PREPARATION AND EVALUATION OF THE SEMINAL DOSES
After collection, the ejaculate is placed at the laboratory of Al-center, where an initial qualitative evaluation is determined: color (gray-white to white), appearance (milky to creamy) and odor (Althouse, 2007). The different methods of semen collection and the environment where it is extracted can affect some parameters of the seminal sample.
Once the ejaculate is suitable for processing after this initial appreciation, the next step is the determination of spermatozoa concentration. Traditionally, the most used method to calculate the cell concentration has been the counting chambers such as Neubauer, Barker, and Thoma (Althouse, 2007; Brito et al., 2016). Nowadays, this method has been suipassed by spectro- photometric teclmiques because manual counting is a slow process (Brito et al., 2016). In an ejaculate, the opacity depends on the number of cells among other elements of the SP that could interfere with the passage of the light through the sample. Therefore, it is recommendable to dilute a small sample for obtaining a more reliable result (depending extender, dilution rates from 1:4—1:25) (Althouse, 2007). Besides, errors in the evaluation can occur if the dilution is not correct or untimely reading (Althouse, 2007; Brito et al., 2016). CASA (Computerized Assisted Sperm Analysis) system can also be used to determine the concentration, although this system is more recommendable to assess spenn motility (Arnann and Waberski, 2014; Brito et al., 2016), as will be explained later. Another method for cell counting is the flow cytometry, which allows rapid and automated counts of many cells. However, the use of flow cytometry is limited due to its high cost and the need for qualified persormel to manage the equipment and interpret the results (Brito et al., 2016).
Then, seminal doses for Al will be elaborated and stored. The first step is to dilute the ejaculate in an adequate extender to conserve the semen and drive the spermatozoa during Al (Fig. 10.2b). The different extenders for the preservation of spermatozoa are divided into three groups according to the storage period (short, medium and long-term based on their ability to preserve spermatozoa from 1 day up to 10 days after collection) (Kara- georgiou et al., 2016; Pezo et al., 2019b). There are numerous extenders commercially available although the exact composition is not fully known due to economic interests. In general terms, extenders are composed by: (1) nutrients: to maintain cell metabolism, being the most common glucose and fructose; (2) pH controller: bicarbonate, sodium citrate, Tris or HEPES; (3) osmotic pressure controller: NaCl and KC1; (4) antibiotics: to inhibit bacterial growth (Yeste, 2017). Bacteriospennia is one of the main problems in Al worldwide (Yeste, 2017). Problems derived by the presence of bacteria affect directly the semen quality impacting fertility rates and causes endometritis in recipient females (Althouse et al., 2000; Maes et al., 2008; Bussalleu and Torner, 2013). Although antibiotics are frequently used in extenders, bacterial resistance has been reported in up to 90% (Schulze et al., 2014) of the seminal doses studied. Then, new alternatives to conventional antibiotics are on the light of researching (Schulze et al., 2014) (see Section 10.8.3.)-
After dilution, a spermatozoa quality control is necessary to ensure that the process has been performed adequately. Spermatozoa quality parameters commonly evaluated in Al-centers are morphoanomalies (commonly classified as normal morphology, proximal and distal cytoplasmic droplet or tails defects, folded and coiled) (Garcia-Vazquez et ah, 2015) and motility (Brito et ah, 2016). In the case of motility, there are several methods for its evaluation (Fig. 10.2): (1) light field or phase contrast microscopy: a subjective maimer to evaluate total motility (values from 0 to 100%) and progressive motility (values from 0 to 5, where 5 score represents the highest level of progressivity), although this method has a high degree of inaccuracy; 2) CASA system: evaluates spermatozoa motility and kinetic parameters through a high-resolution camera mounted on a phase contrast microscope (Brito et ah, 2016). The motility parameters offered by CASA system are the following: Total and progressive motility (%), curvilinear velocity (VCL), rectilinear velocity (VSL), average speed (VAP) (all of them expressed in pm/s), linearity Index (LIN, VSL/VCL in percentage), straightness Index (STR, VSL/VAP in percentage), oscillation Index (WOB, VAP/VCL in percentage), average amplitude of the lateral displacement of the head (ALH, pm), beat frequency (BCF, Hz) (Amann and Waberski, 2014). Although this method is the most objective and evaluates different parameters of sperm kinetics, some factors have to be considered to reduce variability during evaluation. Such factors are sample temperature, dilution factor, frequency and duration of mixing, or chamber depth (Broekhuijse et ah, 2011).
Another technique for accurate assessments of sperm samples is the flow cytometry (Broekhuijse et ah, 2015). This technique permits the evaluation of a huge number of cells in a short time determining special characteristics in the spermatozoa by using fluorochrom.es (Broekhuijse et ah, 2015). Some of the evaluable parameters by flow cytometry are spermatozoa viability, DNA integrity, mitochondrial activity, membrane fluidity, and acrosome reaction (Broekhuijse et ah, 2012).
However, most of the parameters evaluated for the quality of ejaculates have not been fully correlated with fertility (Broekhuijse et ah, 2012). Thus, a multivariate analysis seems to be more efficient in determining a quality ejaculate (Knox, 2016). Nowadays, researchers are putting effort into looking for some new biomarkers for reliable determination of the fertility of a seminal dose (see Section 10.8.1).
Finally, the process of the individual seminal dose starts (Althouse, 2007). The volume and number of spermatozoa per seminal dose will depend on the type of AI that will be performed (see Section 10.6). Subsequently, the seminal dose will be packaged in its final container (bottles, tubes or bags/flat bags) (Fig. 10.2) made of non-toxic plastic material (Althouse, 2007; Nerin et al., 2014). In highly specialized Al-centers the laboratories have automatic packing machines, which optimizes the tune and ensures the same volume in the doses (Yeste, 2017). After packing, seminal doses can be refrigerated for storage (15-17 °C) (Yeste, 2017) (Fig. 10.2).
FACTORS AFFECTING SEMINAL DOSES ELABORATION AND CONSERVATION
The elaboration and conservation of the seminal doses is not an extremely complex process. However, there are several factors that should be known during the procedure to get a final product of enough quality and guarantee of success during AI (Fig. 10.2). One of the main critical points during seminal doses elaboration is the dilution which involves temperature, steps of dilution and storage conditions.
Boar spermatozoa are very susceptible to cold shock; therefore, the temperature of the ejaculate and extender during mixing is a critical point. Drastic cooling from ejaculate temperature (~38°C) to refrigeration temperature (~15°C) reduces spermatozoa viability and motility (Johnson, 2000). Then, the procedure of dilution is focused on decreasing the temperature gradually. The protocol of dilution followed by Al-centers are mainly one-step or two-step dilution, and temperature plays a key role in both (Waberski, 2009). The one-step dilution consists in the mix of the ejaculate with the extender at ~33°C or room temperature. In the two-step protocol, ejaculate is diluted 1:1 (at ~33°C), followed by a second dilution. This second dilution could be carried out using a pre-heated extender (~33°C) or at room temperature (22-23°C) without differences between them in terms of motility and membrane/acrosome integrity (Lopez-Rodriguez et al. 2012). There is also a three-step protocol dilution which consists in a first dilution (1:1, ~33°C), second dilution (adding 50% of the remaining extender volume at ~33°C) and third dilution (adding the remaining 50% of the extender at ~33°C) (Schulze et al., 2018). The one-step dilution at
~33°C is the most recommendable procedure due to less work labor, lower risk of mistakes because of simplicity, and better spermatozoa quality results (Schulze et al., 2018). The orders in which the semen and extender are mixed (extender to the semen or reverse) have not adverse effects on the spermatozoa quality (Schulze et al., 2017). Commonly, once the seminal doses are ready, a period of ~90 min at room temperature is necessary before being transferred to 15-17°C (Waberski, 2009).
Boar spermatozoa quality is impaired during storage and it is determined by the conditions and the length of this storage (Johnson et al., 2000; Waberski et al., 2011; Schulze et al., 2013; Yeste, 2017). Sperm motility and viability has been determined as the main parameters affected during refrigeration (Althouse et al., 1998). Boar sperm membrane alteration, showing as a change in permeability allowing the entry to stains, is considered a great indicator of spermatozoa storage-related damage (Johnson et al., 2000; Roca et al., 2005; Bielas et al., 2017). Spermatozoa DNA is also affected by refrigeration, alterations have been described after the first hour of storage (Bielas et al., 2017) and after a longer period of storage of 3 or 4 days (Fraser and Strzezek, 2004; Boe-Hansen et al., 2005; Perez-Llano et al., 2009).
Supplementation of seminal doses just before Al has been suggested as a good practice to solve the detrimental effect of storage. In this sense, several hormones have been tested, such as oxytocin (Pena et al., 1998; Okazaki et al., 2014), relaxine (Feugang et al., 2015) or prostaglandin F:a (Maes et al., 2003). On the other hand, a significant number of studies have focused their goals in the improvements of seminal doses storage conditions. One of the strategies used has been the supplementation of additives to the extender such as hyaluronic acid (tested to postponed premature capacitation; Yeste et al. 2008) or bovine serum albumin (BSA, used for long-term extender; Zhang et al., 2015; Yeste et al., 2017) among others. More information about new additives in extender will be found in Section 10.8.3.
Other approaches improving spennatozoa conservation conditions have been the rotation and homogenization of semen samples during their storage to prevent spennatozoa sedimentation. Contrary to initial thoughts, rotation of semen samples triggers an increase in pH with negative effects on spermatozoa quality during storage (Schulze et al., 2015). Similarly, the homogenization of doses during storage impaired mitochondrial activity, acrosome and plasma membrane integrity (Menegat et al., 2017).
Accordingly, rotation and homogenization may not be a recommendable action during the storage of seminal doses.
As aforementioned, the recommended temperature of storage ranges from 15 to 17 °C (Yeste, 2017) and storages temperatures below 12 °C induce cold shock with detrimental effects in boar spermatozoa (Althouse et al., 1998; Johnson et al., 2000). According to this, the need for maintaining the temperature in a limited range during shipping of seminal doses from Al-center to the farms represents a logistic problem (Riesenbeck, 2011). Besides keep constant the seminal dose temperature from its elaboration to its use, another aspect that should be considered during seminal doses transportation is vibration emission. It has been proposed that vibration might have a negative impact on pH values during transportation (Johnson et al., 1982). Therefore, vibration emission with 300 rpm frequencies for 6 h resulted in alkalization of semen extender (Schulze et al., 2018) which impairs sperm motility (Gatti et al., 1993; Vyt et al., 2007). Some others spermatozoa quality parameters such as mitochondrial activity, acrosome, plasma membrane integrity, and thermo-resistance showed a negative frequency-dependent effect of vibration emissions during simulated transportation (Schulze et al., 2018). In order to control this factor, a novel monitoring tool for boar semen transport has been developed. This device, established using mobile sensing, works as a data logger of vibration emission from seminal doses (Schulze et al., 2018). Thanks to this new technology, further studies aimed to find the optimal shipping packing for boar seminal doses may be attained.
Apart from refrigeration, there are other strategies for the long-term preservation of boar spermatozoa, such as cryopreservation or lyophiliza- tion. Cryopreservation is performed in the presence of cryoprotectants, to avoid cold shock. In fact, boar spermatozoa are subjected to changes at low temperature because of their plasmatic membrane composition, poor in cholesterol (Rodriguez-Martinez and Wallgren, 2010; Casas and Flores, 2013; Yeste, 2017). Cryopreservation may be performed in two ways: 1) SIon- freezing-, spermatozoa are freezing in an extender containing cryoprotectants, using controlled-rate freezers; in this case, the formation of ice crystals within the suspension, due to the flow of water between intracellular and extracellular space, can induce spermatozoa damage (Johnson et al., 2000; Watson, 2000; Holt and Van Look, 2004); 2) Fast freezing or vitrification', it is performed with very high cooling rates. By this method, water does not form ice crystals, but it reaches the vitreous state. Despite vitrification requires less time and costs than slow freezing, it is not currently used because of the osmotic effect due to high concentrations of cryoprotectants (Arraztoa et al., 2017). A possible solution could be an alternative vitrification method in the absence of cryoprotectants, called spheres method (Arraztoa et al., 2017). This method consists of using microdroplets of spermatozoa samples, dipping in liquid nitrogen and obtaining spheres. Otherwise, lyophilization consists of sperm cell dehydration by evaporation (conventional heat-drying) or by sublimation (freeze-drying) (Zahan et al., 2014). One of the negative aspects of lyophilization is that spermatozoa lose motility after rehydration. However, the spermatozoa acrosome membrane remains intact, so intracytoplasmic speim injection may be used (Zahan et al., 2014).
Nowadays, there are ongoing research to improve long-term preservation method to increase spermatozoa survival. Actually, only 1% of pAI is performed using cryopreserved spermatozoa because of low fertilizing ability of spermatozoa after thawing (Yeste, 2017; Garcia-Vazquez et al., 2019a).