Review Article. New Artificial Insemination Technologies for Swine

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1 Reprod Dom Anim 50 (Suppl. 2), (2015); doi: /rda ISSN Review Article New Artificial Insemination Technologies for Swine FP Bortolozzo 1, MB Menegat 1, APG Mellagi 1, ML Bernardi 2 and I Wentz 1 1 Departamento de Medicina Animal, Faculdade de Veterinaria, Universidade Federal do Rio Grande do Sul, Porto Alegre-RS, Brazil; 2 Departamento de Zootecnia, Faculdade de Agronomia, Universidade Federal do Rio Grande do Sul, Porto Alegre-RS, Brazil Contents Better efficiency in swine artificial insemination (AI) is accomplished by reducing the necessary number of sperm cells and achieving high reproductive performance. Some strategies have been developed for use under field conditions, such as the site of sperm deposition (post-cervical AI), a single AI with control of oestrus and ovulation (fixed-time AI), and the better use of high genetic merit boars, to spread their genes as much as possible. To apply these technologies, it is important to understand some anatomical limits, physiological aspects and farm requirements. In this review, we point out some strategies to achieve higher efficiency on the use of sperm for AI technology application in swine species. Introduction Artificial insemination (AI) is the most common reproductive technology in swine production. Over the past decades (1990s and 2000s), a great development of swine AI has been observed. Both the quality control of semen doses and their commercial use have improved through the years. Better animal management has played a significant role in high reproductive performance. However, besides the progress obtained, the number of sperm cells used per pregnant female is still high, considering that each female is inseminated twice, or even three times per oestrus, using doses that contain billion sperm cells. In practice, doses with high sperm cells number are still used probably to compensate some detrimental effects related to semen processing (temperature oscillation, contamination, etc.), quality of staff or environmental issues. Some new strategies have been developed to optimize the number of females inseminated with each collected ejaculate. Efforts are being applied to spread desirable genes from high-indexed boars. The purpose of this review is to describe some strategies to optimize the use of the AI technique under field conditions and to allow the more efficient use of sperm cells from genetically superior boars. Artificial Insemination Techniques Artificial insemination in pigs has been used since the early 1930s, but its true development and wide commercial application in the pig industry did not take place until the 1980s. Intracervical insemination (CAI) is the standard insemination procedure and involves the deposition of the semen dose into the posterior portion of the cervical canal (Roca et al. 2006). Generally, billion spermatozoa are used per insemination, in a large volume of extender ( ml), limiting the number of doses that can be prepared from one ejaculate. A non-surgical technique for semen deposition into the uterus was first described in the late 1950s by Hancock (1959). Although this technique was successfully approved, it was only commercially reported in the 2000s (Watson and Behan 2002). Post-cervical AI (PCAI) requires minimal training by stockman, does not require extra time for insemination, has no welfare implications and mostly allows the sperm cell number to be reduced, providing substantial productivity gain (Watson and Behan 2002). PCAI can be performed in the absence of a boar in front of the sows, which has shown, in our personal experience, to facilitate introduction of the PCAI catheter. Furthermore, fertility in the subsequent parity has not been impaired in sows submitted to PCAI (Watson and Behan 2002). Post-cervical AI allows sperm cell dose reduction without impairing reproductive performance, as was shown in the pioneering (Watson and Behan 2002) and in a most recent work (Hernandez-Caravaca et al. 2012). The total number of sperm cells was reduced to 1 billion with no significant reduction in fertility (Watson and Behan 2002). Later, PCAI was successfully performed with at least 0.5 billion sperm cells infused at an optimal insemination ovulation interval (Mezalira et al. 2005). Indeed, PCAI resulted in a similar amount of spermatozoa deposited in the sperm reservoir around ovulation time when compared to conventional AI, even with a threefold reduction (1 vs 3 billion) in the number of sperm cells used (Sumransap et al. 2007). Although PCAI is currently used in several countries, the number of sperm cells per dose is not yet standardized. Sows inseminated with PCAI have similar pregnancy and farrowing rates to those with CAI even when the number of sperm cells is reduced to 1.5 (Dallanora et al. 2004a), 1.0 (Watson and Behan 2002) or 0.5 billion (Bennemann et al. 2007). In some cases, sows submitted to PCAI can even have significantly higher fertility results than those inseminated by CAI, with twofold higher sperm cells number (Hernandez-Caravaca et al. 2012). However, litter size decreased by 0.8 piglets with

2 New Artificial Insemination Technologies for Swine billion sperm cells (Bennemann et al. 2007), and a reduction of viable embryos (Mezalira et al. 2005) and foetuses (Wolken et al. 2002) was observed with 0.25 and 0.1 billion sperm cells, respectively. These findings suggest that there is a limit to the reduction of sperm cell number in the PCAI technique, which is probably below the billion that is commonly used. However, most of literature comparisons between CAI and PCAI did not include groups with similar sperm number per dose, low number CAI, or high number PCAI, making it difficult to state whether the results are due to dose or technique used. The interval between insemination and ovulation (AIOV) is considered a major factor influencing the fertility and reproductive performance of sows, regardless of semen quality (Waberski et al. 1994). Several studies have demonstrated that the optimal AIOV interval is up to 24 h for CAI with 3 billion sperm cells (Soede et al. 1995; Bortolozzo et al. 2005). Similarly, it has been reported that PCAI (1.0 or 2.0 billion sperm cells) performed with an AIOV interval up to 24 h does not reduce the pregnancy rate, number of total embryos or embryo survival (Bennemann et al. 2004). The inner PCAI catheter is easy to introduce into the uterus, and more than 95% of sows can be inseminated (Bennemann et al. 2004). Usually, PCAI is performed at a low level of difficulty, that is insertion of the catheter is possible on the first attempt, in a high percentage (~75%) of multiparous sows (Diehl et al. 2006). However, a medium or high level of difficulty is observed during insertion of the inner catheter in 30 45% of firstparity sows (Diehl et al. 2006; Sbardella et al. 2014). Moreover, the inability to introduce the catheter with the consequent impossibility of performing PCAI may be experienced in 13% of primiparous sows (Sbardella et al. 2014). In some studies that included low numbers of primiparous sows and did not consider the parity order as a primary purpose, unsatisfactory performance has been reported for this category of sows (Serret et al. 2005; Diehl et al. 2006). Nevertheless, more than 85% of primiparous sows were successfully post-cervically inseminated without impairing their reproductive performance in a recent trial (Sbardella et al. 2014) designed to use a large number of primiparous sows (n = 165) and to control the risk factors for subsequent reproductive failure, such as locomotor problems, poor body condition or health issues. Studies aiming to evaluate the efficiency of the inner catheter introduction in gilts are scarce. It is usually stated that a low success rate is achieved following introduction of the inner PCAI catheter in nulliparous females. An experimental evaluation concerning the difficulty of inner catheter introduction in gilts was developed by our group. We found that introduction of the inner catheter beyond 10 cm of the cervix, in the first AI, was possible in only 44% of the gilts. Therefore, the use of PCAI in gilts remains limited due to the low success rate of catheter insertion (Ulguim RR, Vier CM, Betiolo FB, Sbardella PE, Bernardi ML, Wentz I, Bortolozzo FP). Traumatic injury and bleeding occurrence are more common in inseminations showing difficulty of inner catheter introduction, especially if force is applied to pass the resistance point. The presence of blood in the catheter tip at its removal or in semen backflow does not occur (Mezalira et al. 2005) or is seen in <2% of all sows or inseminations (Bennemann et al. 2004). However, some authors reported that approximately 9% of the sows had blood present in backflow semen (Dallanora et al. 2004a) or in the vaginal vestibule (Bennemann et al. 2007) within 120 minutes after insemination. Also, the presence of blood during insemination is more frequent in primiparous than in multiparous sows, which was noticed in 23% of first-parity sows, probably due to their smaller reproductive tract (Sbardella et al. 2014). Although reproductive performance is not always affected by bleeding (Bennemann et al. 2004), its occurrence during PCAI has been associated with an increased return to oestrus rate (2.6 vs 13.8%; Dallanora et al. 2004a) and lower litter size (11.6 vs 9.0 piglets; Bennemann et al. 2007). There is great variation in the dose volume used for PCAI in swine. In the early studies involving PCAI, the volume used was similar to that used in CAI, that is approximately 80 ml (Watson and Behan 2002). Together with the reduction in sperm cells number, the volume can also be decreased to 60 ml (Bennemann et al. 2004), 30 ml (Diehl et al. 2006) or even 20 ml (Wolken et al. 2002; Mezalira et al. 2005). Despite the high profitability obtained with the use of low-volume semen doses, an excessive reduction of extended semen may affect the reproductive performance. A significant reduction in farrowing rate and litter size was observed when the PCAI dose volume was reduced from 50 to 25 ml with no variation in sperm cells number (1.5 billion) (Behan and Watson 2004). Nevertheless, the volume of 20 ml has been used without impairing reproductive performance (Mezalira et al. 2005), although this may have been the result of ensuring that the entire volume dose was removed from the inner catheter into the uterus, minimizing spermatozoa losses. Approximately 70% of the volume and 25% of the sperm cell of the infused dose are eliminated by backflow during or after CAI insemination (Steverink et al. 1998). One advantage of the PCAI method is the reduction or even the absence of sperm backflow during insemination (Bennemann et al. 2004; Dallanora et al. 2004b; Mezalira et al. 2005), which allows reducing the number of sperm cells or the number of inseminating doses. Nevertheless, even in PCAI-inseminated sows, the occurrence of semen backflow during insemination was reported in approximately one-quarter of multiparous (Fontana et al. 2014) and primiparous (Sbardella et al. 2014) sows, raising the question of the importance of the boar presence during insemination having a favourable effect on semen transport in female reproductive tract. Backflow volume was observed to range from 0% to close to or over 100% (Mezalira et al. 2005; Hernandez-Caravaca et al. 2012; Sbardella et al. 2014),

3 82 FP Bortolozzo, MB Menegat, APG Mellagi, ML Bernardi and I Wentz even when a low volume (20 ml) is infused (Mezalira et al. 2005). It seems that semen backflow can be more frequent and abundant in primiparous sows (Steverink et al. 1998; Hernandez-Caravaca et al. 2012; Sbardella et al. 2014), perhaps as a result of differences in animal size leading to a facilitated retention of fluid in multiparous sows due to the gravity and position of their larger uterus (Willenburg et al. 2003). As the boar is absent for PCAI, this method can be carried out more rapidly than CAI, mainly due to the lower volume and forced influx of the semen, because the folds of the cervix are overpassed and the sperm is released into the uterus. In addition, when CAI is used, the catheter should remain in the cervix for an additional few minutes after insemination to minimize the occurrence of semen backflow (Hernandez-Caravaca et al. 2012). The duration of CAI and PCAI insemination is and min, respectively (Hernandez-Caravaca et al. 2012), evidencing that the time spent to perform PCAI is almost 2.5-fold lower than CAI. Surgical deposition of semen close to the uterotubal junction allows the number of sperm cells and dose volume to be reduced to 10 million and 0.5 ml, respectively, without a decrease in the fertilizing potential (Krueger et al. 1999). In the light of these results, modifications in the insemination procedure that involve non-surgical deep intrauterine insemination (DUI) in sows have already been considered. A technique using a fibre-optic endoscope was first developed for successful non-surgical DUI in non-sedated sows (Martinez et al. 2001). Considering the promising results with that technique but the unsuitable use of the endoscope under field conditions, a flexible catheter for semen deposition into the upper third of the uterine horn was soon developed (Martinez et al. 2002). In comparison with standard CAI (3 billion spermatozoa in 100 ml), DUI can be performed with a 20-fold reduction in the number of spermatozoa and an 8- to 10- fold reduction in the dose volume without affecting the farrowing rate or litter size in weaned sows (Martinez et al. 2002). DUI is a useful tool for improving the efficiency of the use of weaker spermatozoa, such as those that have been frozen thawed or sex-sorted, although fertility results with the use of weaker spermatozoa are also influenced by AIOV, not only by the site of semen deposition. Also, it would be of great interest for the wide and effective use of semen from genetically superior boars or in sanitary contingencies, when the number of doses to be used must be drastically decreased (Vazquez et al. 2005). The use of PCAI has been shown to be suitable under field conditions, due to the reduction of sperm cell number used for each insemination, labour efficiency and good reproductive results. On the other hand, because the use of this technique is still limited for nulliparous sows, or even for primiparous, farms must keep both techniques (CAI and PCAI) in their routines. Employees must be well trained to avoid bleeding or semen backflow during insemination. The inner catheter cost must be taken into account for PCAI or DUI employment. Also, boar studs need to produce both types of doses (90 and 45 ml) and precision on concentration analysis is essential, mainly for the PCAI procedure, as a lower number of sperm cells will be used. Fixed-time Artificial Insemination As discussed above, the interval between insemination and ovulation affects the fertilization success. It is well established that insemination performed between 0 and 24 h before ovulation has no significant adverse effects on fertilization rate and consequently on farrowing rate and litter size (Waberski et al. 1994; Soede et al. 1995). Insemination performed early relative to ovulation may incur an insufficient number of sperm cells capable of fertilization in the sperm reservoir. When insemination takes place after ovulation, the lifespan of oocytes is limited and the sperm cells cannot capacitate and reach the site of fertilization at the right time (Kemp and Soede 1997). Thereby, a reliable prediction of the time of ovulation would be worthwhile. Although oestrus duration is the best indicator of ovulation time, unfortunately it is highly variable and gives only a retrospective estimate of the time of ovulation (Soede et al. 1995). Taking into account that the time of ovulation cannot be accurately predicted, most sows receive 2 3 inseminations during oestrus, assuring that at least one semen dose is applied within the optimal AIOV interval. The induction and synchronization of oestrus and ovulation could allow a fully integrated breeding schedule with the purpose of directing the AI within the optimal AIOV interval, thereby minimizing the need for multiple inseminations and reducing semen costs (Driancourt et al. 2013). The understanding of endocrine regulation of follicle development and ovulation, and the availability of commercial reproductively active substances (i.e. hormones and analogues) make fixedtime AI (FTAI) possible in sows (Br ussow et al. 2009). The combined use of a follicular development inducer and an ovulation inducer should be sufficient to eliminate the oestrus detection in weaned sows. However, the expenses required for the implementation of this hormonal protocol could make it impracticable. Therefore, protocols considering the oestrus onset as the timing for a single injection of an ovulation inducer have been applied in weaned sows (Zak et al. 2010; Wongkaweewit et al. 2012). The substances used to control the timing of ovulation include hcg, GnRH (or analogue) (Wongkaweewit et al. 2012) and plh (Zak et al. 2010; Fontana et al. 2014). In protocols without oestrus detection, in sows, the time for hormonal application is defined based on the moment of weaning. The hormones used to induce ovulation are GnRH analogues such as buserelin (Driancourt et al. 2013) and triptorelin (Knox et al. 2014). Some protocols may even consider ecg administration at weaning to stimulate follicle development

4 New Artificial Insemination Technologies for Swine 83 and synchronization of the follicular phase (Cassar et al. 2005). For gilts, ecg may be used to achieve a better synchronization effect, as a wide variation in the oestrus cycle is observed among this group of females (Br ussow et al. 2009). For this purpose, protocols for gilts are based on the suppression of follicle development for at least 14 days with progesterone analogues, such as altrenogest, and the subsequent stimulation of follicular growth and ovulation with ecg and GnRH (or analogues) or plh, respectively (Degenstein et al. 2008; Martinat-Botte et al. 2010). The current knowledge of reproductive physiology and regulation of reproductive processes allowed FTAI to be successfully developed in sows. Nevertheless, it seems that reproductive performance is still compromised in sows submitted to FTAI protocols in comparison with multiple AI protocols. The goal for the widespread FTAI use is to achieve an acceptable reproductive performance until it becomes economically viable, considering the savings in labour, genetic gain and fewer doses used per pregnant sow (Driancourt et al. 2013). However, the use of hormones is another limit factor to be considered in swine industry. The public concern about hormones utilization in livestock is an issue for some countries that face debates about food safety and may impair the wide application of this technique. Strategic Use of Genetically Superior Boars The widespread use of pooled semen and the relatively high number of sperm cells per oestrus tend to mask and compensate the suboptimal fertility of boars (Dyck et al. 2011). Differences in relative fertility become evident when low sperm doses (<1.5 billion) are used for AI (Flowers 2002; Mezalira et al. 2005; Ruiz- Sanchez et al. 2006). Some boars have low reproductive performance (litter size) if the sperm cell number in each AI dose is reduced, due to compensable sperm traits (Flowers 2013). However, some boars can have high performance (high farrowing rate and litter size) even with low dose concentration (1.5 billion) (Kummer et al. 2013). The development of improved techniques for evaluating semen characteristics that are effective predictors of relative boar fertility allows the identification and removal of less fertile boars from commercial studs. Furthermore, this will optimize the use of proven high fertility and high genetic merit boars. With a lower number of sperm cells per AI dose and fewer inseminations per sow, it is possible to direct the use of genetically high-indexed boars, hence allowing more sows being inseminated by these superior sires (Foxcroft et al. 2008). Another strategy is driving semen doses of high-indexed boars to insemination that is closer to ovulation for the majority of the sow population. The sperm cells from these boars would have a greater chance of producing the majority of piglets in the herd. Concluding Remarks Reducing the number of spermatozoa used per sow is the main aim of an efficient AI programme. Some boars show acceptable results, even when their semen doses contain low numbers of sperm cells. Moreover, exploring the use of high genetic merit boars assures profitable gains in market pigs. Studies concerning biotechniques such as PCAI and FTAI should be encouraged to be run and applied on farms. Acknowledgements The authors acknowledge the support of Conselho Nacional de Desenvolvimento Cientıfico e Tecnologico CNPq, for their financial support. Conflict of interest None of the authors have any conflict of interest to declare. 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In: Wheeler M, Smith L (eds), Proceedings of the Annual Conference of the International Embryo Transfer Society. Theriogenology 57, 392. Wongkaweewit K, Prommachart P, Raksasub R, Buranaamnuay K, Techakumphu M, De Rensis F, Tummaruk P, 2012: Effect of the administration of GnRH or hcg on time of ovulation and the onset of estrus-to-ovulation interval in sows in Thailand. Trop Anim Health Prod 44, Zak LJ, Patterson J, Hancock J, Hockley D, Rogan D, Foxcroft GR, 2010: Benefits of synchronizing ovulation with porcine luteinizing hormone in a fixed-time insemination protocol in weaned multiparous sows. J Swine Health Prod 18, Submitted: 10 Feb 2015; Accepted: 19 Apr 2015 Author s address (for correspondence): FP Bortolozzo, Departamento de Medicina Animal, Faculdade de Veterinaria, Universidade Federal do Rio Grande do Sul, Avenida Bento Goncßalves, 9090, CEP Porto Alegre-RS, Brazil. fpbortol@ufrgs.br