Reproductive Biotechnologies Applied to Artificial Insemination in Swine

FRANCISCO ALBERTO GARCIA-VAZQUEZ12', CHIARA LUONGO1, GABRIELA GARRAPPA1'3, and ERNESTO RODRIGUEZ TOBON1

department of Physiology, Faculty of Veterinary Science, International Excellence Campus for Higher Education and Research "Campus Mare Nostrum", University of Murcia, Murcia, Spain

institute for Biomedical Research of Murcia (IMIB-Arrixaca), Murcia, Spain

institute of Animal Research of the Semi-Arid Chaco (IIACS), Agricultural Research Center (CIAP), National Institute of Agricultural Technology (INTA), Tucuman, Argentina

*Corresponding author. E-mail: This email address is being protected from spam bots, you need Javascript enabled to view it

ABSTRACT

Artificial insemination (AI) is the most widespread reproductive technique used in porcine. This chapter provides firstly relevant information about the workflow involved in porcine AI, from the collection of boar ejaculate and seminal doses preparation to the sperm deposition in the female by AI devices and spermatozoa journey toward the oocyte. Secondly, we summarize new issues and future perspectives trying to increase the porcine AI efficiency, such as new fertility biomarkers, improving the composition of extenders used for semen dilution, or studying new methods for sperm selection as the use of magnetic nanoparticles to remove damaged spermatozoa.

INTRODUCTION

Advances in reproductive biotechnologies have allowed the increase of efficiency in animal production in several species. Artificial insemination (AI) has been one of the most successful applied reproductive biotechnologies since its implementation several decades ago (reviewed by Soriano-Ubeda et al. 2013). In the case of porcine species, AI (porcine AI, pAI) is routinely used in most of the farms (Riesenbeck, 2011), which among other factors, has allowed to place pork production as one of the most important meat industries worldwide (Knox, 2014; Zhang et al. 2018).

The advantages of the pAI are innumerable (Knox, 2016), although to achieve considerable success, multiple factors must be properly geared. AI methodology perse is a simple technique which requires scarce material to perform, just a single plastic tube ending in a tip that fits within the cervix. AI device has changed slightly through the years, but these changes has also considerably improved its effectiveness (i.e. deeper deposition which reduces the number of sperm per seminal dose increasing the number of seminal doses produced per boar) (Garcia-Vazquez et al. 2019a; Llamas-Lopez et al. 2019). However, AI is not only the process in which spermatozoa are deposited in the female genital tract. The great numbers reached in porcine reproductive performance (farrowing rates, litter sizes) in the last years are attributable to the improvement of different factors (Knox, 2016) (Fig. 10.1). Boars are one of the main pieces in pAI puzzle because they support the initial step in seminal doses production. Boars are usually confined in facilities known as Al-centers or boar studs. Then, sanitary status, genetic, semen collection training or housing in Al-centers are essential factors to contemplate for proficient semen production. After ejaculate collection, the processing of semen starts which includes an initial dilution in an extender, subsequently quality spermatozoa evaluation and packing in bags or bottles for further conservation and/or distribution. Once seminal doses are prepared, the other important piece to complete the pAI puzzle is the female. Even after having the best semen, it is useless if it is deposited into a female without an optimal moment of estrus. So, estrus detection and physiology knowledge of oestrus cycle are key factors for a successful AI. Finally, after seminal dose deposition, spermatozoa start a complicated journey in search of the oocyte. During insemination (natural or artificial) millions of spermatozoa are deposited but only a few of them are able to reach the place of fertilization (Tummamk and Tienthai, 2010). Most of the mechanisms involved in this drastic reduction of spermatozoa within the female genital tract are still unknown.

Scheme of the phases involved in porcine Artificial Insemination (pAI)

FIGURE 10.1 Scheme of the phases involved in porcine Artificial Insemination (pAI).

Although several reproductive biotechnologies associated with Al process have currently been applied in porcine, others are still under study and researchers are constantly trying to solve the reproductive porcine industry demands. Nowadays, some of these new investigations in pAI seems far to be applied but surely, they are the base to continue and increase the efficiency of this methodology even more in the future. These and other factors are going to be treated more extensively along the following sections.

THE ROLE OF THE BOAR IN ARTIFICIAL INSEMINATION

Over the years, the development of reproductive technologies has permitted an increase of farm size and the birth of specialized Al-centers (Knox, 2014). This scenario has allowed to raise the reproductive performance and, in consequence, the economic impact on productive efficiency of pAI (reviewed by Knox, 2016). Al-centers are in charge of producing seminal doses with enough quality to be used in AI programs. Then, seminal doses produced from a single ejaculate of a boar allow inseminating several sows (Maes et al., 2011). The total number of seminal doses generated varies depending on the type of AI used, cervical (CAI) or post-cervical artificial insemination (PCAI), because of the different number of sperm and volume needed. In general terms, the CAI-doses are composed of 3 x Ю9 sperm cells in a final volume of 90 ml, while the PCAI-doses are composed of

1.5 x Ю9 sperm cells in a final volume of 45 ml (Hemandez-Caravaca et al., 2012; Bortolozzo et al., 2015). The number of doses obtained annually per boar is about 2000 for CAI method (Mezalira et al., 2005), and consequently approximately the double of seminal doses in the case of PCAI.

The quality of an ejaculate depends on multiple factors including animal welfare, environmental temperature, light exposure, feeding or housing. These conditions have been improved through the specialization of Al-centers, by using automatic systems for temperature, feeding and watering control (Knox, 2016). Moreover, the design of Al-centers is also focused on preventing the entiy of pathogens. In this sense, Al-centers have two entries, one for vehicles and other for pedestrians. Once inside, each Al-center is divided into different areas such as an isolation area for boar (where health checks are performed), a boar housing area, a semen collection area, and a laboratory, in which semen samples are prepared, stored and shipped. Likewise, it is important to maintain a high level of hygiene since people can be carriers of diseases (Waberski and Weitze, 2010).

When new boars arrive at an Al-center, they should be isolated for 40-45 days before contact with other animals in order to check their health status (Althouse, 2007). Once this time has elapsed, boars are housed individually in pens (Knox, 2016), allowing visual contact and smell with other animals (Lopez-Rodriguez et al., 2017). The pens have an available area per boar of at least 6 m2 according to the European legislation (Commission Directive 2001/93/EC). The type of boar housing can influence boar health and it must be safe and allow easy handling of the animals (Althouse, 2007).

In order to enter the collection rotation, 7 months old boars are subjected to a training period and selected based on good health and semen quality (Bortolozzo et al., 2015; Knox, 2016). The frequency of collection can influence the production and quality of ejaculate, and in this sense a maximum of 3 tunes in two weeks is considered optimal (Knox, 2016). Therefore, a period of rest between extractions is necessary to complete spennatozoa maturation within the epididymis (Pruneda et al., 2005).

The semen collection is perfoimed in specific facilities that allow the operator to safely handle the boar (Althouse, 2007). A collection dummy is placed within the pen, equipped with two lateral supports on which the boar can position the front legs. Moreover, vertical galvanized pipes on 2 or 3 sides of the perimeter of the collection pen can be placed allowing the operator to escape easily when necessary (Althouse, 2007). Apart from the traditional pen, the “Reicks collection pen ” is the most used collection method in Al-centers. This method is similar to the traditional collection facility, but with an additional small pen for the operator. By this way, the operator can handle the boar standing outside by a sliding gate during the collection process (Levis and Reicks, 2005). Additionally, some Al-centers have a warm-up pen to aid with sexual stimulation of the boar. Then, boars will require less tune to mount the dummy and for semen output (Althouse, 2007). Once the boar is situated on the dummy, the process of semen collection starts. But before explaining the methods of semen collection it is necessary to talk briefly about the swine ejaculate. The whole boar ejaculate is about 200-300 ml with a concentration higher than 30 x 109 sperm cells per ml (Rodriguez-Martmez et al., 2005). Apart from sperm cells, the ejaculate is composed of seminal plasma (SP). The ejaculation can last up to 15 min divided in three fractions. The first fraction, called pre-spermatic fraction (10-15 ml), contains secretions from urethral and bulbourethral glands and prostate. It is characterized by the absence of sperm cells (or very poor number) and a high bacterial count, whereby it is discarded (Maes et al., 2011; Lopez-Rodriguez et al., 2017). The second fraction (70-100 ml), the spermatic-rich fraction, contains the majority of spermatozoa (80-90%) and SP, derived from testes, epididymis, and accessory reproductive glands (Garner and Hafez, 2000). The third fraction (120-200 ml) is called post-spermatic rich fraction and contains few sperm cells and more SP than the spermatic-rich fraction. As indicated, SP represents an important part of the whole ejaculate in porcine species. This male biological fluid is a complex milieu in which spermatozoa are immersed during the ejaculation and is mainly composed by inorganic and organic components, such as carbohydrates, lipids, amino acids and proteins (Sancho and Vilagran, 2013). Thr ough the ejaculation, a gelatinous substance (called tapioca) is produced by bulbourethral glands, especially during the emission of the third fraction. Tapioca acts as a plug in the cervix of the sow during natural insemination, to seal the cervix reducing ejaculate backflow (Knox, 2001).

Returning to the methods of semen collection, these can be performed mainly by two methods (Fig. 10.2): 1) the manual method or also called gloved-hand technique; and 2) the automatic technique. Nowadays, the gloved-hand method is the main technique of choice in Al-centers (Knox et al., 2008). In any case, hygiene rules should be followed to avoid bacterial contamination (Lopez-Rodriguez et al., 2017). For this reason, the initial steps are the same for both methods and consist of cleaning the penis and the area surrounding the preputial orifice. During this process, the operator’s hand is covered by a plastic glove to manipulate the boar penis, then is removed after cleaning (Althouse et al., 2000; Aneas et al., 2008). Then the operator can proceed with one of semen collection methods. During the gloved-hand technique, the collection continues using only the iimer glove composed of polyvinyl, due to latex gloves provoke spermatozoa toxicity (Ко et al., 1989). The operator grabs the penis and exerts pressure imitating the pressure exerted by the cervix of the sow (Maes et al., 2011). The automatic method, unlike the manual method, does not allow collecting a single fraction, but the whole ejaculate is collected. In this case, once the penis is externalized, the operator connects the penis of the boar to a system with an artificial vagina. This system is equipped with a disposable sanitary sleeve that allows direct contact between the penis and artificial vagina and avoids ejaculate contamination. After that, the operator activates air pressure to close the artificial vagina with the aim of grabbing the penis (Waberski and Weitze, 2010). In both, manual and automatic methods, the ejaculate is collected in a plastic container equipped with a filter to discard the gelatinous fraction (Aneas et al., 2008). This container is pre-wanned (38 °C) to avoid damage caused by thermal shock, and then placed in warm water to keep the temperature constant (Maes et al., 2011).

The main difference between the two collection methods is that technician efficiency increases using the automatic method, allowing an increase in the number of ejaculates collected per hour (Aneas et al., 2008). Additionally, by the automatic method, bacterial contamination is reduced, and ejaculate volume and total number of sperm are higher than the manual method (Aneas et al., 2008). However, the automatic method is not widely used because the whole ejaculation is collected, including a high SP amount. In fact, it has been proved that better results were obtained when the post-spermatic rich fraction was discarded (Centurion et al., 2003; Okazaki and Shimada, 2012).

Procedure of seminal doses elaboration. (A) Semen collection

FIGURE 10.2 Procedure of seminal doses elaboration. (A) Semen collection: gloved- hand and automatic technique; (B) Dilution in an appropriate extender; (C) Sperm analyses: concentration determination by spectrophotometric, spermatozoa motility evaluation by microscope or computerized assisted sperm analysis system (CASA); (D) Packing: different package for seminal doses (bottles, tubes and bags/flat bags); automatic packing machines; (E) Storage: seminal doses conservation at 15-17 °C; (F) Artificial insemination procedure. (Images provided by Pedro Jose Llamas-Lopez and Minitube).

 
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