Distribution of Spermatozoa and Embryos in the Female Reproductive Tract after Unilateral Deep Intra Uterine Insemination in the Pig

Abstract

The present study was performed to investigate the number of either the spermatozoa or the embryos in the reproductive tracts of sows after unilateral, deep, intra uterine insemination (DIUI). Two experiments were conducted, 10 sows were used in experiment I and eight sows were used in experiment II. Transrectal ultrasonography was used to examine the time when ovulation took place in relation to oestrus behaviour. The sows were inseminated with a single dose of diluted fresh semen 6–8 h prior to expected ovulation, during the second oestrus after weaning. In experimental I, five sows were inseminated by a conventional artificial insemination (AI) technique using 100 ml of diluted fresh semen, containing 3000 × 106 motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 × 106 motile spermatozoa. The sows were anesthetized and ovario-hysterectomized approximately 24 h after insemination. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments, namely ampulla, cranial isthmus, caudal isthmus, utero-tubal junction (UTJ), cranial uterine horn, middle uterine horn and caudal uterine horn. Each segment of the reproductive tracts was flushed with Beltsville thawing solution (BTS) through the lumen. The total number of spermatozoa in the flushing from each segment were determined. In experimental II, eight sows were inseminated by the DIUI technique using 5.0 ml diluted fresh semen containing 150 × 106 motile spermatozoa. The sows were anesthetized 61.1 ± 12 h after insemination (48–72 h) and the embryos were flushed from the oviduct through the proximal part of the uterine horn. It was revealed that, in experimental I, the spermatozoa were recovered from both sides of the reproductive tract in the AI-group, and from unilateral side of the reproductive tract in the DIUI-group (three sows from the left and two sows from the right sides). The number of spermatozoa recovered from the reproductive tracts was higher in the AI- than the DIUI-group (p < 0.001). In experiment II, fertilization occurred in five of eight sows (62.5%) after DIUI. The number of ova that ovulated were 16.4 ± 2.6 per sow and the embryos numbering 11.4 ± 2.3 per sow were recovered from both sides of the reproductive tract. In conclusion, the spermatozoa given by DIUI could be recovered from only one side of the reproductive tract of sows at approximately 24 h after DIUI via the flushing technique. However, embryos were found in both sides of the oviducts and the proximal part of the uterine horns 48–72 h after insemination, indicating that the fertilization occurred in both sides of the oviducts. Artificial insemination (AI) in the pig has been developed since 1926 and nowadays is widely used in the pig industry all over the world (Johnson et al. 2000; Tummaruk et al. 2000, 2004). In practice, 3000 × 106 motile spermatozoa in a volume of 80–100 ml are inseminated into the sow two to four times during standing oestrus. The AI catheter is inserted through the vagina, placed in the cervix of the sows, and the semen is released at the distal part of cervix. Up to 25% of the spermatozoa inseminated are lost due to semen backflow within a few hours of insemination (Steverink et al. 1998). The rest of the spermatozoa are transported through the uterine horn before reaching the sperm reservoir in the caudal part of the oviducts. Most of the spermatozoa are lost due to uterine phagocytosis by polymorphonuclear leucocytes (Rozeboom et al. 1998; Kaeoket et al. 2003). Fertilization takes place at the ampullatory-isthmic junction (AIJ) in the oviduct of the sows soon after ovulation. Less than 1% of the sperm are discovered at the fertilization site during the peri- and post-ovulation period (Mburu et al. 1996). Krueger et al. (1999) found that 10 million spermatozoa per insemination are sufficient for successful insemination surgically placed into the tip of the uterine horns in gilts. Recently, a special designed catheter for AI in pig (180 cm length and 4 mm diameter) has been developed for non-surgical deep intra-uterine insemination (DIUI) with a reduced number of spermatozoa (Martinez et al. 2001; Vazquez et al. 2005). This technique was developed to reduce the losses of spermatozoa and ensure optimal fertilization (Martinez et al. 2001). Similar techniques have already been reported in cattle (Seidal et al. 1997; Hunter 2003; Verberckmoes et al. 2004), horses (Nie et al. 2003), dogs (Tsutsui et al. 1989), cats (Tsutsui et al. 2000) and goats (Sohnrey and Holtz 2005). In pig, the DIUI catheter is inserted through the uterine horn and deposited semen in one horn, at its proximal third, close to the sperm reservoir. Using this technique, a 20-fold reduction in the number of spermatozoa per insemination was possible without any significant effect on the farrowing rate (FR) and litter size (Martinez et al. 2002). The use of the DIUI technique under farm conditions would allow a more efficient use of semen from genetically superior boars. Earlier studies have shown that a flexible catheter could be passed through the cervix completely in 90–95% of multiparous sows (parities 2–6; n = 147) taking approximately 4 min/insemination (Martinez et al. 2001, 2002). The technique is also applicable for some advanced biotechnology procedures such as frozen-thawed semen, sex sorted sperm and embryo transfer (Roca et al. 2003; Vazquez et al. 2003; Martinez et al. 2004). Up to date, data concerning sperm transport and fertilization after DIUI using a small volume of semen as well as a reduced number of spermatozoa are still limited. Martinez et al. (2002) demonstrated that the embryos were found in both side of the tip of the uterine horn 2 days after DIUI in five sows. Investigation on sperm distribution and the fertilization process after the DIUI technique is important if wish to know more about the mechanism of successful DIUI in the pig. The present study was performed to investigate the number of spermatozoa in the female reproductive tract 24 h after DIUI using a low number of spermatozoa per dose as compared with conventional AI and to investigate the number of embryos on each side of the reproductive tract after DIUI. Ten crossbred (Landrace × Yorkshire) multiparous sows (LY) were used in experiment I and eight crossbred LY multiparous sows were used in experiment II. On the day of weaning, they were brought from commercial farms to the Department of Obstetrics, Gynaecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University and were allocated to individual pens adjacent to adult boars. The sows were fed 3 kg/day (twice a day) with a commercial feed (Starfeed176®; BP Feed Co. Ltd, Saraburi, Thailand) containing 15% protein, 2% fat and 10% fibre. Water was provided ad libitum. The sows were observed for pro-oestrus twice a day (am/pm). In experimental I, the sows were randomly assigned to two groups according to ear tag, a control group (conventional AI, n = 5) and a DIUI group (n = 5). After the sows showed signs of pro-oestrus, the sows were examined for the onset of standing oestrus every 6 h by using a back pressure test in the presence of a mature boar. The onset of oestrus was defined as being 3 h before the onset of the standing response. The end of oestrus was defined as 3 h after the last standing response. Transrectal ultrasonography (Echo camera SSD-550; Aloka Co. Ltd, Tokyo, Japan) was performed every 4 h, starting from approximately 10–12 h after the onset of oestrus, using a 5 MHz probe to examine the time when ovulation took place in all sows. The time of ovulation was defined as being 2 h after the last detection of follicles in the ovaries. Previous studies have shown that the interval from the onset of oestrus to ovulation during the first two cycles after weaning was not significantly different and repeated ultrasonographic examination can predict the time of ovulation during the subsequent oestrus (Mburu et al. 1995). In the present study, the time of ovulation in each individual sow was used to estimate the time of insemination during the subsequence oestrus cycle. The ultrasonographic examination was not performed after insemination, so as to not disturb the sows and the process of sperm and/or oocyte transport. The semen was collected from an adult proven Duroc boar by the gloved-hand method. Semen was examined for quality before further processing, i.e. motility, concentration and morphology. Semen with a motility of ≥70%, a concentration of ≥150 spermatozoa/ml and with normal sperm ≥85%, was diluted, using Beltsville thawing solution (BTS) diluent (Pursel and Johnson 1976) at 35°C. The sperm dose contained 3000 × 106 spermatozoa in 100 ml for conventional AI and 150 × 106 spermatozoa in 5 ml for DIUI. The diluted semen was used immediately or kept refrigerated at 18°C for no longer than 2 days before insemination. The refrigerated dilute semen was warmed in water bath at 35°C for 15 min and was checked for motility before being used. For all inseminations, the pre-insemination diluted semen had to have a motility ≥60%. The sows were inseminated with a single dose of the diluted semen during the second oestrus after weaning. The time of ovulation (determined by ultrasonography) during the first oestrus was used to determine the timing of insemination, which was carried out at 6–8 h prior to the expected time of ovulation. In experimental I, the sows were inseminated by conventional AI (n = 5) or the DIUI (n = 5) and in experimental II, the insemination was performed by the DIUI technique (n = 8). The DIUI technique has been described by Martinez et al. (2001). Briefly, the oestrous sows were inseminated in the gestation crates. After cleaning the perineal area of the sows, a commercial AI catheter (Goldenpig®; Minitub, Tiefenbach, Germany) was inserted through the vagina into the cervix. The long flexible catheter (1.8 m) was inserted through the conventional AI catheter. The long catheter was moved forward along one uterine horn (unknown side) for its full length. The diluted fresh semen with 150 × 106 motile sperm in 5.0 ml was deposited in the proximal third of one side of the uterine horn. Subsequently, a warm BTS, 2.5 ml in volume was used to flush the semen into the uterine horn after insemination. In experimental I, the sows were general anaesthetized at approximately 24 h after insemination. General anaesthesia was induced by azaperone (Stressnil®; Janssen Animal Health, Beerse, Belgium), 2 mg/kg, intramuscularly and 30 min later thio-pental sodium, 10 mg/kg, was given intravenously. The ovario-hysterectomy (OVH) was performed by laparotomy. The reproductive organs were removed and immediately transferred to the laboratory. The number of corpora lutea (CL) was recorded. The oviducts and the uterine horns on each side of the reproductive tracts were divided into seven segments as demonstrated in Fig. 1. The division of the sows reproductive tracts (1, ampulla; 2, cranial isthmus; 3, caudal isthmus; 4, utero-tubal junction (UTJ); 5, cranial uterine horn; 6, middle uterine horn; 7, caudal uterine horn) The method of sperm recovery was a modification of a similar method used in gilts and described by Kunavongkrit et al. (2003). Briefly, the ampulla segment was flushed with 1 ml of BTS, while the isthmus and the UTJ segment were flushed with 0.5 ml of BTS through the lumen. Each segment was flushed twice into separate plastic vials. The final volume from each flushing was measured from each plastic vial. Each segment of the uterine horn was flushed with 20 ml of BTS, twice, into a flask. The total number of spermatozoa in the flushing from each segment were determined using Neubauer haemocytometer. If spermatozoa were not found in the counting chamber, the flushing media was centrifuged at 1000 × g for 2 min to remove supernatant and the total number of spermatozoa was recounted. In experimental II, the sows were generally anaesthetized at approximately 48–72 h after insemination using the same methods as described above. A caudal midline incision was made (15–20 cm) with the sows in a dorsal recumbency position. Left and right uterine horns and the ovaries were approached. The number of corpora lutea on each side of the ovary was counted (Fig. 2a). A 1 cm incision was made approximately 20 cm below the utero-tubal junction (UTJ). A Foley catheter were inserted through the incision and moved forward to the tip of the horn and the balloon was made at the tip of the catheter (Fig. 2b). A polyethylene catheter (outer diameter 2.42 mm) was inserted from the end of the oviduct (infundibulum) and was fixed within the oviduct (Fig. 2c). A 100 ml of phosphate buffer solution (PBS) was syringed into the tip of the oviduct through the uterine horn (Fig. 2c). The PBS solution was forced into the Foley catheter, where a flask bottle was used to collect all the flushed solution (Fig. 2d). Using this technique, the embryos were flushed from the oviduct through the proximal part of the uterine horn (Fig. 2e). The flushed solution from each side of the reproductive tract were pour on a plastic plate and the embryos and unfertilized ova were observed under a stereomicroscope with a magnification between 10 and 40 times. The number of embryos and number of ovulations on the left and right sides of the reproductive tract were compared. The recovery rate was defined as the number of embryo, divided by the number of CL, multiplied by 100. The fertilization rate was defined as the number of embryo, divided by number of embryo plus unfertilized ova found (within the animal), multiplied by 100. The number of embryos and the fertilization rate were compared between the left and right side of the uterine horns. Embryo collection procedure (a) approaching the ovaries and the uterus of sows (b) inserting the Foley catheter and making a balloon in the proximal segment of the uterus (c) flushing embryos from the oviduct (d) using a flask bottle to collect all the flushing solution and (e) embryos at the four cell stage after collection Data were analysed using SAS software (Statistical Analysis System, SAS Institute, V. 9.0, Cary, NC, USA) (SAS Institute Inc. 1996). Descriptive statistics including the mean and the standard deviations (SD) of parity number, wean-to-oestrus interval (WOI), oestrus-to-ovulation interval (EOI), oestrus interval, the interval from insemination to operation, the number of CL and the length of the uterine horn in experiments I and II were calculated. The number of CL was compared between groups by the student t-test. The number of CL and the number of spermatozoa were compared between the left and the right sides by the pair t-test. The numbers of spermatozoa were log transformed and were compared between groups and between segments using the general linear model (GLM) procedure of SAS. The number of spermatozoa recovered from the cranial, middle and caudal parts of the uterine horn were pooled in the analyses. The statistical model included the groups (AI vs DIUI), segments of the reproductive tracts (ampulla, cranial isthmus, caudal isthmus, UTJ and the uterine horn) and the interaction between the group and the segments. Least-square means were obtained from each class of the variables and were compared by using the student t-test. In experiment II, the number of ovulations in all sows (n = 8), the number of embryos in the pregnant sows (n = 5) and the number of non-fertilized eggs in the non-pregnant sows (n = 3), between the left and the right side of the uterine horns were compared using the pair t-test. Differences with p < 0.05 were regarded to have statistical significance. The average parity number of sows in experimental I was 8.6 ± 1.1 for AI-group and 6.7 ± 1.8 for DIUI-group, and in experimental II was 7.5 ± 1.7. WOI was 6.4 ± 1.8 and 5.3 ± 1.5 days for AI- and DIUI-groups in experimental I and was 4.8 ± 1.3 days in experimental II. EOI was 30.2 ± 9.1 and 43.5 ± 12.5 h for AI- and DIUI-groups in experimental I and was 41.5 ± 14.1 h in experimental II. The periods of standing oestrus were 52.8 ± 26.9 and 60.0 ± 12.1 h for AI- and DIUI-groups in experimental I and was 63.3 ± 10.7 h in experimental II. In experimental I, the operation was performed at 26.0 ± 3.4 and 24.8 ± 1.8 h after insemination in AI- and DIUI-groups, respectively. In experimental II, the sows were operated at 61.1 ± 12.1 h after insemination. For both experiments, all sows have been ovulated at the operation time. The number of ovulations in the sows in the AI-group and the DIUI-group were not significantly different (17.4 vs 17.2, p = 0.93). Number of ovulation between the left and the right ovaries were not significantly difference (p = 0.13). In experimental II, the number of ovulation was 16.4 ± 2.6. On average, the length of the left/right uterine horns were 121/125 and 125/131 cm for AI- and DIUI-groups in experimental I and was 154/158 cm in experimental II. For the conventional AI-group, the spermatozoa could be recovered from both sides of the reproductive tract in all sows (5/5) (Table 1). The number of spermatozoa in the left and the right side of the conventional AI-group did not differ significantly in all segments of the reproductive tract (p > 0.05). In the DIUI-group, the spermatozoa were recovered from only one side of the reproductive tract (three sows from the left and two sows from the right side) (Table 2). The number of spermatozoa in the ampulla, cranial isthmus, caudal isthmus, UTJ and uterine horns in the AI-group were significantly higher than those in the DIUI-group (p < 0.001) (Fig. 3). In both groups, the number of spermatozoa in the UTJ and the uterine horns were higher than those found in the ampulla, cranial isthmus and caudal isthmus (p < 0.001) (Fig. 3). The number of spermatozoa (log transformation) recovered from different segments of the reproductive tract of sows after conventional artificial insemination (AI, n = 5) and unilateral, deep intra-uterine insemination (DIUI, n = 5), UTJ, uterotubal junction; Ca, caudal; Cr, cranial; ***significant differences between the groups (p < 0.001) The overall recovery rate of the oocytes and the embryos was 66.4% (87/131). The number of ovulations per sow was 16.4 ± 2.6 ova (range 14–22 CL). The number of ovulations on the left and the right side of the ovaries were not significantly different (8.5 vs 7.9; p = 0.75). Fertilization occurred in five of eight sows (62.5%) after DIUI (Table 3). On average, 11.4 ± 2.3 embryos per sow were recovered. The embryos were found in both sides of the reproductive tract in every pregnant sow (Table 3). A total of 57 embryos were recovered. Fifty-four embryos had developed to the 4-cell stage (94.7%) and three embryos had developed to the 8-cell stage (5.3%) (Fig. 2e). The number of embryos recovered from the left and the right side of the reproductive tract were not significantly different (left-right = +4.2, p = 0.20) (Table 3). The number of non-fertilized eggs recovered from the left and the right side of the reproductive tract were not significantly different (left-right = −0.6, p = 0.66) (Table 3). The fertilization rate, based on a few number of pregnant sows, was 100%. The present study demonstrated that the spermatozoa were found in only one side of the reproductive tract, while the embryos were found in both sides of the reproductive tracts after DIUI with a reduced number of spermatozoa. The later finding is in agreement with Martinez et al. (2002), who demonstrated that embryos were found in both sides of the tip of the uterine horn 2 days after DIUI in five sows. However, the distribution of spermatozoa after unilateral DIUI has never been reported. For conventional AI in pig, the spermatozoa were transported along the reproductive tracts of the female and store in the sperm reservoir within a few minutes up to an hour after insemination. The number of spermatozoa stored in the sperm reservoir depended on the number of spermatozoa inseminated. The function of the sperm reservoir are to maintain sperm viability and fertilizing capacity and aiding spermatozoa to escape from phagocytosis in the uterine lumen during pre-ovulation period (Rodriguez-Martinez et al. 2005). The spermatozoa are gradually released toward the site of fertilization at the AIJ in relation to ovulation. Recent data suggested that the release of the spermatozoa from the sperm reservoir may be related to the gradual induction of capacitation following the exposure of the ampulatory fluids (Rodriguez-Martinez et al. 2005). Since the capacitated spermatozoa have a very short survival time, the spermatozoa are continuous released from the reservoir rather than a massive progression. In the present study, the spermatozoa might be released from the sperm reservoir in one side of the reproductive tracts to another side in relation to ovulation via a trans-peritoneal migration pathway. The trans-peritoneal migration of spermatozoa has already been demonstrated in heifers (Larsson 1986). Another possibility was that the spermatozoa may have been transported from one uterine horn to another horn during insemination and a low number of spermatozoa were deposited deeply in the crypts and the interfolds of the mucosa of the sperm reservoir. Chatdarong et al. (2004) demonstrated that more spermatozoa were observed in the tissue section of the reproductive tract than were recovered by flushing technique in the female cat that were naturally mated with 35 × 106 total spermatozoa. The investigation of sperm number using the tissue section technique has not yet been done in the present study. However, in pig, it has been suggested that sperm number in the crypts, interfolds and in the central mucosal surface are not different during ovulation and afterwards because of the redistribution of the spermatozoa affected by ovulation (Rodriguez-Martinez et al. 2005). The mechanism for such sperm transport after DIUI in pig remains to be investigated further. In experimental II, five of eight sows got pregnant and 11.4 embryos/sow were recovered after the DIUI, representing a pregnancy rate of 62.5% with 11.4 embryos. However, these results should be interpreted with caution because of the rather low number of sows studied. In other studies where a sufficient number of sows were included, the FR and the total number of piglets born per litter (TB) after DIUI were approximately 82–86% and 9.7–10.0 piglets/litter, respectively (Martinez et al. 2002; Roca et al. 2003). In addition, Roca et al. (2003) demonstrated that the hormonally treated (eCG/HCG) weaned sows (n = 29), inseminated once with 150 × 106 spermatozoa by the DIUI technique, using fresh semen had a 82.7% FR and 9.96 TB. Spontaneously weaned sows (n = 38), inseminated twice with 150 × 106 sermatozoa by the DIUI technique using fresh semen, resulted in a 84.2% FR and 9.88 TB (Roca et al. 2003). Martinez et al. (2002) found that placing DIUI in one side of the uterine horn, with 150 × 106 spermatozoa, resulted in a 86.3% pregnancy rate, a 82.9% FR and 9.7% TB (n = 117). Reducing number of spermatozoa below 25 × 106 spermatozoa/dose resulted in a significant decrease in the FR (Martinez et al. 2002). In the present study, both 4 and 8-cell embryos were observed. This could be explained by the fact that in the pig, the duration of ovulation, in spontaneous ovulating, is approximately 1–3 h (Soede et al. 1992). Pope et al. (1990) showed that 70% of follicles ovulated during a short period of time, while the remaining follicles ovulated over a protracted period. Oocyte from follicles that ovulated first, became more develop embryos, while oocytes from later ovulating follicles became less develop embryos. Ova may be transported to the fertilization site at different times, and then be fertilized at different times. Unlike the mare, both fertilized and unfertilized ova of the sow are transport via the oviduct and enter the uterus the second day after ovulation (Mwanza et al. 2002). Mwanza et al. (2002) found that there is no difference in the transportation of fertilized and unfertilized ova in the reproductive tract of pigs. Martinez et al. (2006) demonstrated that TB after DIUI in sows that had spontaneous oestrus were significantly fewer than those from conventional AI (9.8 vs 10.9), although the FR was not significantly different. In addition, when embryos on day six after oestrus were observed, it was found that partial fertilization was higher in the DIUI sows (35%) compared with the conventional AI sows (5%). Fertilization after DIUI, took place in both uterine horns, in approximately 80% of the sows, while unilateral fertilization was found in approximately 20% of sows (Martinez et al. 2006). In the present study, all ova from the five pregnant sows were fertilized, while ova from the three non-pregnant sows were not fertilized at all. Partial and/or unilateral fertilization were not found in the present study. The reason might be because the sows in the present study were inseminated once, 6–8 h before ovulation, while in earlier studies (Martinez et al. 2006), the sows were inseminated three times (12, 24 and 36 h after the onset of oestrus) regardless of the timing of ovulation. Variations on the timing of ovulation in spontaneous ovulating sows might cause partial and/or unilateral fertilization. In dogs, a surgical unilateral insemination with ≥20 × 106 spermatozoa in a volume of 0.1 ml resulted in bilateral fertilization, while insemination with ≤10 × 106 spermatozoa in 0.1 ml volume resulted in unilateral fertilization (Tsutsui et al. 1989). In the present study, the recovery rate of the embryo was relatively low; therefore, few embryos may be loss during the surgical collection. The embryos collection technique somehow needed improvement. In conclusion, DIUI in multiparous sows resulted in a significantly lower number of spermatozoa in the female reproductive tract 24 h after insemination, compared with conventional AI, and the spermatozoa were recovered from only one side of the sperm reservoir via the flushing technique. However, embryos were found in both the oviducts and the proximal part of the uterine horns 48–72 h after insemination, indicating that the fertilization occurred in both the oviducts. This study was financed by the Research and Development Center for Livestock Production Technology, Chulalongkorn University. Our thanks to Mrs Wanpen Adulyanubap and Mr Jinda Singlor for technical support. Thanks to Prof. Heriberto Rodriquez-Matinez for helpful collaboration between Thailand and Sweden. We also very much appreciated Prof. Juan M. Vazquez for providing the DIUI catheter for the experiment. We would like to thanks Dr Terry W. Heard for linguistic scrutinizing.