Ability of the ISAS3Fun Method to Detect Sperm Acrosome Integrity and Its Potential to Discriminate between High and Low Field Fertility Bulls.

The objective of the present study was to investigate whether fertility differences in bulls are reflected in variations of sperm quality when analysing only one ejaculate per male. Two experiments were performed. In the first experiment, frozen semen samples from 20 adult bulls were tested; 10 bulls had high field fertility and 10 bulls had low field fertility. Analyses of sperm motility, membrane integrity, and membrane-acrosome integrity with the ISAS3Fun method were performed. Sperm morphometry of the fluorescence sperm subpopulations obtained with the ISAS3Fun method was also analysed. Significant differences between high- and low-fertility groups were only found with the ISAS3Fun technique, specifically in sperm acrosome integrity, the proportion of spermatozoa with an intact acrosome and damaged membrane, and in sperm head width of spermatozoa with intact structures. Discriminant analyses allowed us to correctly classify 90% of sperm samples in their fertility group using sperm quality parameters. Given that only the results obtained with the ISAS3Fun technique were related to bull fertility, we performed a second experiment aimed to validate the efficacy of this technique to detect the acrosomal integrity of bull spermatozoa, comparing them with the conventional FITC-PNA/propidium iodide (PNA/PI) combination under capacitating conditions. The results indicated that the ISAS3Fun combination provided an accurate assessment of both viability and acrosomal integrity for ejaculated spermatozoa, while the PNA/PI combination underestimated the extension of acrosomal damage due to false negatives. It was concluded that the simultaneous assessment of sperm plasma membranes and acrosome integrity with the ISAS3Fun method is precise and seems to have a greater potential to discriminate between high- and low-fertility bulls than more conventional in vitro sperm quality tests.

Expanding the Limits of Computer-Assisted Sperm Analysis through the Development of Open Software.

Computer assisted sperm analysis (CASA) systems can reduce errors occurring in manual analysis. However, commercial CASA systems are frequently not applicable at the forefront of challenging research endeavors. The development of open source software may offer important solutions for researchers working in related areas. Here, we present an example of this, with the development of three new modules for the OpenCASA software (hosted at Github). The first is the Chemotactic Sperm Accumulation Module, a powerful tool for studying sperm chemotactic behavior, analyzing the sperm accumulation in the direct vicinity of the stimuli. This module was validated by comparing fish sperm accumulation, with or without the influence of an attractant. The analysis clearly indicated cell accumulation in the treatment group, while the distribution of sperm was random in the control group. The second is the Sperm Functionality Module, based on the ability to recognize five sperm subpopulations according to their fluorescence patterns associated with the plasma membrane and acrosomal status. The last module is the Sperm Concentration Module, which expands the utilities of OpenCASA. These last two modules were validated, using bull sperm, by comparing them with visual counting by an observer. A high level of correlation was achieved in almost all the data, and a good agreement between both methods was obtained. With these newly developed modules, OpenCASA is consolidated as a powerful free and open-source tool that allows different aspects of sperm quality to be evaluated, with many potential applications for researchers.

Relationship of sperm plasma membrane and acrosomal integrities with sperm morphometry in Bos taurus.

To date, sperm morphometric studies have assessed whole sperm populations without considering sperm function. The aim of this study was to evaluate the relationship of sperm membrane and acrosomal integrity with sperm morphometry in liquid semen samples collected from bulls. To this end, sperm morphometry was performed on cryopreserved semen samples from 16 bulls by a combination of fluorescent dyes, including Hoechst 33343, carboxyfluorescein diacetate, and propidium iodide. This allowed discrimination of different subpopulations on the basis of sperm membrane and acrosomal integrity and analysis of the morphometrics of the sperm head, nucleus, and acrosome using a specific plug-in module created on ImageJ. Acrosomal integrity was related to sperm morphometry as the heads of spermatozoa with a damaged acrosome were significantly smaller than those with a normal acrosome (P < 0.001). In the case of spermatozoa with an intact acrosome, those with a damaged plasma membrane had a larger sperm head and acrosome than spermatozoa with an intact plasma membrane (P < 0.001). No significant differences in the sperm head size were observed between sperm subpopulations without an acrosome or in the nuclear sperm morphometry of the different subpopulations. There was a positive correlation between the sperm motility values of the samples and the morphometric parameters for intact spermatozoa. These correlations were particularly strong for the morphometric parameters of the sperm acrosome. We conclude that there are clear differences in the sperm morphometry depending on the status of the sperm membrane and acrosome and this should be considered when performing this kind of analysis.

OpenCASA: A new open-source and scalable tool for sperm quality analysis.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson's correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

Failure to establish and maintain a pregnancy in undernourished recipient ewes is associated with a poor endocrine milieu in the early luteal phase.

Embryos from undernourished and control donor ewes were transferred to undernourished and control recipient ewes. Progesterone and metabolic hormones were investigated in recipient ewes to determine their association with pregnancy success. Forty-five donor and 52 recipient Rasa Aragonesa ewes were fed 1.5 (control group; donor n=20; recipient n=25) or 0.5 (low group; donor n=25; recipient n=27) times the daily requirements for maintenance from the onset of estrous synchronization treatment to embryo collection and transfer. The embryos were collected 7days after the onset of estrus (day 0), and two good-quality embryos were transferred into each recipient ewe. The percentage of pregnant ewes on day 18 and 40 did not differ between the two groups, although the recipient undernourished ewes tended to have greater late embryonic mortality (from days 18-40) than the control recipient ewes (P=0.11). No effect of the nutrition of the donor was found. Recipients that became pregnant had a higher ovulation rate than non-pregnant ewes (P=0.02). Undernourished ewes had lower plasma insulin concentrations than control ewes (P=0.03), and those that suffered late embryo mortality (from days 18-40) tended to have lower insulin and progesterone concentrations than their counterparts that remained pregnant (P=0.06 and P=0.07, respectively). In this study, pregnancy in control and undernourished recipient ewes was not associated with the origin of the embryo (undernourished and control donors). In conclusion, failure to establish and maintain a pregnancy was associated with lower progesterone and insulin levels one week after estrus in recipient ewes.