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Pollution due to microplastics and nanoplastics is one of the major environmental issues of the last decade and represents a growing threat to human and animal health. In aquatic species, there is a large amount of information regarding the perturbation of marine organisms; instead, there are only a few studies focusing on the pathophysiological consequences of an acute and chronic exposure to micro- and nanoplastics in mammalian systems, especially on the reproductive system. There are several studies that have described the damage caused by plastic particles, including oxidative stress, apoptosis, inflammatory response, dysregulation of the endocrine system and accumulation in various organs. In addition to this, microplastics have recently been found to influence the evolution of microbial communities and increase the gene exchange, including antibiotic and metal resistance genes. Special attention must be paid to farm animals, because they produce food such as milk, eggs and meat, with the consequent risk of biological amplification along the food chain. The results of several studies indicate that there is an accumulation of microplastics and nanoplastics in human and animal tissues, with several negative effects, but all the effects in the body have not been ascertained, especially considering the long-term consequences. This review provides an overview of the possible adverse effects of the exposure of livestock to micro- and nanoplastics and assesses the potential risks for the disruption of reproductive physiological functions.

The objectives of the study aimed to evaluate the effect of weather conditions and scrotal circumference (SC) on standard semen characteristics of Italian Simmental young bulls (n = 577), all raised in the same performance station and sampled by the artificial vagina (AV) method. Considering the increasing SC, the results showed a significant increase in quantitative semen parameters (p < 0.05 and p < 0.0001, for sperm concentration and total number of spermatozoa, respectively); for every extra centimeter of SC, 17.5 × 106 spermatozoa/mL and 0.102 × 109 of total spermatozoa were produced. The age of the animal at semen collection (395 and 465 days) had similar positive effects. The effect of the average temperature humidity index (THI limits ≤ 40 and ≥70) in the previous 60, 30, and 10 days before the semen collection was also considered. Sixty days before the semen collection, the increasing THI increased both primary (p < 0.0001) and secondary (p < 0.0001) abnormalities while the percentage of morphologically normal spermatozoa decreased (p < 0.0001). Thirty and ten days before collection, the same effect on morphological traits was maintained, but total and progressive motility was also influenced (p < 0.01) with an unexpected increasing pattern. Thus, environmental conditions can influence semen quality during the entire spermatogenesis and results can guide future research on this breed.

A study was performed in Umbria, central Italy, to find out whether different culling strategies adopted by farms to control Johne’s disease (JD) infection exerted effects on the seroprevalence in dairy cattle. Fifty Fresian dairy herds in the Perugia and Assisi districts were visited and an audit of herd management was conducted. Among the 50 herds, 20 were selected for the consistency of management practices and, according to the culling strategy, two groups were created: group A (aggressive culling protocol, with average herd productive life <1100 days) and group B (lower culling rate, with productive life greater than 1500 days). The presence of antibodies to Mycobacterium avium subspecies paratuberculosis (Map) in the serum was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit. It was found that 3.3% (n = 14) of the cows of group B (n = 422, from 17 herds) were positive for Map antibodies, in comparison with 5.7% (n = 21) of the cows from group A (n = 366, from three herds). The odds ratio from multiple logistic regression (adjusted odds ratio 2.446, 95% confidence interval 0.412 to 14.525) showed that Johne’s disease prevalence in herds with a greater productive life was not higher than in herds with typical modern management characterized by more aggressive culling. This is a significant finding, indicating that aggressive culling may not be necessary. Current JD control recommendations are derived from data obtained in high-prevalence paratuberculosis areas (northern Europe, including northern Italy), while methods of information transfer to dairy farms in low-prevalence areas should be reassessed to ensure that the correct measures, including basic calving management and calf-rearing practices, are thoroughly implemented. Using the manufacturer’s suggested cut-off for a positive ELISA test and the sensitivity and specificity claimed, the overall true prevalence in Umbria dairy cattle was calculated as 7% (95% confidence interval 5.2% to 8.8%).

Cattle farming is facing an increase in number of animals that farmers must care for, together with decreasing time for observation of the single animal. Remote monitoring systems are needed in order to optimize workload and animal welfare. Where the presence of personnel is constant, for example in dairy farms with great number of lactating cows or with three milking/day, calving monitoring systems which send alerts during the prodromal stage of labor (stage I) could be beneficial. On the contrary, where the presence of farm personnel is not guaranteed, for example in smaller farms, systems which alert at the beginning of labor (stage II) could be preferred. In this case, time spent observing periparturient animals is reduced. The reliability of each calving alarm should also be considered: automatic sensors for body temperature and activity are characterized by a time interval of 6-12 h between the alarm and calving. Promising results have been shown by devices which could be placed within the vaginal canal, thus identifying the beginning of fetal expulsion and optimizing the timing of calving assistance. However, some cases of non-optimal local tolerability and cow welfare issues are reported. Future research should be aimed to improve Sensitivity (Se), Specificity (Sp) and Positive Predictive Value (PPV) of calving alert devices in order to decrease the number of false positive alarms and focusing on easy-to-apply, re-usable and well tolerated products.

One critical point of dairy farm management is calving and neonatal first care. Timely calving assistance is associated with the reduction of calf mortality and postpartum uterine disease, and with improved fertility in dairy cattle. This study aimed to evaluate the performance and profitability of an intravaginal sensor for the prediction of stage II of labor in dairy farms, thus allowing proper calving assistance. Seventy-three late-gestating Italian Holstein cows were submitted to the insertion of an intravaginal device, equipped with light and temperature sensors, connected with a Central Unit for the commutation of a radio-signal into a cell phone alert. The remote calving alarm correctly identified the beginning of the expulsive phase of labor in 86.3% of the monitored cows. The mean interval from alarm to complete expulsion of the fetus was 71.56 ± 52.98 min, with a greater range in cows with dystocia ( p = 0.012). The sensor worked correctly in both cold and warm weather conditions, and during day- or night-time. The intravaginal probe was well tolerated, as any cow showed lesions to the vaginal mucosa after calving. Using sex-sorted semen in heifers and beef bull semen in cows at their last lactation, the economic estimation performed through PrecisionTree™ software led to an income improvement of 119 € and 123 €/monitored delivery in primiparous and pluriparous cows, respectively. Remote calving alarm devices are key components of "precision farming" management and proven to improve animal welfare, to reduce calf losses and to increase farm incomes.

The use of assisted reproductive technologies (ART) still requires strategies through which to maximize individual fertility chances. In vitro folliculogenesis ( iv F) may represent a valid option to convey the large source of immature oocytes in ART. Several efforts have been made to set up iv F cultural protocols in medium-sized mammals, starting with the identification of the most suitable gonadotropic stimulus. In this study, Equine Chorionic Gonadotropin (eCG) is proposed as an alternative to Follicle Stimulating Hormone (FSH) based on its long superovulation use, trans-species validation, long half-life, and low costs. The use of 3D iv F on single-ovine preantral (PA) follicles allowed us to compare the hormonal effects and to validate their influence under two different cultural conditions. The use of eCG helped to stimulate the in vitro growth of ovine PA follicles by maximizing its influence under FBS-free medium. Higher performance of follicular growth, antrum formation, steroidogenic activity and gap junction marker expression were recorded. In addition, eCG, promoted a positive effect on the germinal compartment, leading to a higher incidence of meiotic competent oocytes. These findings should help to widen the use of eCG to iv F as a valid and largely available hormonal support enabling a synchronized in vitro follicle and oocyte development.

In vitro folliculogenesis ( iv F) has been proposed as an emerging technology to support follicle growth and oocyte development. It holds a great deal of attraction from preserving human fertility to improving animal reproductive biotechnology. Despite the mice model, where live offspring have been achieved,in medium-sized mammals, iv F has not been validated yet. Thus, the employment of a network theory approach has been proposed for interpreting the large amount of iv F information collected to date in different mammalian models in order to identify the controllers of the in vitro system. The WoS-derived data generated a scale-free network, easily navigable including 641 nodes and 2089 links. A limited number of controllers (7.2%) are responsible for network robustness by preserving it against random damage. The network nodes were stratified in a coherent biological manner on three layers: the input was composed of systemic hormones and somatic-oocyte paracrine factors; the intermediate one recognized mainly key signaling molecules such as PI3K, KL, JAK-STAT, SMAD4, and cAMP; and the output layer molecules were related to functional iv F endpoints such as the FSH receptor and steroidogenesis. Notably, the phenotypes of knock-out mice previously developed for hub.BN indirectly corroborate their biological relevance in early folliculogenesis. Finally, taking advantage of the STRING analysis approach, further controllers belonging to the metabolic axis backbone were identified, such as mTOR/FOXO, FOXO3/SIRT1, and VEGF, which have been poorly considered in iv F to date. Overall, this in silico study identifies new metabolic sensor molecules controlling iv F serving as a basis for designing innovative diagnostic and treatment methods to preserve female fertility., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bernabò, Di Berardino, Capacchietti, Peserico, Buoncuore, Tosi, Crociati, Monaci and Barboni.)

Endogenous retroviruses (ERVs) are proviral phases of exogenous retroviruses, which have coevolved with vertebrate genomes for millions of years. The conservation of ERV genes throughout evolution suggests their beneficial effects on their hosts' survival. An example of such positive selection is demonstrated by the syncytin gene, which encodes a protein with affinity for various mammalian placentas that is involved in the formation of syncytiotrophoblasts. Although the horse has an epitheliochorial placenta, in which the fetal trophoblasts are simply apposed to the intact uterine epithelium, we have previously demonstrated that the equine ERV (EqERV) env RNA is unexpectedly expressed in placental tissue. In the present study, we investigated the mRNA expression pattern of the EqERV env gene in different parts of the equine placenta, to gain more insight into its putative role in the fetal-maternal relationship. To this end, we used reverse transcription-quantitative PCR (RT-qPCR) and in situ hybridization assays to analyze different target areas of the equine placenta. The retroviral env gene is expressed in the equine placenta, even though there is no syncytium or erosion of the uterine endometrium. The gene is also expressed in all the sampled areas, although with some quantitative differences. We suggest that these differences are attributable to variations in the density, height, and degree of morphological complexity of the chorionic villi forming the microcotyledons. The involvement of the EqERV env gene in different functional pathways affecting the fetus-mother relationship can be hypothesized., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Stefanetti, Pascucci, Wilsher, Cappelli, Capomaccio, Reale, Passamonti, Coletti, Crociati, Monaci and Marenzoni.)

Buffalo breeding is common in Southern Italy. Dystocia compromises dam's and newborn health and welfare. Difficult parturition could be solved through prompt calving assistance, even if the identification of the beginning of delivery is challenging. Herein, we aimed to evaluate a remote calving alarm system in 15 Mediterranean buffalo heifers. An intravaginal probe was placed close to the external cervical os once premonitory signs of delivery were observed. No vaginal discharge nor signs of discomfort were notified in the days following the insertion of the probe. Heifers calved from 48 to 72 hr after the alarm was activated. The system correctly warned the farm personnel at the beginning of stage II of parturition, except for 2 cases. In the former, the intravaginal probe was expelled but the poor carrier network coverage negatively affected phone's signal quality; in the latter, recurrent vaginal prolapse was responsible for non-retention of the probe. Overall median expulsive phase was 68 ± 8 min, while the expulsion of a female calf took 54 ± 22.0 min and 90 ± 34.0 min in males, with significant difference (p =.02). Deliveries were homogeneously distributed across a 24-hr interval. No retention of foetal membranes nor metritis was identified at postpartum clinical examination. The calving alarm system used in this work was well tolerated in buffalo heifers. The introduction of smart technology in buffalo farming could contribute to the overall farm net return by reducing calf losses, especially for calves born from sexed-sorted semen, and by increasing animal welfare through quick resolution of dystocia. Further studies will be necessary to evaluate the net return in buffalo farms which will implement a remote calving alarm system on a wider population., (© 2020 Wiley-VCH GmbH.)

The aim of the study was to evaluate the net return of the implementation of a remote calving monitoring system for obstetrical and neonatal assistance on the herd economy in a dairy farm model. A total of 680 parturitions over a 7-yr period were evaluated. Age at first calving was restricted from 23 to 27 mo for primiparous cows to be included. Among groups of cows that were ready to calve in a 15-d interval, primiparous and multiparous were randomly assigned to the experimental group and monitored through a calving alarm system, whereas the others accounted for controls. Final parturition groups were as follows: control primiparous (CPP, n = 218), control multiparous (CM, n = 345), monitored primiparous (MPP, n = 56), and monitored multiparous (MM, n = 61). Monitored groups received prompt calving assistance and first neonatal care, whereas the presence of farm personnel was discontinuous for controls. A biological model was built considering significant differences in calf loss, early culling, milk production, and days open between groups. Then, a partial budget model was used to estimate costs and net return on a simulated herd of 100 lactating cows. Incidence of calf death was greater in control groups (11.06% and 10.73% in CPP and CM, respectively) compared with monitored cows (0.00% and 1.69% in MPP and MM, respectively). Multiparous cows with calf loss had increased relative risk (relative risk = 3.487) for early culling compared with multiparous counterparts with no neonatal loss. Daily milk production in the first 2 mo was 3.79 kg greater in multiparous cows with no dead calf, compared with their counterparts. A significant difference in median days open was found in MPP and CPP (118 and 148 d, respectively). In the final economic model, different simulations were analyzed. They were created assuming different prices or hypothesizing calving monitoring only in primiparous animals. The model estimated different, but always positive, net return. In conclusion, implementing a calving alarm system led to a net return from €37 to 90 per cow per year (€1 = US$1.15 at the time of the study). However, the device alone is not sufficient: it must be supported by qualified calving monitoring and assistance. Optimized personnel presence in the calving area at the right time leads to prompt calving and neonatal calf assistance and colostrum feeding within the first hours of life, thus reducing calf death and days open, and increasing milk production., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

During the transition period, high-yielding dairy cows suffer from negative energy balance, intense lipomobilization and impaired lipid metabolism; this metabolic condition can lead to overburdened triglycerides accumulation in the liver, known as liver lipidosis, which has been associated to impaired fertility in dairy cows. The mechanisms of this impairment can be in principle correlated with the presence and the extent of ectopic fat depots. However, current methods for evaluating fat accumulation in liver and in the reproductive tract suffer from low resolution, sensitivity, and specificity. Confocal microscopes are equipped with Gallium arsenide phosphide detectors, thus enabling the acquisition of intense signals from tissue biopsies. This method could differentiate whether fat deposition occurred without requiring sample sectioning. Here, we examined with this technique liver, uterine and ovarian samples of heifers and regularly slaughtered repeat breeder and overconditioned dairy cows, to quantify lipid droplets and depots at a submicrometer scale with high specificity. With the aid of this technique, we found lipid depots in uterine and ovarian specimens. Moreover, we found that the size and number of depots increased with the degree of liver lipidosis. Further studies are needed to elucidate the relationship between the severity and extent of these deposits and the fertility of lactating dairy cows. Since tissues other than liver display different characteristic lipid droplet distributions, this technique can be potentially employed to shed new light on the pathogenesis of lipidosis and to assess new risk factors for infertility., (Copyright © 2018 Elsevier Inc. All rights reserved.)

High-yielding dairy cows are metabolically challenged during transition, when intense mobilization and hepatic oxidation of lipids is achieved, thus leading to fatty infiltration, ketosis and generalized inflammation. The condition is associated to periparturient diseases and poor fertility. The aim of this study was to assess whether serum lipoprotein concentrations in the dry period could influence the occurrence of postpartum diseases and reproductive performance in dairy cows. The study was carried out on 30 multiparous Holstein Friesian cows. Blood samples were collected at dry-off (-60 days), 30 days after dry-off and within 12 h after parturition for biochemical and serum lipoprotein assays. From 10 to 60 days after parturition milk was collected twice weekly after feeding, for milk whey progesterone assay. The Optimal Cutpoint package identified a threshold of 89% for serum High Density Lipoprotein (HDL) concentration at the beginning of the dry period with 95% of confidence interval. Cows with serum HDL greater than 89% (High group, n = 10) showed better reproductive performance when compared to those with low values (Low group, n = 18). The odds ratio for reproductive disorders in High group was 0.6875, however, differences were not significant probably due to both the reduced number of animals per group and overall low incidence of postpartum reproductive disease. First postpartum luteal activity occurred around day 23, while the second one between days 40 and 48. The average calving to first AI interval was 64.00 ± 3.95 days and 94.50 ± 12.32 days in High and Low group, respectively (P < 0.05). The calving-conception interval was 129.86 ± 24.42 days and 199.18 ± 24.73 days in High and Low groups, respectively (P < 0.05). Low group displayed an increase in liver markers, that is total bilirubin, with 0.46 ± 0.09 mg/dL and 0.23 ± 0.08 mg/dL, in Low and High group respectively (P < 0.05), and NEFA/cholesterol ratio, with 0.30 ± 0.06 and 0.14 ± 0.03, in Low and High groups, respectively (P < 0.05), at parturition. Concentrations of HDL >89% at dry-off could be suggestive of improved liver adaptation to the transition, and probably of enhanced fertility in High group., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Traditional superovulation protocols that include multiple gonadotropin treatments are time-consuming and labor intensive. These protocols require multiple handling and restraining of embryo donors. This will likely increase the risks of injuries in both animals and humans and induce stress that may lead to a reduced superovulatory response. These are more evident when working with cattle that are rarely handled or raised on extensive grazing. The objectives of this experiment were to compare the efficacy of a split-injection protocol of porcine pituitary-derived porcine FSH (pFSH) preparation (slow release [SR] group) to the traditional 4-day treatment with pFSH administered twice daily (C group) and to determine the concentrations of cortisol in the hair as a marker of activation of the hypothalamic-pituitary-adrenal (HPA) axis during the two superovulatory treatments. Thirty-two heifers were stimulated twice in a 2 × 2 crossover design and compared for ovarian response and numbers and characteristics of recovered ova-embryo among treatments. No differences between SR and C groups were found in terms of percentage of responsive animals (100% vs. 93.8%) and ovulation rate (83.7 ± 1.1 vs. 79.5 ± 1.0%). A positive correlation was found between the number of follicles responsive to pFSH (2-8 mm) at the beginning of treatments and the superovulatory response, and no differences were found in these follicular populations between the two treatment groups. The numbers of CLs, ova-embryos, fertilized ova, transferable and freezable embryos recovered per cow were found to be significantly higher in SR compared with C group (14.0 ± 1.6 vs. 10.6 ± 1.0, 12.1 ± 1.6 vs. 7.6 ± 1.0, 11.1 ± 1.1 vs. 7.3 ± 1.0, 9.6 ± 1.4 vs. 6.6 ± 1.0, and 9.4 ± 1.4 vs. 6.0 ± 1.0 for SR and C group, respectively). The SR group produced also a significantly greater number of excellent- and/or good-quality embryos compared with the C group. The concentrations of cortisol in the hair at Days 14 and 21 were significantly greater in the C compared with the SR group (P < 0.05). In conclusion, these results indicate that the dilution of gonadotropin in a 0.5% hyaluronan solution, reducing the administration frequency, improves the quantitative and qualitative superovulatory response of Marchigiana heifers [corrected]. Further studies using other breeds of cattle are needed to verify the results herein obtained and to confirm the lower activation of the hypothalamic-pituitary-adrenal axis caused in the donor by the split-injection protocol., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although semen quality is one of the major traits that influence breeding soundness examination outcomes in bulls, field conditions occasionally do not allow for the collection of semen samples by means of an artificial vagina. The aims of the present study were to report the results of a large number of semen collections that were performed via the transrectal massage (TRM) of the accessory sexual glands of Italian yearling beef bulls and compare this semen collection method to the artificial vagina (AV) method in term of breeding soundness examination outcomes; furthermore, we determined whether the breed affected the semen characteristics. In the TRM group (n = 475), the semen samples were collected via TRM of the accessory sexual glands, and in the AV group (n = 502), the AV method was used. In the TRM group, semen samples were obtained from 81.3% of the bulls and penile protrusion was observed in 87.6% of the animals during semen collection. The sperm concentrations (920.5 ± 439.0 vs. 281.0 ± 259.8 × 10(6)/mL) and the percentages of total abnormal spermatozoa (22.8 ± 15.0 vs. 18.8 ± 12.9) were significantly higher in the AV group than those in the TRM group. The percentage of bulls that did not meet the minimum requirement for normal cells (≥70%) was 6.2% higher in the AV group than that in the TRM group (P < 0.05). Moreover, the samples collected from Chianina bulls by TRM exhibited a lower percentage of motile sperm and a higher percentage of abnormal spermatozoa when compared with the other two breeds. The major drawbacks of the TRM technique were the inability to conduct complete evaluation of the libido and mating ability of the yearling bulls, a significant reduction of the number of spermatozoa collected, and an increase in the variability of the semen characteristics due to breed. In conclusion, despite the drawbacks, TRM guarantees that semen evaluation can be conducted in cases in which the semen samples cannot be collected with the AV method., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Proper calving management of dairy herds is a crucial aspect of the bovine life cycle, as it has profound effects on calf viability and on the post-partum course of the dam. The objectives of this study were to monitor the calving process through the use of a remote alarm system and to determine the impact of prompt emergency obstetric procedures in case of dystocia for the prevention of stillbirths and post-partum reproductive pathologies, and for improving herd fertility. Six groups of experimental animals were studied: monitored heifers (n = 60) and multiparous cows (n = 60) were compared with non-observed animals (n = 60 heifers and n = 60 multiparous) giving birth during the same time period and housed in the calving barn, and with unmonitored animals placed in a dry zone (n = 240 heifers and n = 112 multiparous cows)., Results: The incidence of dystocia ranged from a minimum of 23.4% (monitored multiparous cows) to a maximum of 33.3% (monitored heifers), and there were no differences compared with control groups. However, the rate of stillbirth was higher in control groups than in the monitored groups (P < 0.01). Among both heifers and multiparous cows, the incidence of post-partum uterine infections was higher in the unmonitored animals both in the calving barn (P < 0.01) and in the dry zone (P < 0.05) compared with monitored animals. Among both heifers and multiparous cows, the control groups showed higher rates of foetal membrane retention than did the monitored groups (P < 0.001). The calving-to-conception interval was shorter; in particular, observed heifers showed a significant advantage of approximately 46 days compared with the unmonitored group (P < 0.001) and 32 days compared with the group in the calving barn (P < 0.05). Multiparous cows also had a reduction in the number of days open., Conclusions: The remote alarm system used to monitor the calving process assured the prompt presence of personnel, improving both the cow's reproductive efficiency and neonatal viability.

The zona pellucida (ZP) is an extracellular membrane surrounding mammalian oocytes. The so-called zona hardening plays a key role in fertilization process, as it blocks polyspermy, which may also be caused by an increase in the mechanical stiffness of the ZP membrane. However, structural reorganization mechanisms leading to ZP's biomechanical hardening are not fully understood yet. Furthermore, a correct estimate of the elastic properties of the ZP is still lacking. Therefore, the aim of the present study was to investigate the biomechanical behaviour of ZP membranes extracted from mature and fertilized bovine oocytes to better understand the mechanisms involved in the structural reorganization of the ZP that may lead to the biomechanical hardening of the ZP. For that purpose, a hybrid procedure is developed by combining atomic force microscopy nanoindentation measurements, nonlinear finite element analysis and nonlinear optimization. The proposed approach allows us to determine the biomechanical properties of the ZP more realistically than the classical analysis based on Hertz's contact theory, as it accounts for the nonlinearity of finite indentation process, hyperelastic behaviour and material heterogeneity. Experimental results show the presence of significant biomechanical hardening induced by the fertilization process. By comparing various hyperelastic constitutive models, it is found that the Arruda-Boyce eight-chain model best describes the biomechanical response of the ZP. Fertilization leads to an increase in the degree of heterogeneity of membrane elastic properties. The Young modulus changes sharply within a superficial layer whose thickness is related to the characteristic distance between cross-links in the ZP filamentous network. These findings support the hypothesis that biomechanical hardening of bovine ZP is caused by an increase in the number of inter-filaments cross-links whose density should be higher in the ZP inner side.

Polyspermy is a common problem in bovine in vitro fertilization (IVF) and has a still unclear etiology. In this specie, after IVF, despite the lack of a biochemical post-fertilization hardening, the stiffness of the outer ZP layer is significantly increased. Therefore, polyspermy might be related to an incomplete or insufficient stiffening of the ZP. We obtained, by using atomic force spectroscopy in physiological conditions, a complete characterization of the biomechanical changes of the inner and outer ZP layers occurring during oocyte maturation/fertilization and correlated them to the ultrastructural changes observed by transmission electron microscopy using ruthenium red and saponin technique. In both the inner and outer ZP layers, stiffness decreased at maturation while, conversely, increased after fertilization. Contextually, at the nanoscale, during maturation both ZP layers displayed a fine filaments network whose length increased while thickness decreased. After fertilization, filaments partially recovered the immature features, appearing again shorter and thicker. Overall, the observed biomechanical modifications were substantiated by ultrastructural findings in the ZP filament mesh. In fertilized ZP, the calculated force necessary to displace ZP filaments resulted quite similar to that previously reported as generated by bovine sperm flagellum. Therefore, in bovine IVF biomechanical modifications of ZP appear ineffective in hindering sperm transit, highlighting the relevance of additional mechanisms operating in vivo.

We have investigated the changes in the mechanical properties of the zona pellucida (ZP), a multilayer glycoprotein coat that surrounds mammalian eggs, that occur after the maturation and fertilization process of the bovine oocyte by using atomic force spectroscopy. The response of the ZP to mechanical stress has been recovered according to a modified Hertz model. ZP of immature oocytes shows a pure elastic behavior. However, for ZPs of matured and fertilized oocyte, a transition from a purely elastic behavior, which occurs when low stress forces are applied, towards a plastic behavior has been observed. The high critical force necessary to induce deformations, which supports the noncovalent long interaction lifetimes of polymers, increases after the cortical reaction. Atomic force microscopy (AFM) images show that oocyte ZP surface appears to be composed mainly of a dense, random meshwork of nonuniformly arranged fibril bundles. More wrinkled surface characterizes matured oocytes compared with immature and fertilized oocytes. From a mechanical point of view, the transition of the matured ZP membrane toward fertilized ZP, through the hardening process, consists of the recovery of the elasticity of the immature ZP while maintaining a plastic transition that, however, occurs with a much higher force compared with that required in matured ZP.

Granulosa-theca cell tumors (GTCTs) are able to secrete variable amounts of sex steroids and immunoreactive inhibin (ir-INH). Although the pituitary appears to be affected by the presence of a GTCT, pituitary responsiveness to exogenous GnRH has not been examined. The aims of the present study were to: (i) assess the plasma hormone concentrations of ir-INH, gonadotropins and sex steroids in eight mares with GTCT and (ii) assess the responsiveness of pituitary gonadotroph cells to exogenous GnRH stimulus both before and after tumor removal. In seven mares, the contralateral ovary was firm, small and inactive. Histopathological observations of the tumors confirmed the presumptive diagnosis of a GTCT. Four mares, judged to be in vernal transition period (n=2) and in the breeding season (n=2), were used as controls. A single intravenous injection of 40 microg of GnRH agonist was given to each mare and blood samples were collected every 15 min from 2 h before to 4 h after injection. In four GTCT mares, this procedure was repeated 20 (n=2) and 90 (n=2) days after tumors removal. All plasma samples were analyzed for concentrations of ir-INH, LH, FSH, estradiol-17beta (E2), testosterone (T) by RIA and progesterone (P) by EIA. Results showed that E2 levels were significantly higher (P<0.001) in control animals compared to E2 levels in GTCT mares before and after surgery. P and T concentrations were not statistically different between the groups. Baseline levels of ir-INH were greater (P<0.05) in GTCT mares before surgery than in control mares, and decreased to undetectable levels after neoplasia ablation. Baseline FSH did not differ between control and GTCT animals either before or after the ovaries were removed. LH baseline values appeared to be higher for affected mares, but the difference was not statistically significant. Maximum release (MR) and area under the gonadotrophin release curve (AUC) after the GnRH challenge for both the gonadotrophins were similar between the groups.

The effect of orally administered l-carnitine on the quality of semen obtained from stallions with different semen qualities was investigated. Four stallions with proven fertility (high motility group, HM) and with normal seminal characteristics (>50% progressive motility and > 80 x 10(6) spermatozoa/ml), and four questionable breeders (low motility group, LM) with <50% of sperm progressive motility and < 80 x 10(6) spermatozoa/ml, received p.o. 20 g of l-carnitine for 60 days. Blood and semen samples were collected before treatment (T0) and after 30 (T1) and 60 days (T2). Semen evaluation were performed on five consecutive daily ejaculates (n = 120 ejaculates) and conventional semen analysis was carried out on each ejaculate, both at collection and after refrigeration for 24, 48, and 72 h. Furthermore l-carnitine, acetylcarnitine, pyruvate, and lactate concentrations, and carnitine acetyltransferase activity (CAT) were determined both in raw semen and seminal plasma. There were an increase in progressive motile spermatozoa only in the LM group (26.8 +/- 12.9, 39.1 +/- 15.5, and 48.8 +/- 8.6 for T0, T1, and T2, respectively). Free seminal plasma carnitine concentration was higher in the LM group compared to the HM one. Both pyruvate and lactate were higher in the LM group. Raw semen and seminal plasma carnitine and acetylcarnitine levels correlate positively with both sperm concentration and progressive motility; moreover, acetylcarnitine content was positively correlated with total motile morphologically normal spermatozoa. In conclusion, oral administration of l-carnitine to stallions with questionable seminal characteristics may improve spermatozoa kinetics and morphological characteristics; whereas, it seem to be ineffective in normospermic animals.