**15. Boar semen cryopreservation, experiences in Thailand**

yond the tip of the outer catheter). The IUI catheter can be used with minimal training and it does not seriously delay the process of insemination, although it can only be safely used in sows [83]. Under commercial conditions, use of the IUI catheter with extended fresh semen

ble effect on both FR and LS (89% FR and 12 LS) compared with 91% FR and 12.5 LS after

sperm, but IUI sows had significantly less piglets born per litter (1.5 to 2 smaller LS). The reasons for the loss in LS have not been clarified. Rozeboom and co-workers suggested that several factors such as aged sperm, improper semen handling or insemination-ovulation in‐ terval can cause decreases in reproductive performances when low numbers of sperm are used, and in order to obtain consistently high fertility results, a slightly higher number of

Non-surgical DIUI has been performed in non-sedated pigs using a flexible fiber optic endo‐ scope (1.35 m length, 3.3 mm outer diameter) inserted via the vagina and cervix to reach the upper segment of one uterine horn [84]. The procedure required 3 to 5 min in 90% of the females. After this DIUI, only 1% of the sows showed signs of uterine infection. However, the endoscope is a highly expensive instrument and unpractical for routine use. A flexible catheter was therefore developed on the basis of the propulsion force and flexibility of the fibro-endoscope [85]. The method allows deposition of low sperm doses of either fresh or FT sperm. Moreover, the technology can be successfully used to produce piglets with sex-sort‐

Using fresh semen, FR and LS were not statistically different between DIUI with 150x106 sperm per dose and conventional AI with 3x109 sperm, ranging from 83 to 87% FR and 9.2 to 10.4 LS [88]. Nonetheless, LS was always lowest in the DIUI sows. Similarly, although no differences in FR were found (83% and 90% for DIUI and conventional AI, respectively), DIUI sows had less LS (10.5 and 12.9, respectively). The low LS achieved in the DIUI sows

incomplete bilateral fertilization, and could be overcome by increasing the number of in‐

of DIUI sows (83% and 9.7) were not different from those of conventional AI sows (83% and

obtained. With hormonally induced ovulation and a single DIUI, the FR was 77.5% and LS was 9.3, while with spontaneous ovulation and two DIUIs, the FR was 70% and LS was 9.3. The lower fertility obtained in the latter group resulted from the suboptimal inseminationovulation period [91]. Bolarin and staff working with spontaneously ovulating sows (n=407) obtained FR of over 80% and about 10 piglets born per litter when two DIUIs, at 6 h interval,

sperm was performed in hormonally induced ovulating sows, both FR and LS

FT sperm per dose were conducted at the peri-ovulatory period [92]. It has

sperm probably resulted from the high incidence of unilateral or

sperm per dose [90]. On the other hand, when a single DIUI

sperm per dose) was used for DIUI, promising results were

sperm per insemination dose and results in a compara‐

sperm and conventional AI with 3x109

sperm. However, in the field trials carried out by references

can reduce sperm numbers to 1x109

[86,87], FR were similar between IUI with 1x109

26 Success in Artificial Insemination - Quality of Semen and Diagnostics Employed

**14. Deep intra-uterine insemination (DIUI) (Figure 1b)**

conventional AI with 3x109

sperm should be considered.

ed sperm [88], or for embryo transfer [89].

inseminated with 150x106

with 150x106

with only 1x109

seminated sperm to 600x106

10) [85]. When FT semen (1x109

In tropical countries including Thailand, cryopreservation of boar semen is nowadays per‐ formed in a very limited scale and it has yet to be conducted for the commercial purpose. Our studies undertaken between 2004 and 2009 therefore aimed to develop boar semen cry‐ opreservation in Thailand. Effects of straw volume, Equex STM paste added to a freezing extender and of the individual differences on boar sperm quality after cryopreservation were investigated. In addition, in vivo fertility results such as fertilization rate, FR and LS of FT boar semen after DIUI and IUI in multiparous sows were evaluated.

Using a lactose-egg yolk extender with 9% glycerol as a freezing extender of boar semen, it was demonstrated that after thawing the motility, viability and NAR of sperm evaluated with conventional methods were improved when 1.5% Equex STM paste was added into the freezing media [94]. This finding confirms beneficial effects of the detergent on preventing/ diminishing cell damage during the freeze-thawing process [68,95]. Equex STM paste im‐ proves post-thaw survival of sperm by acting as a surfactant to stabilize cell membranes, particularly acrosomal membranes, and to protect sperm against the toxic effects of glycerol during cryopreservation [73]. However, since the positive effects of this substance are only observed in the present of egg yolk in the semen extender, it is suggested that Equex STM paste exerts its beneficial action through the alteration of low-density lipoproteins in egg yolk rather than directly affects sperm membranes [69].

In theory, post-thaw sperm loaded in 0.5-ml straws which have smaller surface-to-volume ratio should not have a better quality than those in 0.25-ml straws. Nevertheless, based on the results of 12 ejaculates from 4 boars evaluated in our study [94], the viability and normal morphology of FT sperm packaged in 0.5-ml straws were superior to those in 0.25-ml straws despite being frozen and thawed with their own optimal protocols. The reason behind this is unknown, but it is interesting that similar results have also been observed in dog semen [96]. Therefore, in order to find the reason and draw conclusions with boar sperm, more in‐ vestigations in this aspect might have to be performed.

With regard to effect of individual variations on the FT sperm quality, 45 ejaculates of 15 boars from three breeds (Landrace (L), Y and Duroc (D); 5 boars each) were studied [97]. It was found that the breed of boar and the individual boars within the same breed significant‐ ly influenced most of the FT sperm parameters evaluated. For instance, the post-thaw sperm viability in D and L boars was significantly higher than Y boars. The motility and the normal morphology of FT sperm were lowest in Y boars. L boars seemed to have the most varia‐ tions in many of the FT sperm parameters. The difference in sperm quality among individu‐ al boars that was found in our study was in agreement with previous findings [52,98], suggesting that such individual variation may be correlated with difference in physiological characteristic of the sperm plasma membrane among boars. Additionally, the genomic dif‐ ferences between individual boars may be responsible for freezability and post-thaw quality of their sperm [55].

According to the results of our studies, it could be indicated that timing of insemination in relation to ovulation and sperm numbers per insemination dose are important factors for successful insemination regardless of insemination procedures and types of semen used. The time of insemination factor becomes more essential when using FT semen because the life span of FT sperm in the female reproductive tract is relatively short compared with the fresh cells, i.e. 4 to 8 h vs about 24 h after insemination, respectively [103,104]. It has been demonstrated that the number of sperm per insemination dose is related to both the number of functional sperm colonized in the oviductal sperm reservoir and fertilization rate [49,101]. Insufficient sperm numbers in the DIUI group might account for the lower fertilization rate

Improvement of Semen Quality by Feed Supplement and Semen Cryopreservation in Swine

The feed supplement containing the rich of PUFAs, vitamins and minerals can improve the sperm motility, vitality and number of sperm per ejaculation in boar. The success of feed supplement depends on the initial performance of the boar. They may not improve the se‐ men quality if the boars are the good performance of semen producers. Moreover, taking all of our researches, we can conclude that the production of cryopreserved boar semen and AI with FT boar semen could be successfully performed in Thailand and its application in com‐ mercial farm is undergoing. An IUI procedure was considered to be suitable for FT boar se‐ men to produce acceptable fertility rates. This is very useful for the conservation and/or

1 Department of Obstetrics Gynaecology and Reproduction, Faculty of Veterinary Science,

[1] Am-in N, Kirkwood RN, Techakumphu M, Tantasuparuk W. Lipid profiles of sperm and seminal plasma from boars having normal or low sperm motility. Theriogenolo‐

2 Institute of Molecular Biosciences (MB), Mahidol University, Thailand

, Wichai Tantasuparuk1

and

http://dx.doi.org/10.5772/51737

29

[99] and thus smaller LS [102].

production of animal with high genetic merits.

Mongkol Techakumphu1\*, Kakanang Buranaamnuay2

Chulalongkorn University, Thailand

gy 2011;75: 897-903.

**16. Conclusion**

**Author details**

Nutthee Am-In1

**References**

Cervical AI with FT semen usually results in suboptimal fertility; thereby, deep AI using IUI and DIUI procedures was developed. We evaluated fertility (fertilization rate, FR and LS) of FT boar semen after IUI, with 2x109 total sperm per dose, and DIUI, with 1x109 per dose, in spontaneously ovulating weaned sows. The results revealed that at approximately 2 days following inseminations either with IUI or DIUI, embryo(s) could be recovered from both sides of the oviducts. This observation, the first report in FT semen [99], was consistent with previous studies where the extended fresh semen was used [85,100,101]. It was demonstrat‐ ed that both transuterine and transperitoneal migrations were involved in transport of sperm inseminated using DIUI to reach the other side of the oviduct [85]. Nonetheless, com‐ paring between techniques, fertilization rate in the IUI group was significantly higher than the DIUI group. The reason for this finding might not associate with the insemination tech‐ niques, but rather it was a result of insemination time relative to the moment of ovulation which was not appropriate in the DIUI group (≥ 8 h before ovulation).

After AI using the same procedures (IUI and DIUI) and same numbers of FT sperm (1 to 2 x109 per dose), acceptable fertility (67% FR and 7.7 to 10.5 LS) were obtained in both groups (P>0.05); however, TB in the DIUI group was about 3 piglets fewer than the IUI group. This was probably the consequence of inadequate numbers of functional sperm used for DIUI (400x106 motile sperm) which leaded to the unilateral and/or incomplete bilateral fertiliza‐ tion and resulted in the low LS [102] (Table 2)


**Table 2.** Non-return rate, farrowing rate, number of total piglets born per litter and number of piglets born alive per litter after intra-uterine insemination (IUI) and deep intra-uterine insemination (DIUI) with frozen-thawed boar semen [102]

According to the results of our studies, it could be indicated that timing of insemination in relation to ovulation and sperm numbers per insemination dose are important factors for successful insemination regardless of insemination procedures and types of semen used. The time of insemination factor becomes more essential when using FT semen because the life span of FT sperm in the female reproductive tract is relatively short compared with the fresh cells, i.e. 4 to 8 h vs about 24 h after insemination, respectively [103,104]. It has been demonstrated that the number of sperm per insemination dose is related to both the number of functional sperm colonized in the oviductal sperm reservoir and fertilization rate [49,101]. Insufficient sperm numbers in the DIUI group might account for the lower fertilization rate [99] and thus smaller LS [102].
