The influence of pre- and post-ovulatory insemination on sperm distribution in the oviduct, accessory sperm to the zona pellucida, fertilisation rate and embryo development in sows

Animal Reproduction Science 71 (2002) 239–248

The influence of pre- and post-ovulatory insemination on sperm distribution in the oviduct, accessory sperm to the zona pellucida, fertilisation rate and embryo development in sows K. Kaeoket a,c,∗ , E. Persson b,c , A.-M. Dalin a,c a

Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), S-750 07 Uppsala, Sweden b Department of Anatomy and Histology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), S-750 07 Uppsala, Sweden c Centre for Reproductive Biology in Uppsala (CRU), S-750 07 Uppsala, Sweden

Received 15 October 2001; received in revised form 14 February 2002; accepted 14 February 2002

Abstract The aim of present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the accessory sperm counts on the zona pellucida and early embryonic development. Thirty-six crossbred multiparous sows (Swedish Landrace × Swedish Yorkshire) were artificially inseminated once either at 20–15 h before (group AIB) or at 15–20 h after (group AIA) ovulation by using a pooled semen of two boars. Thereafter, they were randomly allocated to one of five groups: slaughter at 5–6 h after AI (group I-AIB), at 20–25 h after ovulation (groups II-AIB and II-AIA), at 70 h after ovulation (groups III-AIB and III-AIA), on day 11 (groups IV-AIB and IV-AIA, first day of standing oestrus = day 1) and on day 19 (groups V-AIB and V-AIA). The plasma levels of oestradiol-17␤ and progesterone differed significantly (P ≤ 0.05 and P ≤ 0.001, respectively) between AI before (group AIB) and after (group AIA) ovulation. All sows inseminated before ovulation (group I-AIB) had spermatozoa in the UTJ but this was found only in one of the late inseminated sows (group II-AIA). The number of oocytes with spermatozoa in the ZP differed significantly (P ≤ 0.001) between ‘group II-AIB’ and ‘group II-AIA’. Comparing fertilisation rate, the fertilisation rate in ‘group III-AIB’ was significantly (P ≤ 0.001) higher than in ‘group III-AIA’. A larger number of recovered embryos (on days 11 and 19) per sow in groups IV-AIB and V-AIB compared with IV-AIA and V-AIA were found. The embryos in ∗ Corresponding author. Present address: Department of Obstetrics and Gynaecology, Faculty of Veterinary Medicine, Swedish University of Agricultural Sciences (SLU), S-750 07 Uppsala, Sweden. Tel.: +46-1867-2323; fax: +46-1867-3545. E-mail address: [email protected] (K. Kaeoket).

0378-4320/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 4 3 2 0 ( 0 2 ) 0 0 0 2 2 - 2

240

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

group IV-AIB were also larger than embryos in group IV-AIA. In group V-AIA, no embryos were found. In conclusion, the results of this study showed that if insemination was performed at 15–20 after ovulation in sows that still were in standing oestrus, the transport of spermatozoa to the UTJ and oviduct was impaired and a lower proportion of oocytes with accessory spermatozoa to the zona pellucida was found compared with insemination before ovulation. In the late inseminated sows, fertilised oocytes and developed embryos were observed up to day 11 but no embryos were found at day 19. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Sow; Artificial insemination; Spermatozoa; Embryo development

1. Introduction At insemination in pigs, spermatozoa are deposited into the cervix/uterus and transported to a natural sperm reservoir (utero-tubal junction or UTJ), where they can be arrested in mucosal diverticulae for up to 36–40 h (Viring et al., 1980; Hunter, 1984; Raycoudhurry and Saurez, 1991). In addition, their viability can be maintained beyond the reach of neutrophils, which enter the uterine lumen after insemination (Lovell and Getty, 1968; Pursel et al., 1978; Rozeboom et al., 1998) but not the lumen of the UTJ and oviductal isthmus (Hunter et al., 1987; Rodriguez-Martinez et al., 1990). The spermatozoa are released from the UTJ to the fertilisation site (ampullary-isthmic junction or AIJ) (Hunter, 1984). Mburu et al. (1996) reported that the release of spermatozoa from the UTJ and distal part of the isthmus to the fertilisation site (AIJ) is influenced by the ovulation event. The insemination to ovulation interval influences the fertilisation rate and number of accessory sperm bound to the zona pellucida (Waberski et al., 1994; Soede et al., 1995a,b). It has also been shown that the optimal insemination time in order to achieve a good fertilisation rate is within 24 h before ovulation (Soede et al., 1995a; Nissen et al., 1997), while insemination after ovulation results in a lower farrowing rate and litter size in pigs (Nissen et al., 1997; Rozeboom et al., 1997; Terqui et al., 2000). No study has been published on the sperm distribution in the porcine oviduct at post-ovulatory insemination. Therefore, the aim of the present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the number of accessory spermatozoa bound to the zona pellucida and early embryo development. 2. Materials and methods 2.1. Animals and general management Thirty-six crossbred multiparous sows (Swedish Landrace × Swedish Yorkshire) with an average parity number of 3.5 ± 0.6 (mean ± S.D., range 3–5) were purchased from a commercial herd and brought to the Department of Obstetrics and Gynaecology on the day of weaning. Prior to this study, the sows had shown a normal reproductive performance. Their body weights ranged from 174 to 261 kg. The sows were kept in individual pens and boars were housed in the same stable throughout the experimental period. The sows were

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

241

fed twice a day according to the Swedish breeding stock standard for dry sows (Simonsson, 1994). The feed allowance was 4–5 kg per day (barley-based sow diet, 14.5% protein and 12.5 MJ/kg metabolisable energy) until first oestrus after weaning; thereafter they were fed about 2–2.5 kg per day. Water was available ad libitum. Two sows were excluded due to a weaning to oestrus interval of more than 10 days and udder health problems, respectively. 2.2. Oestrous detection and monitoring of ovulation Oestrous detection was performed by inspection of the vulva for reddening and swelling (prooestrus) as well as by control of the standing reflex (oestrus) in the presence of a boar. The oestrous detection was carried out twice daily and every 4 h after the onset of prooestrus. Ovulation was followed every 4 h by transrectal ultrasonography as described earlier (Soede et al., 1992; Dalin et al., 1995). 2.3. Insemination and slaughter All sows were inseminated once by the same person either at 20–15 h before expected ovulation (estimated from the first oestrus after weaning) or 15–20 h after ovulation in their second oestrus after weaning with a dose of pooled semen (two boars of proven fertility), containing 10 × 109 spermatozoa in 100 ml Beltsville thawing solution (BTS) (Pursel and Johnson, 1976). After dilution, the semen was stored at 16–18 ◦ C and used within 48 h. All sows were allocated to slaughter in different groups, see Table 1. The genital organs were removed immediately after slaughter. The number of mature follicles (group I) or corpora lutea (groups II–V) were counted (Table 1). 2.4. Recovery of oocytes and spermatozoa from flushed oviducts The flushing technique allows a more accurate assessment of the number and distribution of oviductal spermatozoa than in situ observation with a scanning electron microscope (Mburu et al., 1996). For that reason, the UTJ (1 cm of the tip of the uterine horn and 1 cm of the isthmus) were flushed twice with 0.5 ml and isthmus and ampulla separately twice with Table 1 Distribution of the sows, and numbers (means ± S.D.) of large follicles, corpora lutea (CL) in the different experimental groups Groups

AI before ovulation (AIB)

Number of follicles or CL

AI after ovulation (AIA)

Number of CL

Time of slaughter

I II III IV V

n=4 n=4 n=4 n=3 n=3

17.3 ± 6.0a 19.3 ± 2.2 21.0 ± 5.4 21.0 ± 4.6 17.0 ± 2.6

– n = 4b n=4 n=4 n=4

– 16.5 ± 3.4 20.3 ± 3.3 20.8 ± 1.9 20.3 ± 3.9

5–6 h after AI 20–25 h after ovulation 70 h after ovulation Day 11c Day 19c

a

Number of follicles. Group II-AIA: slaughter 5–6 h after AI. c First day of standing oestrus = day 1. b

242

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

10 ml of a phosphate buffer saline (PBS) at 37 ◦ C (both sides). All the flushings were made directly into plastic Eppendorf vials (UTJ) or petri dishes (isthmus and ampulla). Spermatozoa from the flushed UTJ (groups I-AIB and II-AIA) were fixed with formal-saline solution and evaluated under the light microscope by using a haemocytometer (Bürker chamber, magnification 400×). The oocytes (groups II-AIB and II-AIA) were recovered under a stereomicroscope and examined under an inverted phase contrast microscope (magnification 200×) for quality and presence of spermatozoa in the zona pellucida. Thereafter, the oocytes (groups II-AIB and II-AIA) were fixed in 25% (v/v) acetic alcohol, stained with 1% (w/v) orcein in 45% (v/v) acetic acid, and examined under a phase contrast microscope (magnification 400×) to confirm the presence of spermatozoa in the zona pellucida and pronuclear development. The oviducts (isthmus and ampulla) of group III sows were also flushed to recover oocytes. 2.5. Recovery of unfertilised and cleaved oocytes from flushed uterine horns The uterine horns (20 cm from the tip of the horns) from group III sows were flushed twice with 20 ml of PBS at 37 ◦ C and the fluid was collected in petri dishes. The oocytes were then isolated and examined under a stereomicroscope and an inverted phase contrast microscope (Olympus, Japan; magnification 200×) for their morphology and developmental stage. An oocyte was considered as non-fertilised when no cleavage was observed. A cleaved oocyte was considered normal when a clear perivitelline space was seen and the blastomeres were distributed with no sign of disintegration. 2.6. Recovery of spherical and filamentous embryos from flushed uterine horns The uterine horns from group IV sows were equally divided into three parts and flushed twice with 20 ml of phosphate buffer saline (PBS) at 37 ◦ C. The fluid was collected in petri dishes and the embryos were examined in PBS under a stereomicroscope. Degeneration of embryos was evaluated using criteria as described earlier by Nissen et al. (1997), i.e. retarded growth after hatching and markedly smaller than their littermates, or with a surface covered with villous-like structures. In group V sows, the total length of the uterine horns was cut open to enable macroscopical recovery of embryos. The horns were further immersed in PBS at 37 ◦ C as an extra control for the presence of additional embryos. Ages of embryos recovered from groups IV and V sows and calculated from the ovulation were 10 and 18 days, respectively. 2.7. Definition of recovery rate Recovery rate was determined as the percentage of oocytes/embryos recovered as a proportion of the total number of corpora lutea. 2.8. Blood collection and hormone assays Within 6 h after AI, blood samples were collected from the jugular vein on restrained animals (12 out of 18 sows from group AIB and 13 out of 16 sows from group AIA), using

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

243

vacutainer heparin-tubes. The blood samples were centrifuged immediately after collection at 3000 rpm, for 10 min, and the plasma was stored in plastic tubes at −20 ◦ C until analysed. The hormones were analysed at the Department of Clinical Chemistry, Swedish University of Agricultural Sciences, Uppsala, Sweden. The plasma oestradiol-17␤ (E2 ) level was determined by radioimmunoassay (Double antibody oestradiol, Diagnostic Products Corporation, Los Angeles, USA) as previously described for analysis of bovine plasma (Duchens et al., 1994) and validated for oestradiol-17␤ analyses in the pig (Mwanza et al., 2000). The progesterone (P4 ) level was determined by a luminescence immunoassay (Amerlite, Kodak Clinical Diagnostics, Amersham, England). The kit was used according to the manufacturer’s instructions and the method had earlier been validated for progesterone analyses in the pig (Rojkittikhun et al., 1993). 2.9. Statistical analyses Data were analysed by using SAS Programme (1989). Differences in mean plasma levels of oestradiol-17␤ and progesterone in two groups (AIB and AIA) were tested by using analysis of variance (GLM) with the effect of insemination time included in the model. The proc FREQ (Fisher’s exact test, two tails) was used to compare the distribution of spermatozoa (groups I-AIB and II-AIA), the number of oocytes with accessory sperm to the zona pellucida (group II) and fertilised oocytes (group III) between AI before and after ovulation. 3. Results 3.1. Clinical observations The average weaning to oestrous interval was 4.0±0.5 (means±S.D.) days, with a range of 3–6 days. Ovulation took place at 39.8 ± 2.8 and 40.3 ± 3.3 h after onset of the first and second oestrus after weaning, respectively, with a range of 32–48 h. The average oestrous cycle length was 22.6 ± 1.1 days. The average insemination times before (AIB) and after (AIA) ovulation were 18.6 h (range 15–23) and 17.5 h (range 15–18.5), respectively. When insemination after ovulation was performed, altogether 87.5% (14/16) of the sows were still in standing oestrus at insemination. The plasma levels of oestradiol-17␤ and progesterone around the time of insemination are presented in Fig. 1. The plasma levels of oestradiol-17␤ and progesterone differed significantly (P ≤ 0.05 and P ≤ 0.001, respectively) between AI before and after ovulation. 3.2. Recovery of ova and spermatozoa For groups I-AIB and II-AIA, mean times of 5.8 ± 0.6 and 5.5 ± 0.4 h after insemination, respectively, the numbers of sows with UTJ and isthmus containing spermatozoa, are presented in Table 2. All sows inseminated before ovulation had spermatozoa in the UTJ, compared with one of the late inseminated sows. Furthermore, no late inseminated sows had spermatozoa in the isthmus.

244

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

Fig. 1. Plasma levels of oestradiol-17␤ (䊏, pmol/l) and progesterone ( , nmol/l) of sows in blood samples collected at insemination before (n = 12) and after (n = 13) ovulation (means + S.D., ∗P ≤ 0.05, ∗∗∗P ≤ 0.001). Table 2 Numbers of sows with oviductal segments containing spermatozoa at 5–6 h after AI Groups

UTJ

Isthmus

I-AIB II-AIA

4/4 1/4

3/4 0/4

In groups II-AIB and II-AIA (slaughtered 21.1 ± 1.7 and 21.9 ± 1.5 h after ovulation, respectively), the recovery rates of oocytes/cleaved oocytes ranged between 64.7–90.0 and 27.8–100.0%, respectively. The numbers of oocytes and cleaved oocytes are presented in Table 3. The number of oocytes with spermatozoa in the ZP differed significantly (P ≤ 0.001) between ‘group II-AIB’ and ‘group II-AIA’. In group II-AIB, the oocytes/cleaved oocytes were found in ampulla (38.1%) and in isthmus (61.9%). Out of 44 one-cell stage oocytes (the eight degenerated ones are excluded), 15 oocytes had two pronuclei (male and female). All other oocytes (29/44) were in the second metaphase of the meiotic division. In group II-AIA, 22.2% of oocytes were found in ampulla and 77.8% in isthmus. All oocytes were in the one-cell stage. In one sow, three oocytes with spermatozoa in the zona pellucida were found. This sow also had spermatozoa in the UTJ (Table 2). All oocytes (degenerated ones excluded) were in the second metaphase of the meiotic division. In groups III-AIB and III-AIA (slaughtered 70.0 ± 3.4 and 69.0 ± 1.1 h after ovulation, respectively), the recovery rates of unfertilised and cleaved oocytes ranged between 29.2–71.0 and 50.0–80.9%, respectively. Most of unfertilised and/or cleaved oocytes were found in the upper part of uterine horns. The numbers of recovered unfertilised and cleaved Table 3 Numbers of oocytes and their cleavage stage at 20–25 h after ovulation Groups

One-cell

Two-cell

Four-cell

Total

Degenerated oocytes

Oocytes with spermatozoa in the ZP

II-AIB II-AIA

52 45

10 0

1 0

63 45

8/63 2/45

63/63 3/45

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

245

Table 4 Numbers of oocytes and cleaved oocytes at 70 h after ovulation Groups

One-cell

Two-cell

Four-cell

Six-cell

Eight-cell

Total

Fertilised

III-AIB III-AIA

2 25

0 6

38 4

2 11

2 2

44 48

42/44 23/48

Table 5 The numbers and size of 10-day-old embryos in each sow Group IV

Numbers of embryos

Recovery rate (%)

AIB/sow1 AIB/sow2 AIB/sow3

21 6 25

95.5 37.5 100

Total

52

82.5

AIA/sow1 AIA/sow2 AIA/sow3 AIA/sow4

7 19 8 5

31.8 86.4 44.4 23.8

Total

39

47

a

Size of the embryos, diameter (mm) 1–1.5 1 – –

5 (2a ) 19a 4 4

2–3

≥4

11 6 25

9 – –

– – 4 1

– – – –

Degenerated morphology.

oocytes are presented in Table 4. Comparing fertilisation rate, the fertilisation rate in ‘group III-AIB’ was significantly (P ≤ 0.001) higher than in ‘group III-AIA’. 3.3. Recovery of embryos In groups IV-AIB and IV-AIA (slaughtered 10 days after ovulation), the number of recovered embryos and morphology of the embryos are presented in Table 5. All embryos had a spherical shape but differed in size. The recovery rate in group IV-AIA was lower than in group IV-AIB. In group V-AIB, filamentous embryos (11.0 ± 2.6) were found in all sows. The recovery rate of live embryos as a proportion of the total number of corpora lutea was 64.7%. In group V-AIA, no sows were pregnant. In one out of four sows, small pieces of fetal membrane with degenerated morphology and without embryonic bodies were observed.

4. Discussion The results of the sperm distribution study showed that in sows inseminated before ovulation, spermatozoa were found in all UTJ and in the majority of isthmus. However, in late inseminated sows, spermatozoa were found in only one out of four sows. In agreement with Mburu et al. (1996), the present study showed that the distribution of spermatozoa in the UTJ–isthmus region was influenced by the ovulation event. This may be due to a

246

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

changed oviductal motility when late AI was performed owing to the lower plasma level of oestradiol-17␤ and a higher level of progesterone observed when compared with AI performed prior to ovulation. Mwanza et al. (2000) reported that the decline in the isthmic frequencies of phasic pressure fluctuations and amplitudes seen after ovulation might have been due to the rising plasma levels of progesterone. The ability of an oocyte to be fertilised has been considered to be as short as 8–12 h after ovulation in the pig (Hunter, 1967a; Hunter and Dziuk, 1968). Successful fertilisation has been associated with higher numbers of accessory spermatozoa to the zona pellucida. Hunter and Dziuk (1968) reported that a lower proportion of oocytes with accessory spermatozoa to the zona pellucida could be observed when insemination was performed after ovulation, which is in accordance with the present study. In principle, factors that influence the fertilisation results of sows may be related to several factors, such as the fertile life span of spermatozoa and oocytes, sperm distribution, the binding of spermatozoa to oocytes and sperm loss. In gilts inseminated up to 12 h (Waberski et al., 1994), or up to 16 h in sows (Soede et al., 1995a,b; Nissen et al., 1997), after ovulation, fertilised ova were found. However, in the present study, fertilisation was also observed when AI was performed up to 18.5 h after ovulation. One explanation could be that Soede et al. (1995a) inseminated sows once with 3 × 109 spermatozoa while we used an insemination dose of 10 × 109 spermatozoa. However, Steverink et al. (1997) reported that there was no effect of sperm dose on fertilisation rate and accessory spermatozoa to zona pellucida when AI was performed during 24–12 h (1 × 109 versus 3 × 109 spermatozoa) and 36–24 h (3 × 109 versus 6 × 109 ) before ovulation. Therefore, it is likely that in the study by Soede et al. (1995a) the sperm number per dose was not a limiting factor. Low recovery rates in groups II and III might be due to degeneration of oocytes/cleaved oocytes. Another reason could be that some oocytes/cleaved oocytes have remained in the oviduct or uterine horns after flushing. The number of recovered embryos on days 11 and 19 clearly show that AI 15–20 h after ovulation led to an increased rates of embryonic loss during early pregnancy as compared with AI 15–20 h before ovulation. Low recovery rate in group IV-AIA might be due to fertilisation failure or embryonic death before day 11. It has been reported that delayed mating (43 h post-hCG; approximately during ovulation at late stage (Dziuk and Baker, 1962; Hunter, 1967b)) resulted in decreased embryonic size and embryo oestradiol-17␤ secretion (in vitro) when embryos were collected 12 days post-hCG injection (Cárdenas and Pope, 1993). This is consistent with the present study showing that the embryos on day 11 of sows inseminated before ovulation were larger than embryos in sows inseminated after ovulation. However, the embryos in the latter sows probably had less development potential because their oocytes were aged when fertilised (Nissen et al., 1997). On day 19, no embryos were found in sows inseminated after ovulation. This might be due to a non-parallel development of the embryos and the endometrium (Pope, 1988; Geisert et al., 1991), i.e. embryonic loss due to failure in placentation which normally starts around days 13–14 and is completed after day 18 (Dantzer, 1985). Another explanation might be that more than four embryos are required in the pig to establish and maintain pregnancy (Polge et al., 1966). In addition, Nissen et al. (1997, one insemination), Rozeboom et al. (1997, three inseminations) and Terqui et al. (2000, two to five inseminations), all reported a decrease in farrowing rate in sows inseminated after ovulation.

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

247

5. Conclusion The results of this study showed that if insemination was performed at 15–20 h after ovulation in sows that still were in standing oestrus, the transport of spermatozoa in the UTJ and oviduct was impaired and a lower proportion of oocytes with accessory spermatozoa to the zona pellucida was found compared with insemination before ovulation. In the late inseminated sows, fertilised oocytes and developed embryos were observed up to day 11 but no embryos were found at day 19.

Acknowledgements The authors are grateful to Prof. Stig Einarsson, Department of Obstetrics and Gynaecology, for his constructive comments during preparation of this manuscript and Dr. Nils Lundeheim, Department of Animal Breeding and Genetics, for advice in statistical analysis. The present study received financial support and research funds from Swedish Meats. The Faculty of Veterinary Science, Mahidol University, is thanked for the scholarship and study leave granted to Dr. Kampon Kaeoket.

References Cárdenas, H., Pope, W.F., 1993. Effect of time of mating relative to ovulation on morphological diversity of swine blastocysts. Biol. Reprod. 49, 1015–1018. Dalin, A.-M., Nanda, T., Hulten, F., Einarsson, S., 1995. Ovarian activity at naturally attained oestrus in the sow: an ultrasonographic and LH study. Acta Vet. Scand. 36, 377–382. Dantzer, V., 1985. Electron microscopy of the initial stages of placentation in the pig. Anat. Embryol. 172, 281–293. Duchens, M., Forsberg, M., Edqvist, L.-E., Gustafsson, H., Rodriguez-Matinez, H., 1994. Effect of induced suprabasal progesterone levels around estrus on plasma concentrations of progesterone, estradiol-17␤ and LH in heifers. Theriogenology 42, 1159–1169. Dziuk, P.J., Baker, R.D., 1962. Induction and control of ovulation in swine. J. Anim. Sci. 21, 697–699. Geisert, R.D., Morgan, G.L., Zavy, M.T., Blair, R.M., Gries, K.L., Cox, A., Yellin, T., 1991. Effect of asychronous transfer and oestrogen administration on survival and development of porcine embryos. J. Reprod. Fert. 93, 475–481. Hunter, R.H.F., 1967a. The effect of delayed insemination on fertilisation and early cleavage in the pig. J. Reprod. Fert. 13, 133–147. Hunter, R.H.F., 1967b. Porcine ovulation after injection of human chorinic gonadotrophin. Vet. Rec. 81, 21–23. Hunter, R.H.F., 1984. Pre-ovulatory arrest and peri-ovulatory redistribution of competent spermatozoa in the isthmus of the pig oviduct. J. Reprod. Fert. 72, 203–211. Hunter, R.H.F., Dziuk, P.J., 1968. Sperm penetration of pig eggs in relation to the timing of ovulation and insemination. J. Reprod. Fert. 15, 199–208. Hunter, R.H.F., Fléchon, B., Fléchon, J.-E., 1987. Pre- and peri-ovulatory distribution of viable spermatozoa in the pig oviduct: a scanning electron microscope study. Tissue Cell 19, 423–436. Lovell, J.E., Getty, R., 1968. Fate of semen in the uterus of the sow: histologic study of endometrium during the 27 h after natural service. Am. J. Vet. Res. 29, 609–625. Mburu, J.N., Einarsson, S., Lundeheim, N., Rodriguez-Martinez, H., 1996. Distribution, number and membrane integrity of spermatozoa in the pig oviduct in relation to spontaneous ovulation. Anim. Reprod. Sci. 45, 109–121. Mwanza, A.M., Englund, P., Pettersson, A., Einarsson, S., 2000. Oviductal isthmic motility patterns as monitored by PolyviewTM in unrestrained sows around ovulation. Anim. Reprod. Sci. 62, 309–320.

248

K. Kaeoket et al. / Animal Reproduction Science 71 (2002) 239–248

Nissen, A.K., Soede, N.M., Hyttel, P., Schmidt, M., D’Hoore, L., 1997. The influence of time of insemination relative to time of ovulation on farrowing frequency and litter size in sows, as investigated by ultrasonography. Theriogenology 47, 1571–1582. Polge, C., Rowson, L.E.A., Chang, M.C., 1966. The effect of reducing the number of embryos during early stages of gestation on the maintenance of pregnancy in the pig. J. Reprod. Fert. 12, 395–397. Pope, W.F., 1988. Uterine asynchrony: a cause of embryonic loss. Biol. Reprod. 39, 999–1003. Pursel, V.G., Johnson, L.A., 1976. Frozen boar spermatozoa: methods of thawing pellets. J. Anim. Sci. 42, 927–931. Pursel, V.G., Schulman, L.L., Johnson, L.A., 1978. Distribution and morphology of fresh and frozen-thawed sperm in the reproductive tract of gilts after insemination. Biol. Reprod. 19, 69–76. Raycoudhurry, S.S., Saurez, S.S., 1991. Porcine sperm binding to oviductal explants in culture. Theriogenology 36, 1059–1070. Rodriguez-Martinez, H., Nicander, L., Viring, S., Einarsson, S., Larsson, K., 1990. Ultrastructure of the uterotubal junction in preovulatory pigs. Anat. Histol. Embryol. 19, 13–36. Rojkittikhun, T., Einarsson, S., Zilinskas, H., Edqvist, L.-E., Uvnäs-Moberg, K., Lundeheim, N., 1993. Effect of insulin administration at weaning on hormonal patterns and reproductive performance in primiparous sows. J. Vet. Med. A 40, 161–168. Rozeboom, K.J., Troedsson, M.H.T., Shurson, G.C., Hawton, J.D., Crabo, B.G., 1997. Late estrus or metestrus insemination after estrual inseminations decreases farrowing rate and litter size in swine. J. Anim. Sci. 75, 2323–2327. Rozeboom, K.J., Troedsson, M.H.T., Crabo, B.G., 1998. Characterisation of uterine leukocyte infiltration in gilts after artificial insemination. J. Reprod. Fert. 114, 195–199. SAS Programme, 1989. SAS User’s Guide, Statistic Version 6.12. SAS Institute, Inc., Cary, NC, USA. Simonsson, A., 1994. Näringsrekommendationer och fodermedel till svin, Vol. 75. Swedish University of Agricultural Sciences, Research Information Centre, Uppsala, Husdjur, p. 71. Soede, N.M., Noordhuizen, J.P.T.M., Kemp, B., 1992. The duration of ovulation in pigs, studied by transrectal ultrasonography, is not related to early embryonic diversity. Theriogenology 38, 653–666. Soede, N.M., Wetzels, C.C.H., Zondag, W., de Koning, M.A.I., Kemp, B., 1995a. Effect of time of insemination relative to ovulation, as determined by ultrasonography, on fertilisation rate and accessory sperm count in sows. J. Reprod. Fert. 104, 99–106. Soede, N.M., Wetzels, C.C.H., Zondag, W., Hazeleger, W., Kemp, B., 1995b. Effect of a second insemination after ovulation on fertilisation rate and accessory sperm count in sows. J. Reprod. Fert. 105, 135–140. Steverink, D.W.B., Soede, N.M., Bouwman, E.G., Kemp, B., 1997. Influence of insemination–ovulation interval and sperm cell dose on fertilisation in sows. J. Reprod. Fert. 111, 165–171. Terqui, M., Guillouet, P., Maurel, M.-C., Martinat-Botté, F., 2000. Relationship between peri-oestrus progesterone levels and time of ovulation by echography in pigs and artificial insemination (AI) on litter size. Reprod. Nutr. Dev. 40, 393–404. Viring, S., Einarsson, S., Nicander, L., Larsson, K., 1980. Localization of the sperm reservoir at the uterotubal junction of the pig. In: Proceedings of the Ninth International Congress on Animal Reproductive and AI, Vol. 5, Madrid, p. 331. Waberski, D., Weitze, K.F., Gleumes, T., Schwarz, M., Willmen, T., Petzoldt, R., 1994. Effect of time of insemination relative to ovulation on fertility with liquid and frozen boar semen. Theriogenology 42, 831– 840.