Implantation in both of the uterine horns after the unilateral intrauterine insemination in rabbits.

Artificial insemination was carried out by injecting semen into the upper part of the left uterine horn in rabbits with a duplex uterus. Ovulation was then induced by administration of hCG. As a result, implanted fetuses were observed not only in the left uterine horn but also in the right uterine horn. However, when similar insemination was carried out after ligation of the right utero-tubal junction, fertilization did not occur in the right oviduct. From these finding, we conclude that some of the spermatozoa injected into the left uterine horn was discharged into the vagina, entered the right uterine cervical canal, and reached the right oviduct to fertilize the ova.     

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.     

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.     

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.     

Experimental evidence for the influence of cumulus-oocyte-complexes on sperm release from the porcine oviductal sperm reservoir

In the pig, a temporal relationship is suggested between sperm release from the sperm reservoir (SR) and ovulation, but the mechanism(s) is still under discussion. In two experiments, the influence of transferred ova on the release of SR-spermatozoa at ovulation and the effect of supplementation with non-sulfated glycosaminoglycan hyaluronan (HA) on embryo development and the number of accessory spermatozoa, respectively, were examined. PMSG/hCG primed ovectomized gilts that had previously received endoscopic low-dose insemination into the cranial uterine horn were used as an experimental model. After salpingectomy, tubal segments (ampulla, cranial, and caudal isthmus) were flushed and sperm numbers or respective accessory spermatozoa were counted. In Experiment 1, the distribution of the sperm population was altered in the presence of cumulus-oocyte-complexes (COCs). A higher proportion of spermatozoa was found after transfer of COCs into one oviduct in the ampulla and cranial isthmus segments compared with the controls (17.5 vs. 4.9%, p<0.05). In Experiment 2, the quality of the transferred ova and treatment influenced the presence of accessory spermatozoa. Transfer of COCs together with HA increased (p<0.05) the number of accessory spermatozoa compared with the other treatment groups and was similar to those in the "undisturbed" controls. No modifications were obtained regarding mean blastomere numbers (2.6 +/- 0.2 to 3.1 +/- 0.2). In summary, this study was demonstrated that cumulus-oocyte-complexes may be involved in triggering sperm release from the pig oviductal SR and that HA might be related to sperm release.     

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.     

Influence of Boar Seminal Plasma on the Distribution of Spermatozoa in the Genital Tract of Gilts

The main purpose of the present study was to investigate whether boar seminal plasma affects the transport of spermatozoa in the genital tract of oestrous pigs or not, with special reference to the sperm transport into the oviducts. Altogether 17 gilts were used in three experiments.Experiment I. In nine gilts one uterine horn was injected surgically with 10¹⁰ spermatozoa suspended in seminal plasma and the other uterine horn with 10¹⁰ spermatozoa suspended in TESNaK-glucose buffer solution. The sperm deposition was performed under general anaesthesia. The gilts were slaughtered 1–2 or 4–6 h after insemination. The genital tract was removed and the numbers of spermatozoa determined in oviducts and in uterine horns.Experiment II. The insemination doses were prepared exactly as in Experiment I. Approx. 24 h before insemination Polyvinylchloride cannulas were inserted into the uterine lumen of the horns, drawn via the midventral incision at linea alba subcutaneously to cutaneous incisions ventral to the vulva opening. One cannula was placed in each uterine horn. At standing heat the insemination doses were slowly injected through the cannulas. The gilts were slaughtered 1 h after insemination and the numbers of spermatozoa within the genital tract were counted.Experiment III. In three gilts under general anaesthesia the uterine horns were ligated 10 cm from the uterotubal junction. The semen doses (containing 2 × 10⁹ spermatozoa), prepared as in Experiment I, were deposited into the uterine horns anterior to the ligatures through a cannula. The gilts were slaughtered 1 h after insemination, and the numbers of spermatozoa within the oviducts and ligated part of the uterine horns were counted.In all three experiments more spermatozoa were, on average, recovered in the oviducts connected to uterine horns inseminated with spermatozoa suspended in seminal plasma. In Experiments I andII this was the case for 10 of 14 gilts and in Experiment III for all the three gilts. It is therefore suggested that boar seminal plasma pro¬motes sperm transport into the oviduct of oestrous pigs. The back¬ground mechanism for this is discussed.     

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.     

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.     

Intrauterine and intravaginal insemination with frozen canine semen using an extender consisting of orvus ES paste-supplemented egg yolk tris-fructose citrate

Our previous report indicated that addition of Orvus ES Paste (OEP) to the extender of frozen canine semen protected acrosomes and maintained sperm motility after thawing. In this study, artificial insemination (AI) using the frozen semen was carried out. The frozen semen was prepared using egg yolk Tris-fructose citrate, and the final concentrations of glycerol and OEP were 7% (v/v) and 0.75% (v/v), respectively. AI was performed during the optimal mating period predicted from the peripheral plasma progesterone level. In intrauterine insemination (IUI), the bitches were laparotomized and 1 x 10(8) spermatozoa were infused into one of the uterine horns. In insemination of non-OEP supplemented semen, 3 x 10(8) spermatozoa were inseminated. In intravaginal insemination (IVI), 10-40 x 10(8) spermatozoa were inseminated. Conception was obtained in nine of 10 bitches (90.0%) that underwent IUI. The number of newborns was from 1 to 7 (mean 3.6 +/- 0.9). The mean ratio of the number of puppies to the number of ovulations in the inseminated uterine horn was 71.8%. The number of puppies did not exceed the number of ovulation in the inseminated uterine horn. Conception using non-OEP supplemented frozen semen was unsuccessful in all four bitches. In IVI, conception was not obtained in any of the six bitches that received insemination of 10 x 10(8) or 40 x 10(8) spermatozoa, but two of three bitches that received insemination of 20 x 10(8) spermatozoa were fertilized. It was shown that a high conception rate can be obtained by IUI using OEP-supplemented frozen canine semen. Developmenmt of a non-surgical method of IUI and a method of freezing canine sperm applicable to IVI is necessary.     

Effect of unilateral cornual insemination upon fertilization rate in superovulating and single-ovulating cattle

In Exp. 1, 21 first-service cattle and seven repeat-breeder cattle, averaging 4.7 infertile services, were brought into estrus and superovulated by treatment with follicle-stimulating hormone and prostaglandin F2 alpha. At insemination, semen was deposited in the greater curvature of one uterine horn, about midway between the utero-cervical junction and the utero-tubal junction. Cattle were necropsied 2 to 7 d after estrus and ova were recovered and examined. The fertilization rate for first-service cows was 74% of 362 intact ova and for repeat-breeders, 43% of 128 intact ova (P less than .001). Fertilization rate in first-service cows was 81% on the side of semen deposition and 68% on the opposite side (P less than .01); the rates in repeat-breeders were 54% and 32% (P less than .025). Differences between sides were due mostly to four cows that averaged 93% fertilization on the side of semen deposition and 19% on the opposite side. The proportion of fertilized ova with accessory sperm (17%) did not differ between sides of the reproductive tract. In Exp. 2, 60 first-service and 32 repeat-breeder cows in natural estrus had semen deposited in the uterine body or in the greater curvature of one uterine horn, either on the side of impending ovulation or on the opposite side. At necropsy, 55 ova were recovered from first-service cows, of which 42 (76%) were intact and 13 (24%) were ruptured or fragmented. Of the 42 intact ova, 41 (98%) were cleaved. From the 32 repeat-breeders, 30 ova were recovered, of which 26 (87%) were intact and 4 (13%) were ruptured; 23 of the 26 intact ova (88%) were cleaved. Site of semen deposition had no significant effect on either fertilization rate or number of accessory sperm in either type of cow. First-service cows averaged more accessory sperm (40) than did repeat-breeders (19, P less than .01). Overall results indicated that sperm deposited deep in one uterine horn fertilized ova nearly as frequently in the opposite oviduct as in the adjacent oviduct except in 14% of superovulating cattle.     

Sperm localization in the oviducts of artificially inseminated dairy cattle

Eight animals, 3 heifers and 5 primiparous cows, were artificially inseminated by intrauterine deposition of frozen-thawed semen. The insemination dose comprised 20×10⁶ or 200 × 10⁶ spermatozoa, frozen in French mini straws. Four animals were inseminated at fixed time interval (72 or 84 h) after cloprostenol injection. The remaining 4 animals were inseminated in spontaneous oestrus. Slaughter took place 2 or 12 h after insemination. After fixation the oviducts were cut into segments, which were serial-sectioned and stained. Six sections per segment were examined under the microscope for sperm recovery.The number of spermatozoa recovered from the oviducts varied considerably among animals. Recovery was poor (less than 50 spermatozoa) in 4 animals. Recovery was low when insemination took place in induced oestrus and with the lower sperm number (20×10⁶). In animals in which more than 50 spermatozoa were found the distribution varied both between animals and between oviducts within the same animal. Overall, more spermatozoa were found in the lower (UTJ, isthmus and AIJ) than in the upper (ampulla) parts of the oviducts. In 3 out of 4 animals more spermatozoa were recovered from the left than from the right oviduct. Only in 1 animal were the majority of spermatozoa found in the oviduct ipsilateral to the follicle-bearing ovary.     

Site of Intrauterine Artificial Insemination in the Bitch does not Affect Sperm Distribution within the Uterus

The aim of this study was to evaluate the distribution of frozen–thawed spermatozoa within the uterine lumen and oviducts following intrauterine laparoscopic deposition at two sites. Twelve bitches of unknown reproductive history were randomly distributed into two groups. Semen (3 ml containing 300 × 10⁶ frozen–thawed spermatozoa) was infused at the uterine body (UB group) or at the cranial tip of the left uterine horn. A 22‐G catheter was used to access the uterine lumen. Sperm cell distribution was evaluated after ovariohysterectomy performed 3 h after artificial insemination (AI). There was no difference between groups in mean time to perform AI. Spermatozoa were detected in all uterine segments, including the tip of both horns, but none was detected in the oviduct. The 22‐G catheter facilitated deposition of semen in the uterine lumen, particularly at the UB site. Sperm cell distribution occurred evenly along both horns, independent of the site of semen deposition.     

Use of a novel double uterine deposition artificial insemination technique using low concentrations of sperm in pigs

Currently, the three most important non-surgical artificial insemination systems used in pigs are the conventional, the post-cervical (IUI), and the deep-intrauterine (DIUI) methods. In this study, a new system, termed double uterine deposition insemination (DUDI), which combines aspects of both IUI and DIUI, was evaluated. This method used a thinner, shorter and more flexible catheter than those normally used for DIUI and resulted in the deposition of semen post-cervically, approximately half-way along the uterine horn, thus potentially by-passing the threat of 'unilateral' insemination or pregnancy when using sperm of low concentration. The experiment was carried out over 8 weeks on a group of 166 sows, which were divided into seven groups, inseminated with semen of varying concentration, using the conventional system (control group) or by DUDI. There were no significant differences in fertility at day 35 post-insemination between the controls and the various DUDI sub-groups. Only sows inseminated with 500 million viable spermatozoa in a total of 30 mL of fluid using the DUDI system demonstrated decreased total litter sizes when compared to conventional insemination (P<0.001). While conventional insemination normally uses 2.5-3.5 billion sperm, the findings of this study suggest that DUDI can be used under 'field' conditions with sperm concentrations as low as 750 million spermatozoa in 50-30 mL without any detrimental effect on fertility or litter size. DUDI may provide a viable, robust alternative to IUI and DIUI, and has the potential to become incorporated into on-farm insemination systems.     

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.     

A new alternative for embryo transfer and artificial insemination in mares: ultrasound-guided intrauterine injection

A transvaginal ultrasound-guided intrauterine injection (IUI) technique was developed for embryo transfer and for injection of small quantities of sperm in mares. The target area of a horn was positioned by transrectal manipulation against the wall of the vaginal fornix over the face of a transvaginal transducer. A needle with a catheter containing the embryo or semen was inserted through the needle guide of the transducer into the uterine lumen. The tips of the needle and catheter, the movement of the catheter in the uterine lumen, and the ejection of fluid was monitored on the ultrasound screen. Pregnancy rate 15 days after ovulation for the IUI embryo transfer technique (30/39, 77%) was similar to the pregnancy rate for transcervical (TC) embryo transfer (30/38, 79%). The pregnancy rate for IUI insemination of 20 × 10⁶ progressively motile sperm into the tip of the uterine horn ipsilateral to ovulation was 5/10 (50%). Results indicated that the IUI approach is a viable alternative for embryo transfer. Results also supported the potential of IUI for insemination of low numbers of sperm, but more extensive studies with various doses of sperm are needed.     

Distribution of Live and Dead Spermatozoa in the Genital Tract of Gilts at Different Times After Insemination

Twenty-four gilts were inseminated pair-wise with live or dead spermatozoa from the same ejaculate. The insemination dose was 100 ml undiluted semen containing, on average, 19×10⁹ spermatozoa. The gilts were slaughtered 1, 2, 6 and 12 h after insemination. The numbers of spermatozoa were counted in the uterus, uterotubal junction and in four equally long segments of the oviduct, called I–IV, with a haemocytometer. IV was adjacent to the uterotubal junction. The numbers of spermatozoa recovered in the uterus diminished significantly during the first 12 h after insemination. From gilts inseminated with live spermatozoa more spermatozoa were recovered in the uterotubal junction than from gilts inseminated with dead spermatozoa. Two h after insemination spermatozoa were recovered in all oviducts. Significantly more live than dead spermatozoa were recovered in Segments III and IV of the oviduct, regardless of time. In gilts inseminated with live spermatozoa the sperm count in Segment I varied significantly with time, being hiigest 2 h after insemination. At 6 and 12 h there were no distinct differences in the distribution of live spermatozoa between the various oviduct segments. The numbers of spermatozoa recovered in the oviduct were at these times apparently related to the sperm depots in the uterotubal junction.     

Distribution of spermatozoa in the genital tract of artificially inseminated heifers

Eight heifers were artificially inseminated in the uterine body with 160×10⁶ spermatozoa frozen in French mini-straws. The heifers were slaughtered 2 (n = 4) or 12 (n = 4) h after insemination and spermatozoa were recovered by flushing defined segments of the reproductive tract. The efficiency of the method was checked in different ways. There was a slight underestimation of the number of recovered spermatozoa. This underestimation was randomly distributed among heifers and genital tract segments.The total number of spermatozoa recovered was higher at 2 than at 12 h (14.6 vs 0.6 % of the total number inseminated). Most spermatozoa were found in the vagina both at 2 and 12 h after insemination and in greater number at 2 h. In uterus there was a slight decline in the number of spermatozoa recovered at 2 versus 12 h after insemination. The number of spermatozoa recovered from the oviducts were similar at 2 (89.6 × 10³) and 12 h (71.5 × 10³) after insemination. At 2 h spermatozoa were found in all parts of the oviduct with the majority located in the utero tubal junction, whereas at 12 h the most were recovered from isthmus.More spermatozoa were recovered from the left than from the right side of the tract in 6 of the 8 heifers. Only in 1 heifer were the majority of spermatozoa found in the oviduct ipsilateral to the follicle bearing ovary.     

Relationship between the sperm count and the fertilization rate of ova ovulated from the contralateral ovary in intrauterine horn insemination in cats

Unilateral intrauterine horn insemination (UIUI) was carried out in cats, and we investigated the fertilization rate of ova ovulated from the contralateral ovary. Various numbers of sperm were used to inseminate the uterine horn on the side where ovulation was inhibited. The rates of conception were 1/11 (9.1%), 2/11 (18.2%), and 5/7 (71.4%) in the 2 x 10(6), 4 x 10(6), and 8 x 10 (6) groups, respectively. Furthermore, the fertilization rate was 70.7% in the 8 x 10(6) group. Thus, ova ovulated from the contralateral ovary were not fertilized or the fertilization rate was low in some cats even when UIUI was performed with a large number of sperm.     

Low Dose Insemination in the Sow – A Review

Artificial insemination (AI) in pigs has been established for about four decades but ejaculates are still used insufficiently. Higher demand of semen for AI and new techniques that involve low sperm concentration require the optimization of insemination protocols. Based on the knowledge of the physiology of sperm transportation and events in the female genital tract prior to fertilization, new strategies are under development to minimize sperm losses. One goal is to deposit the semen into the uterine horn rather than into the proximal cervix. It was shown that the minimal number of spermatozoa necessary for surgical AI at the utero‐tubal junction (UTJ) were at least 1 × 10⁶ diluted in 0.5 ml of a special extender. Artificial insemination into the distal part of the uterine horn required about 1 × 10⁷ million sperm in 20 ml of extender. Meanwhile, first insemination devices for non‐surgical intra‐uterine AI are commercially available. Using similar sperm concentrations as for surgical AI, non‐surgical uterine insemination did not differ significantly from control inseminations in terms of pregnancy rate and litter size. With respect to the fertilizing capacities of their ejaculates, boars have to be selected more strictly for sperm quality parameters as most of the compensatory effects of sperm cells disappear in maximally extended semen samples.