase by enzymatic assays. Vasectomy increased protein concentration and the intensity of high molecular weight bands (p < 0.001), with no differences between breeds. DIGE revealed the absence of six proteins in vasectomized rams: angiotensin-converting enzyme, lactotransferrin, phosphoglycerate kinase, sorbitol dehydrogenase, epididymal secretory glutathione peroxidase and epididymal secretory protein E1. Vasectomy also decreased melatonin concentrations in seasonal rams, and testosterone in all of them (p < 0.001), but did not affect antioxidant enzyme activity. Equatorial rams showed lower melatonin and testosterone concentration (p < 0.01) and catalase, but higher GPx activity (p < 0.05). In conclusion, vasectomy modifies the protein profile and hormonal content of ram seminal plasma, whereas the exposure to a constant photoperiod affects hormonal concentration and antioxidant enzymes activity

Improvement of the Seminal Characteristics in Rams Using Agri-Food By-Products Rich in Phytomelatonin

The aim of this study was to evaluate the effect of a phytomelatonin-rich diet, including by-products from the food industry, on ram sperm quality and seminal plasma composition. Melatonin content in several by-products before and after in vitro ruminal and abomasal digestion was determined by HPLC-ESI-MS/MS. Finally, 20% of a mix of grape pulp with pomegranate and tomato pomaces was included in the rams’ diet, constituting the phytomelatonin-rich diet. Feeding the rams with this diet resulted in an increase in seminal plasma melatonin levels compared with the control group (commercial diet) in the third month of the study. In addition, percentages higher than those in the control group of morphologically normal viable spermatozoa with a low content of reactive oxygen species were observed from the second month onwards. However, the antioxidant effect does not seem to be exerted through the modulation of the antioxidant enzymes since the analysis of the activities of catalase, glutathione reductase and glutathione peroxidase in seminal plasma revealed no significant differences between the two experimental groups. In conclusion, this study reveals, for the first time, that a phytomelatonin-rich diet can improve seminal characteristics in ram

Melatonin MT1 and MT2 receptors in the ram reproductive tract

Some melatonin functions in mammals are exerted through MT1 and MT2 receptors. However, there are no reports of their presence in the reproductive tract of the ram, a seasonal species. Thus, we have investigated their existence in the ram testis, epididymis, accessory glands and ductus deferens. Real-time polymerase chain reaction (qPCR) revealed higher levels of m-RNA for both receptors in the testis, ampulla, seminal vesicles, and vas deferens, than in the other organs of the reproductive tract (p < 0.05). Western blot analyses showed protein bands compatible with the MT1 in the testis and cauda epididymis, and for the MT2 in the cauda epididymis and deferent duct. Immunohistochemistry analyses revealed the presence of MT1 receptors in spermatogonias, spermatocytes, and spermatids, and MT2 receptors in the newly-formed spermatozoa in the testis, whereas both receptors were located in the epithelial cells of the ampulla, seminal vesicles, and ductus deferens. Indirect immunofluorescence showed significant differences in the immunolocation of both receptors in spermatozoa during their transit in the epididymis. In conclusion, it was demonstrated that melatonin receptors are present in the ram reproductive tract. These results open the way for new studies on the molecular mechanism of melatonin and the biological significance of its receptors

One-step in vitro generation of ETV2-null pig embryos

Each year, tens of thousands of people worldwide die of end-stage organ failure due to the limited availability of organs for use in transplantation. To meet this clinical demand, one of the last frontiers of regenerative medicine is the generation of humanized organs in pigs from pluripotent stem cells (PSCs) via blastocyst complementation. For this, organ-disabled pig models are needed. As endothelial cells (ECs) play a critical role in xenotransplantation rejection in every organ, we aimed to produce hematoendothelial-disabled pig embryos targeting the master transcription factor ETV2 via CRISPR-Cas9-mediated genome modification. In this study, we designed five different guide RNAs (gRNAs) against the DNA-binding domain of the porcine ETV2 gene, which were tested on porcine fibroblasts in vitro. Four out of five guides showed cleavage capacity and, subsequently, these four guides were microinjected individually as ribonucleoprotein complexes (RNPs) into one-cell-stage porcine embryos. Next, we combined the two gRNAs that showed the highest targeting efficiency and microinjected them at higher concentrations. Under these conditions, we significantly improved the rate of biallelic mutation. Hence, here, we describe an efficient one-step method for the generation of hematoendothelial-disabled pig embryos via CRISPR-Cas9 microinjection in zygotes. This model could be used in experimentation related to the in vivo generation of humanized organs