The Effect of Post-ovulatory Insemination on the Subsequent Embryonic Loss, Oestrous Cycle Length and Vaginal Discharge in Sows

The aim of present study was to study the effect of post-ovulatory insemination on the subsequent embryonic loss, oestrous cycle length and vaginal discharge in sows. Ten Large White multiparous sows were divided into two groups. Group A sows were inseminated once at 15 h after ovulation. Thereafter, they were ovariohysterectomized on day 11 (n = 5, first day of standing oestrus = day 1) and flushed for recovery of embryos. Group B sows were also inseminated once at 15 h after ovulation. They were further observed for return to oestrus and vaginal discharge (n = 5) after insemination. The endometrium tissues were biopsied from sows with vaginal discharge, embedded with paraffin, stained with haematoxylin and eosin and examined under light microscope. Only two embryos were observed in one of four sows from group A. All embryos had a spherical shape but differed in size (range 1-2 mm). In group B, only one sow had a regular return to oestrus (i.e. on day 23) and another sow had an irregular return to oestrus (i.e. on day 27). The other two sows in this group had shown vaginal discharge on days 20 and 38 after standing oestrus. For the number of leucocytes in the endometrium of sows with vaginal discharge, a large number of lymphocytes and plasma cells were observed in the connective tissue of the subepithelial layer. In conclusion, post-ovulatory insemination resulted in early embryonic loss, a subsequent prolonged oestrus interval and also vaginal discharge (i.e. endometritis) in sows.     

The Influence of Pre- and Post-ovulatory Insemination and Early Pregnancy on the Infiltration by Cells of the Immune System in the Sow Oviduct

The aim of this study was to investigate the influence of pre- and post-ovulatory insemination and early pregnancy on the distribution of immune cells in the oviduct. Eighteen sows were pre-ovulatory and sixteen sows were post-ovulatory inseminated and slaughtered at different times, 5-6 h after insemination, 20-25 h and approximately 70 h after ovulation, day 11 and day 19. Immediately after slaughter, oviductal samples of three different segments (isthmus, ampulla and infundibulum) were fixed, embedded in plastic resin and stained with toluidine blue or cryofixed and stored in a freezer at -70 degrees C until analysed by immunohistochemistry (pre-ovulatory inseminated sows) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. After pre- or post-ovulatory insemination, neutrophils were not observed in the oviductal epithelium from any of the segments or groups. The numbers of intraepithelial lymphocytes of all sows as well as CD2- and CD3-positive cells of the pre-ovulatory inseminated sows were higher in the infundibulum than in the other segments (p < or = 0.001). In the subepithelial connective tissue of the pre-ovulatory inseminated sows, significantly higher numbers of lymphocytes (p < or = 0.001) and plasma cells (p < or = 0.001) were found in infundibulum than in isthmus. Neutrophils were found mainly in infundibulum, the number approximately 40 h after pre-ovulatory insemination was significantly higher (p < or = 0.05) than in the other groups and segments. Significantly higher numbers of CD2 than CD3-positive cells were found for all groups and segments. In the subepithelial connective tissue of post-ovulatory inseminated sows, the numbers of lymphocytes was higher (p < or = 0.001) at day 19 than up to 50 h after insemination and lower (p < or = 0.001) in isthmus than in ampulla and infundibulum. Neutrophils were found in infundibulum in almost all groups and the number was significantly higher (p < or = 0.05) in the infundibulum up to 50 h after insemination than in other segments. In the oviductal epithelium, no influence of insemination was found on the presence of phagocytes, i.e. neutrophils and macrophages, but on lymphocytes. In the infundibular connective tissue, pre-ovulatory insemination had an effect on neutrophil distribution, indicating an active immune response to insemination in the upper segment. Post-ovulatory insemination changed the oviductal immune cell pattern.     

Infiltration of local immune cells in the sow reproductive tracts after intra-uterine and deep intra-uterine insemination with a reduced number of spermatozoa is less than conventional artificial insemination

The present study investigated the infiltration of leukocyte subpopulations in the utero-tubal junction (UTJ) and each part of the oviducts at about 24 hr after intra-uterine insemination (IUI) and deep intra-uterine insemination (DIUI) compared to conventional artificial insemination (CAI) in sows. Fifteen crossbred Landrace x Yorkshire multiparous sows were used (CAI, n=5; IUI, n=5; DIUI, n=5). The sperm dose contained 3,000 × 10(6) (100 ml), 1,000 × 10(6) (50 ml) and 150 × 10(6) (5 ml) motile spermatozoa for CAI, IUI and DIUI, respectively. The sows were inseminated with extended fresh semen at 6 to 8 hr prior to the expected time of ovulation. At 25.2 ± 1.6 hr after insemination, the oviducts and the UTJ were collected. The tissue samples of UTJ, caudal isthmus, cranial isthmus and ampulla were transversely cut to a thickness of 5 μm and stained with H&E. The total numbers of lymphocytes, neutrophils, macrophages, eosinophils and plasma cells were determined under light microscope. It was found that the numbers of lymphocytes, eosinophils and macrophages after CAI, IUI and DIUI were not significantly different (P>0.1) in both epithelial and sub-epithelial connective tissue layer of the UTJ, caudal isthmus, cranial isthmus and ampulla. Intra-epithelial neutrophils in the UTJ were higher than cranial isthmus (P<0.05) and ampulla (P<0.05). In the UTJ, the intra-epithelial neutrophil in the CAI group was higher than DIUI group (P<0.01). Plasma cells in sub-epithelial layer of the endosalpinx in the CAI group were higher than DIUI group (P<0.05) and tended to be higher than the IUI group (P=0.08). In conclusion, compared to CAI, IUI and DIUI do not influence the infiltration of lymphocytes, macrophages and eosinophils in the UTJ and the oviduct prior to fertilization. But a lower number of neutrophils in the intra-epithelial layer of the UTJ and plasma cells in the sub-epithelial layers of the oviduct was observed in the DIUI group compared to CAI.     

Expression of Progesterone Receptor in the Utero‐tubal Junction After Intra‐uterine and Deep Intra‐uterine Insemination in Sows

The aim of this study was to investigate the expression of progesterone receptor (PR) in the utero‐tubal junction (UTJ) of sows at 24 h after intra‐uterine insemination (IUI) and deep intra‐uterine insemination (DIUI) compared with conventional artificial insemination (AI) in pigs. Fifteen multiparous sows were used: AI (n = 5), IUI (n = 5) and DIUI (n = 5). The sows were inseminated with a single dose of diluted semen during the second oestrus after weaning at 6–8 h prior to ovulation (AI: 3000 × 10⁶ spermatozoa, IUI: 1000 × 10⁶ spermatozoa and DIUI: 150 × 10⁶ spermatozoa). The UTJ was collected and subject to immunohistochemical staining using avidin‐biotin immunoperoxidase technique with mouse monoclonal antibody to PR. In the oviductal part of the UTJ, the intensity of PR in the tunica muscularis and the proportion of PR‐positive cells in the surface epithelium after DIUI were lower than AI (p < 0.05). The intensity and the proportion of PR‐positive cells between AI and IUI in all compartments of the UTJ did not differ significantly (p > 0.05). When comparing between tissue compartments, prominent staining was observed in the muscular layer of the UTJ. It could be concluded that the expression of PR in the UTJ prior to fertilization after DIUI with a reduced number of spermatozoa was lower than that after AI. This might influence sperm transportation and the fertilization process.     

Sperm distribution in the reproductive tract of sows after intrauterine insemination

The purpose of the present study was to compare the number of spermatozoa obtained from different parts of the oviducts and the uterine horns of sows after intrauterine insemination (IUI) and conventional artificial insemination (AI), 24 h after insemination. Twelve crossbred (Landrace x Yorkshire) multiparous sows were used in the experiment. The sows were examined for standing oestrus using a back pressure test and were examined every 4 h after standing oestrus by real-time B-mode ultrasonography to estimate the time of ovulation. The sows were allocated to two groups, group I sows (n = 6) were inseminated by a conventional AI technique with 3 x 10(9) motile spermatozoa in 100 ml of extended semen, and group II sows (n = 6) were inseminated by an IUI technique using 1 x 10(9) motile spermatozoa in 50 ml of extended semen. A single dose of AI or IUI was given using the same boar, 8-10 h before the expected time of ovulation during the second oestrus after weaning. Twenty four hours after insemination, the sows were ovario-hysterectomized. The oviducts and the uterine horns were removed and divided into seven parts, the cranial, middle and caudal uterine horns, the utero-tubal junction (UTJ), the cranial and caudal isthmus, and the ampulla. All parts of the reproductive tract were flushed and the spermatozoa were counted using a haemocytometer. The results revealed that the spermatozoa were found in both the oviducts and the uterine horns in all animals. The number of flushed spermatozoa in the UTJ of groups I and II, was 142,500 and 131,167 (p > 0.05), and in the caudal isthmus was 1411 and 1280 (p > 0.05), respectively. The proportion of spermatozoa in different parts of the reproductive tract in relation to the total number of spermatozoa within the tract was not significantly different between groups I and II (p > 0.05). It could be concluded that IUI, with a three-time reduction in the number of spermatozoa used resulted in the same number of spermatozoa to be deposited in the sperm reservoir around ovulation time.     

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

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 x 10(6) motile spermatozoa and five sows were inseminated by the DIUI technique with 5 ml of diluted fresh semen, containing 150 x 10(6) 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 x 10(6) 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.     

Study of the distribution of inflammatory cells in the sow endometrium: effect of intravenous administration of adrenocorticotropin hormone

Seventeen multiparous cross-bred sows (Swedish Land-race x Swedish Yorkshire) were inseminated in their second oestrus after weaning and divided into two groups. One group (ACTH, n = 9) was given an intravenous injection of adrenocorticotropin hormone (ACTH) every 6 h commencing 4-8 h after ovulation, whereas another group (control, n = 8) was given saline solution at the same times. The sows were slaughtered 35-53 h after ovulation. Uterine samples, taken from the mesometrial side of the uterine horns immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue. The endometrium was then examined by light microscopy. There was no significant effect of the ACTH treatment on the distribution of lymphocytes and macrophages, but there was a tendency of an effect on the distribution of neutrophils (P = 0.1) in the sow endometrium.     

Immunohistochemical Studies on Oestrogen Receptor Alpha (ERα) and the Proliferative Marker Ki-67 in the Sow Uterus at Oestrus and Early Pregnancy

Oestrogen receptor alpha (ERalpha), the main subtype in the uterus, is involved in the regulation of uterine growth/proliferation. A relationship between ERalpha and proliferative activity has been shown in the cyclic sow uterus, but to our knowledge, no study has been carried out on early pregnant sows. Therefore, by means of immunohistochemistry and use of mouse monoclonal antibodies to ERalpha and a proliferative marker, Ki-67, the localization of these proteins was investigated in the sow uterus during early pregnancy. Eighteen crossbred multiparous sows were artificially inseminated once at 20-15 h before expected ovulation. After artificial insemination (AI), they were slaughtered at five different times: at oestrus, 5-6 h after AI (n = 4), 20-25 h after ovulation (n =4), 70 h after ovulation (n = 4), on day 11 (the first day of standing oestrus = day 1, n = 3) and on day 19 (n = 3). Immediately after slaughter, uterine samples were collected at the mesometrial side of the uteri, fixed in 10% formaldehyde and embedded in paraffin. Immunohistochemistry was performed by using mouse monoclonal antibodies to ERalpha (C-311) and Ki-67 (MM1). All sows slaughtered after ovulation were pregnant. In general, positive immunostaining for ERalpha and Ki-67 was found in the nuclei. Variations in staining intensity and proportion of positive nuclei were observed in different uterine compartments and stages of early pregnancy. The highest level of ERalpha presence in the surface epithelium and myometrium was found at oestrus (5-6 h after AI), and low levels of ERalpha in these compartments were observed as early as 20-25 h after ovulation. In the glandular epithelia, presence of ERalpha was highest at 70 h after ovulation. The largest number of ERalpha-positive cells in the stroma was observed at oestrus and early after ovulation. Low proliferation was observed, and with no significant difference in tissue compartments except in the glandular epithelium. High proliferative activity in the glandular epithelium at 70 h after ovulation indicated involvement in preparation for secretory activity and growth during pregnancy establishment. Significant positive correlation was found between the number of ERalpha-positive cells in the stroma and Ki-67-positive cells in the surface epithelium. In conclusion, the present study showed differences in immunolocalization of ERalpha and the proliferative marker Ki-67 in different tissue compartments of the sow uterus at oestrus and early pregnancy. In some uterine compartments, the patterns of ERalpha and Ki-67 immunostaining seemed to be influenced by insemination and the presence of embryos, in addition to the effects of steroid hormones.     

Number of Spermatozoa in the Crypts of the Sperm Reservoir at About 24 h After a Low‐Dose Intrauterine and Deep Intrauterine Insemination in Sows

The aim of this study was to investigate the number of spermatozoa in the crypts of the utero‐tubal junction (UTJ) and the oviduct of sows approximately 24 h after intrauterine insemination (IUI) and deep intrauterine insemination (DIUI) and compared with that of conventional artificial insemination (AI). Fifteen crossbred Landrace × Yorkshire (LY) multiparous sows were used in the experiment. Transrectal ultrasonography was performed every 4 h to examine the time of ovulation in relation to oestrous behaviour. The sows were inseminated with a single dose of diluted fresh semen by the AI (n = 5), IUI (n = 5) and DIUI (n = 5) at approximately 6–8 h prior to the expected time of ovulation, during the second oestrus after weaning. The sperm dose contained 3000 × 10⁶ spermatozoa in 100 ml for AI, 1,000 × 10⁶ spermatozoa in 50 ml for IUI and 150 × 10⁶ spermatozoa in 5 ml for DIUI. The sows were anaesthetized and ovario‐hysterectomized approximately 24 h after insemination. The oviducts and the proximal part of the uterine horns (1 cm) on each side of the reproductive tracts were collected. The section was divided into four parts, i.e. UTJ, caudal isthmus, cranial isthmus and ampulla. The spermatozoa in the lumen in each part were flushed several times with phosphate buffer solution. After flushing, the UTJ and all parts of the oviducts were immersed in a 10% neutral buffered formalin solution. The UTJ and each part of the oviducts were cut into four equal parts and embedded in a paraffin block. The tissue sections were transversely sectioned to a thickness of 5 μm. Every fifth serial section was mounted and stained with haematoxylin and eosin. The total number of spermatozoa from 32 sections in each parts of the tissue (16 sections from the left side and 16 sections from the right side) was determined under light microscope. The results reveal that most of the spermatozoa in the histological section were located in groups in the epithelial crypts. The means of the total number of spermatozoa in the sperm reservoir (UTJ and caudal isthmus) were 2296, 729 and 22 cells in AI, IUI and DIUI groups, respectively (p < 0.01). The spermatozoa were found on both sides of the sperm reservoir in all sows in the AI and the IUI groups. For the DIUI group, spermatozoa were not found on any side of the sperm reservoir in three out of five sows, found in unilateral side of the sperm reservoir in one sow and found in both sides of the sperm reservoir in one sow. No spermatozoa were found in the cranial isthmus, while only one spermatozoon was found in the ampulla part of a sow in the IUI group. In conclusion, DIUI resulted in a significantly lower number of spermatozoa in the sperm reservoir approximately 24 h after insemination compared with AI and IUI. Spermatozoa could be obtained from both sides of the sperm reservoir after AI and IUI but in one out of five sows inseminated by DIUI.     

Uterine Insemination with a Standard AI Dose in a Sow Pool System

The effect of uterine AI with a standard dose of spermatozoa on fertility of the sow was studied in a field trial. The trial involved a sow pool system with 440 sows using AI as the primary method of breeding. Sows were twice a day checked for oestrus symptoms by back pressure test in front of a boar on days 3–6 after weaning. When in standing heat, sows were randomly allocated into either a uterine insemination group (UTER, n = 157) or standard AI group (CONT, n = 169) and bred accordingly using 3 billion spermatozoa in 80 ml of extender. In both treatment groups, insemination was repeated once if the sow was still receptive 24 h later. Using pregnancy (farrowed or not) and live‐born litter size as the outcome variables, a logistic and linear regression approach, respectively, was taken to study the effect of the following factors: treatment (UTER vs CONT), AI operator, breed, satellite herd preceding weaning, parity, weaning‐to‐oestrus interval and length of lactation. Overall, live‐born litter size was 11.3 ± 2.9, repeat breeding rate 4.2% and farrowing rate 91.2%. In the UTER group, 93.6% of inseminated sows farrowed, whereas farrowing rate for the CONT group was 88.8% (p = 0.13). Intrauterine insemination with a standard AI dose did not result in a significant improvement in the live‐born litter size (11.5 ± 2.8 for the UTER and 11.1 ± 3.0 for the CONT sows, respectively, p = 0.13). However, the preceding satellite herd had a highly significant effect on the live‐born litter size (12.4 ± 2.6; 11.1 ± 2.9; 10.8 ± 2.9 and 10.9 ± 2.9 for the four satellite herds, p < 0.01). We conclude that uterine insemination did not have a significant effect on live‐born litter size and farrowing rate and we also conclude that satellite herd appears to have a major effect on fertility in a sow pool system.     

采用深部输精与常规输精研究不同剂量和有效精子数对经产母猪繁殖力的影响

为探讨不同输精剂量与不同有效精子数对经产母猪繁殖力的影响,使用深部输精和常规输精作为主要配种方式。将发情母猪随机分为2组,A组为深部输精组(n=60),B组为常规输精组(n=60)。对A、B组内母猪分别用不同的输精剂量(Ⅰ:30亿/80 mL、Ⅱ:15亿/80 mL、Ⅲ:15亿/40 mL;n=20)进行输精。结果表明:与常规输精相比,深部输精组的受胎率和分娩率分别为93.33%和88.33%,略高于常规输精组的86.67%和83.33%,但差异不显著(P>0.05);但深部输精可显著提高经产母猪的产仔数和产活仔数,分别提高1.07头和1.15头。无论是深部输精还是常规输精,15亿/80 mL的输精量,均可获得与30亿/80 mL输精量一致的产仔效果,这一结果可以使优秀种公猪的利用率提高1倍,降低了最新国标《GB23238-2009》中对外种猪输精剂量的要求,建议扩大实验并推广使用。     

Ultrastructure of the Uterotubal Junction in Preovulatory Pigs

The ultrastructure of the surface epithelia from the uterotubal junction (UTJ), and the adjacent tubal isthmic and endometrial regions, was studied in preovulatory oestrus gilts, either unmated or inseminated 12 h before with fresh boar semen. The simple columnar epithelium of the UTJ consisted of non-ciliated (secretory) and ciliated cells. Secretory vesicles occurred in the secretory cells, especially in inseminated gilts. Lymphocytes, monocytes and macrophages were found dispersed basally among the epithelial cells. Phagocytosis of epithelial cells undergoing apoptosis was seen throughout the UTJ at oestrus, increasing after insemination. Neutrophilic granulocytes were found in the lamina propria of the uterine component of the UTJ, but only occasionally in the epithelium. After insemination, neutrophils invaded the uterine epithelium, to actively participate in intraepithelial phagocytosis or move into the lumen, engulfing spermatozoa. Neutrophils were absent from the UTJ proper and the isthmic epithelium, irrespective of the presence of spermatozoa in the lumen. Those spermatozoa in the uterine lumen that escaped phagocytosis had severely damaged plasma membranes, whereas those in the UTJ proper--concentrated towards the deep furrows of the diverticulae--mostly showed normal sperm ultrastructure.     

Peri-oestrus hormone profiles and follicle growth in lactating sows with oestrus induced by intermittent suckling.

This study describes follicle dynamics, endocrine profiles in multiparous sows with lactational oestrus compared with conventionally weaned sows (C). Lactational oestrus was induced by Intermittent Suckling (IS) with separation of sows and piglets for either 12 consecutive hours per day (IS12, n = 14) or twice per day for 6 h per occasion (IS6, n = 13) from day 14 of lactation onwards. Control sows (n = 23) were weaned at day 21 of lactation. Pre-ovulatory follicles (> or =6 mm) were observed in 100% of IS12, 92% of IS6 and 26% of C sows before day 21 of lactation and in the remaining 74% C sows within 7 days after weaning. All sows with pre-ovulatory follicles showed oestrus, but not all sows showed ovulation. Four IS6 sows and one IS12 sow developed cystic follicles of which two IS6 sows partially ovulated. Follicle growth, ovulation rate and time of ovulation were similar. E(2) levels tended to be higher in IS sows (p = 0.06), the pre-ovulatory LH surge tended to be lower in IS12 (5.1 +/- 1.7 ng/ml) than in C sows (8.4 +/- 5.0 ng/ml; p = 0.08) and P(4) levels were lower in IS12 and IS6 than in C sows (at 75 h after ovulation: 8.8 +/- 2.4 ng/ml vs 7.0 +/- 1.4 ng/ml vs 17.1 +/- 4.4 ng/ml; p < 0.01). In conclusion, sows with lactational oestrus induced by IS are similar to weaned sows in the timing of oestrus, early follicle development and ovulation rates, but the pre-ovulatory LH surge and post-ovulatory P(4) increase are lower.     

Postovulatory Effect of Intravenous Administration of Lipopolysaccharide (E. coli,O55:B5) on the Contractile Activity of the Oviduct,Ova Transport,Binding of Accessory Spermatozoa to the Zona Pellucida and Embryo Development in Sows

The effect of lipopolysaccharide (LPS) (E. coli, O55:B5), administered 18 h after ovulation in the second oestrus after weaning, on the contractile activity of the oviduct, ova transport, sperm binding to zona pellucida (ZP) and embryo development, was studied in 14 Swedish crossbred (Landrace Yorkshire) multiparous sows. The endotoxin group (E‐group) sows were administered with 300 ng/kg of LPS while the control group (C‐group) sows were administered with 5 ml of saline i.v. via an indwelling jugular cannula. Immediately after evidence of standing oestrus, a Millar^® pressure transducer was placed intraluminally about 3 cm into the mid‐isthmus, via laparotomy. Pressure recordings of the oviduct were collected from all conscious sows until slaughter. After slaughter, the genital tract opposite to the side with the transducer was retrieved, and three equal isthmic segments and the first third of the uterine horn part adjacent to the utero‐tubal‐junction (UTJ) were flushed separately to recover the ova. The intervals (mean±SD) from ovulation to slaughter (OS) and insemination to ovulation (IO) were not different between the E‐group (44.5±5.7 h; 13.3±6.5 h) and the C‐group (42.7±5.9 h; 14.8±4.1 h), respectively. Ova recovery rate (RR) in the E‐group (80.2±22.9%) did not differ from that in the C‐group (85.2±4.5%). The frequency distribution of ova recovered in the different segments did not significantly (p>0.05) differ between the groups. The E‐group showed higher cleavage rate than controls. A higher proportion of spermatozoa bound to the ZP was also found in the E‐group compared with controls. The isthmic intraluminal pressure slightly increased (p=0.07) 18 h after ovulation and immediately following LPS in the E‐group, compared with the C‐group. The frequencies of phasic pressure fluctuations were significantly (p<0.05) lower at 30 and 38 h after ovulation in the E‐ than in the C‐group. It can be concluded from the present study that a single i.v. administration of LPS (300 ng/kg body weight) to sows, 18 h after ovulation might be associated with changes in isthmic pressure and the frequency of phasic pressure fluctuations, increased numbers of spermatozoa attached to the ZP and an enhanced embryo development but not with ova transport rates.     

A study on the number of recovered spermatozoa in the uterine horns and oviducts of gilts,after fractionated or non-fractionated insemination

The objective of this study was to compare the number of recovered spermatozoa, in different parts of the uterine horn and oviduct in gilts, after insemination with fractionated (experiment) and non-fractionated (control) liquid stored semen. The number of spermatozoa and volume of backflow was also investigated. Twenty three cross-bred gilts were used in the study. They were divided into 2 groups, a control group (non-fractionated liquid stored semen, n=10) which were inseminated with 100 ml of liquid stored semen containing 3,000 million spermatozoa per dose and an experimental group (fractionated liquid stored semen, n=10) which were inseminated with 50 ml of liquid stored semen, with 3,000 million spermatozoa per dose and followed by another 50 ml of semen dilutor (Beltsville Thawing Solution, BTS). Thereafter, backflow semen was collected and measured every 15 min for a period of 1 hr. Three or 12 hr after insemination, 5 gilts from each group had the uterus, the horn of the uterus, the oviducts and the ovaries removed under general anaesthesia. The horn of uterus and the oviducts were seperated by ligation into 6 segments. All 6 segments were flushed with BTS to collect all spermatozoa within the segment. Recovered spermatozoa were counted, using a haemocytometer and the volume recorded. It was seen that the percentage of spermatozoa in the backflow semen in the experimental group was less than in the control group. The difference was not significant in the gilts that were operated on 3 hr after insemination, the mean number of spermatozoa in the uterine horn and the utero-tubular junction (UTJ) was more in the experimental than in the control group, but less in the isthmus and the ampulla of the oviduct. The gilts which were operated on 12 hr after insemination, had relativity more ovulating gilts in the control group than in the experimental group (3 of 4 gilts compare to 3 of 5 gilts). The control group had more spermatozoa in the oviduct than the experimental group, but less in UTJ and in the horn of the uterus. Again the difference was not significant. It can be concluded that fractionated (experimental) or non-fractionated (control) insemination of semen with the same number of spermatozoa provides no significant difference in the number of spermatozoa either in the horn of the uterus, the UTJ or the oviduct of gilts.     

The Effect of Intravaginal Applied GnRH-agonist on the Time of Ovulation and Subsequent Reproductive Performance of Weaned Multiparous Sows

In order to prove the effect of 'fixed time insemination' and insemination at standing oestrus after post-weaning application of GnRH, in a Croatian large breeding unit, 502 sows were assigned to three groups and were artificially inseminated (AI) at their first post-weaning oestrus as many times as they stand, in 24-h intervals. The groups were treated as follows: group 1 (control, n = 160) were AI during their standing reflex; group 2 ['GnRH-fixed time insemination' (GnRH-FT-AI), n = 175] were AI, independent of detection of oestrus and following administration of GnRH-agonist at 96 h post-weaning; group 3 [GnRH insemination at standing oestrus (GnRH-OE-AI), n = 167] the animals were GnRH-agonist treated as group 2 and were AI at their standing reflex. Pre-trial daily average lactational feed intake, average daily feed intake from weaning to oestrus, oestrus within 6 days post-weaning (%), ovulation within 6 days post-weaning (%), weaning-to-oestrus interval (h), duration of oestrus (h), follicle size (mm), interval from oestrus to ovulation (h), subsequent day 24 pregnancy rate (%), farrowing rate (%) and total pigs born were evaluated. Pre-trial average daily lactational voluntary feed intake was 7.1 +/- 0.08 kg in group 1, 7.0 +/- 0.07 kg in group 2 and 7.1 +/- 0.17 kg in group 3 (p > 0.05). Average voluntary daily feed intake from weaning to oestrus was 5.1 +/- 0.3 kg in group 1, 5.2 +/- 0.5 kg in group 2 and 5.2 +/- 0.19 kg in group 3 (p > 0.05). Oestrus was detected within 6 days post-weaning in 134 (83.8%) in control, 164 (93.7%) in GnRH-FT-AI and 155 (92.8%) animals in GnRH-OE-AI groups (p = 0.05). Follicle size did not differ (p > 0.05) among the groups. In control 82.8%, in GnRH-FT-AI 91.5% and in GnRH-OE-AI 91.0% of the sows ovulated within 6 days post-weaning (p = 0.04), and had 80.6, 90.9 and 89.7% 24-day pregnancy rates (p = 0.16), respectively. In GnRH-FT-AI group 90.2%, in GnRH-OE-AI sows 89.7%, in control animals 79.9% farrowing rates were recorded (p = 0.17). Weaning to oestrus interval was 113.1 h in control, 114.1 h in GnRH-FT-AI and 112.6 h GnRH-OE-AI (p > 0.05). Duration of oestrus was significantly shorter in GnRH-FT-AI (44.9 h) and GnRH-OE-AI (48.1 h) animals, compared with the control (62.9 h) sows (p = 0.001). Similarly, the interval from oestrus to ovulation revealed significant (p = 0.004) differences between the groups (control 44.1 h, GnRH-OE-AI 34.1 h and GnRH-FT-AI 32.9 h). GnRH-FT-AI (12.5) and GnRH-OE-AI (12.6) sows had significantly higher (p = 0.01) number of total pigs born (n = 10.4) compared with control sows. GnRH-agonist-gel treatment to the sow shortens duration of oestrus, the interval from oestrus to ovulation, and may eliminate the need for oestrus detection in the hands of skilled personnel.     

Distribution and viability of spermatozoa in the canine female genital tract during post-ovulatory oocyte maturation

Background

Unlike other domestic mammals, in which metaphase-II oocytes are ovulated, canine ovulation is characterized by the release of primary oocytes, which may take 12 to up to 36 hours. Further 60 hours are needed for maturation to secondary oocytes which then remain fertile for about 48 hours. Oestrus takes 7 to 10 days on average and may start as early as a week before ovulation. This together with the prolonged process of post-ovulatory oocyte maturation requires an according longevity of spermatozoa in the female genital tract in order to provide a population of fertile sperm when oocytes have matured to fertilizability. Therefore the distribution and viability of spermatozoa in the bitch genital tract was examined during post-ovulatory oocyte maturation.

Methods

Thirteen beagle bitches were inseminated on the day of sonographically verified ovulation with pooled semen of two beagle dogs containing one billion progressively motile spermatozoa. Ovariohysterectomy was performed two days later (group 1, n = 6) and four days later (group 2, n = 7). The oviduct and uterine horn of one side were flushed separately and the flushing’s were checked for the presence of gametes. The oviducts including the utero-tubal junction and the uterine horns, both the flushed and unflushed, were histologically examined for sperm distribution.

Results

The total number of spermatozoa recovered by flushing was low and evaluation of viability was limited. Prophase-I oocytes were collected from oviduct flushing in group 1, whereas unfertilized metaphase-II oocytes were detected in group 2. From day 2 to day 4 after ovulation a significant decrease in the percentage of glands containing sperm (P<0.05) and a marked reduction of the mean sperm number in uterine horn glands were observed. A concomitant diminution of spermatozoa was indicated in the utero-tubal junction accompanied by a slight increase in sperm numbers in the mid oviduct.

Conclusions

Oocyte maturation to metaphase-II stage is accompanied by a continuous sperm detachment and elimination in the uterine horns. Entrance of spermatozoa into the caudal oviduct seems to be steadily controlled by the utero-tubal junction thus providing a selected sperm population to be shifted towards the site of fertilization when oocyte maturation is completed.     

Early embryo survival and development in sows with lactational ovulation.

During lactation, daily separation of sow and piglets, intermittent suckling (IS), can induce lactational oestrus and ovulation. This study examined effects of IS on subsequent early embryo survival and development. Multiparous Topigs40 sows were separated from their piglets for either 12 consecutive hours per day (IS12, n = 13) or two times for 6 h per day (IS6, n = 10) from day 14 of lactation onwards until 23 days after ovulation. Control sows (C, n = 17) were weaned at day 21 of lactation. Oestrus was shown in all treatments within 5 days after the start of treatment. Sows were inseminated each day of oestrus and slaughtered at D23 after ovulation. Intermittent suckling did not significantly affect pregnancy rates of sows (75% IS12 vs 78% IS6 vs 94% C; p > 0.10). Embryo survival was not significantly affected by IS (IS12: 57%; IS6: 51%; p > 0.10) although it seemed to be lower than in C sows (70%). Some parameters of embryo, placental and uterine development were affected by IS, especially in the IS6 group. IS6 embryos had shorter placentas (17.5 +/- 1.2 cm; p < 0.05) than C (20.3 +/- 1.4 cm) and IS12 sows (20.9 +/- 0.7 cm) were smaller and less developed than C sows (p < 0.05). In conclusion, embryo survival does not seem significantly affected by IS, although numerical differences were great. Embryo development, however, was negatively affected in IS6 sows possibly due to a combination of high milk production, stress and lactational effects on uterine development.     

Effect of Insemination–Ovulation Interval and Addition of Seminal Plasma on Sow Fertility to Insemination of Cryopreserved Sperm

In swine, the use of frozen-thawed (FT) sperm for artificial insemination (AI) is limited because of poor sow fertility, possibly associated with a post-thaw capacitation-like status resulting in fewer fully viable sperm. Sow fertility to AI with FT sperm may improve with deeper deposition of sperm within the female tract, insemination very close to ovulation, or reversal of cryocapacitation by seminal plasma (SP). We performed two experiments to examine these suggestions. In experiment 1, 122 multiparous Yorkshire sows received 600 IU equine chorionic gonadotrophin at weaning and 5 mg pLH 80 h later to control time of ovulation. The predicted time of ovulation (PTO) was 38 h after pLH injection. Thereafter, sows were assigned on the basis of parity to a single AI of FT sperm at 2 h before PTO, or at 12 h before PTO, or FT sperm supplemented with 10% SP at 12 h before PTO. Control sows received fresh semen at 12 h before PTO. All semen doses were adjusted to 3 x 10(9) live cells and deposited into the cervix. Experiment 2 employed 99 multiparous crossbred sows and repeated the treatments of experiment 1 except that all FT inseminations were intrauterine. In both experiments, farrowing rates were lower (p < 0.01) following FT inseminations with no effect of time of insemination or of supplemental SP. In experiment 1, litter size was smaller following FT insemination (p < 0.05), but no effect on litter size was evident in experiment 2. Supplemental SP had no effect on litter size in either experiment. The lack of effect of either SP or timing of FT insemination on sow fertility suggests that the non-lethal sperm cryoinjury affecting fertility involves more than just cryocapacitation.     

Sperm migration in pigs after deep intrauterine and intraperitoneal insemination

Deep intrauterine insemination in pigs allows sperm deposition only into one uterine horn, but bilateral fertilization of oocytes occurs. How the sperm reach the contralateral oviduct remains disputable. The aim of this experiment was to study possible transperitoneal and/or transuterine sperm migration ways. Follicle growth and ovulation were induced in 24 peripubertal gilts with eCG and hCG 72 h after eCG. Endoscopic intrauterine insemination (IUI) was performed 32 h after hCG with 20 ml of extended semen (60 × 10(6) spermatozoa) as follows: Group CONTROL (n=8) received IUI into the right horn, and the left horn served as non-treated control; Group LIGATURE (n=8) received IUI into the right horn, and the left horn was closed by endoscopic double ligature close to the bifurcation; Group INTRAPERITONEAL (IPI; n=8) received IUI into the right uterine horn, the left horn was closed by double ligature and semen was deposited intraperitoneally at the surface of the left ovary. Genital tracts were removed 65-66 h after hCG, the oviducts were flushed and ova (n=299) were analyzed for fertilization and cleavage. Furthermore, the accessory spermatozoa count/oocyte was graded as 0, without spermatozoa, 1, <5 spermatozoa, 2, 5-50 spermatozoa, 3, 50-100 spermatozoa and 4, >100 spermatozoa. The results indicate that low dose IUI into one horn provides a lower grade of accessory spermatozoa in the contra-lateral side (1.6 vs. 2.8). No spermatozoa were found in ova flushed from oviducts of the ligated uterine horn, even after intraperitoneal insemination (P<0.05), and no fertilization occurred, respectively. Our results clearly indicate that after low dose IUI into one uterine horn, spermatozoa reach the contralateral oviduct via transuterine migration.