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.     

Birth of puppies after intrauterine and intratubal insemination with frozen-thawed canine semen

The present study was performed to assess the fertility of frozen-thawed dog semen prepared by freezing with 6% glycerol and thawing at 70℃ for 8 sec, and to evaluate the least number of post-thaw spermatozoa necessary to achieve pregnancy by intrauterine or intratubal artificial insemination. It was found that the pregnancy rate of intrauterine artificial insemination was 100% using 6% glycerol buffer and thawing at 70℃ for 8 sec with 5 × 10⁷ spermatozoa. Even though the pregnancy rate (80%) and the whelping rate (24.5%) in the 5 × 10⁶ spermatozoa inseminated group were lower than those of the 5 × 10⁷ spermatozoa group, conception was confirmed with 5 × 10⁶ spermatozoa. Although the pregnancy rate of intratubal insemination was low (20%) with 4 × 10⁶ spermatozoa, this study is the first report to show the pregnancy rate of intratubal insemination with frozen-thawed ejaculated canine semen. In order to improve the pregnancy rate with intratubal insemination of canine spermatozoa, it is necessary to investigate the optimal insemination site of the uterine tube, the appropriate number of sperm, and the direct effect of buffer on oocytes.     

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 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.     

Semen quality and fertility after heat stress in boars

Sperm morphology and the fertilizing capacity of ejaculated spermatozoa were examined in 6 Swedish Landrace boars before and after heat stress. The boars were exposed to 35° C during 100 h in a climatic room. Fertility was measured by insemination of gilts before and at various times after heat stress. Each gilt (n = 44) was inseminated with a total of 5×10⁹ spermatozoa diluted to 10O ml with EDTA-glucose diluent and fertilization was assessed by examining recovered ova 2 days after insemination.Changes in semen quality varied among the boars from a very weak response in 2 boars to pronounced semen alterations occurring 2–6 weeks after heat stress in the other boars. A close relationship was found between seminal changes and fertilization rates, all ejaculates which had high fertilization rates being of the same quality as the pre-exposure ejaculates. The ejaculates that had poor fertility were characterized by lowered sperm motility and increased numbers of spermatozoa with abnormal heads, proximal cytoplasmic droplets and nuclear pouch formations.     

Intraperitoneal insemination and retrograde sperm transport in dairy cows

To examine the efficiency of retrograde sperm transport following intraperitoneal insemination, live and dead spermatozoa were used at different concentrations, and sperm recovery from cervical mucus (0.5 ml) 2, 6, 12 and 24 h following insemination was evaluated. Forty lactating Friesian cows, in their second to fourth lactation period, were used in this experiment. Thirty-six cows received intraperitoneally either live or dead spermatozoa. Each group of six cows received one of three total sperm numbers of 30, 45 and 90 million. Four cows were inseminated with 90 million spermatozoa into the uterus and served as a control group. All cows were inseminated towards the end of oestrus. After intrauterine insemination sperm recovery declined, but motile and/or immotile spermatozoa were recovered from all cows at any time. In cows inseminated intraperitoneally, sperm was recovered from the cervix at 6-24 h when 90 million were inseminated. A greater number of spermatozoa was recovered after dead rather than after live sperm inseminations. Only immotile, intact or broken spermatozoa and tail-less heads were recovered after intraperitioneal insemination using either live or dead spermatozoa. No sperm was recovered for 30 and 45 million inseminations. Our results show that, following intraperitoneal insemination, there is passive sperm transport from the peritoneal cavity to the genital tract close to the time of ovulation, and suggest a higher sperm retention in the genital tract when live as opposed to dead spermatozoa are used.     

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.     

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.     

Relationship between sperm quality traits and field-fertility of porcine semen

An investigation involving seven boars, active in artificial insemination, and 1,350 multiparous sows was conducted at a private farm and aimed at examining the relationship between sperm quality traits and boar fertility in terms of farrowing rate and litter size. This experiment was done for 6 months. The semen samples were evaluated for subjective sperm motility and concentration. Ejaculates with at least 1 × 10⁸ sperm/mL and 70% sperm progressive motility were extended with a commercial medium to 30 × 10⁶ sperm/mL and used for artificial insemination (AI). AI dose was 100 mL semen containing 3 × 10⁹ spermatozoa. Aliquots of diluted semen were assessed for live morphologically normal spermatozoa (LMNS, eosin-nigrosin stain exclusion assay) and sperm chromatin instability (SCI, acridine orange assay). Farrowing rates according to different boar sperm varied (p < 0.001) from 59.3 to 88.92%. The mean values of LMNS (47.2~76.5%) and SCI (0.16~4.67%) differed significantly among boars. LMNS (r = 0.79, p < 0.05) and SCI (r = -0.90, p < 0.02) accounted for 62.2 and 81.7% of the variability in farrowing rates, respectively. After the combination of sperm traits, the relationship between percentage of LMNS with stable chromatin structure and farrowing rate was significant (r = 0.86, p < 0.05). The number of live piglets per parturition was not significantly correlated with sperm quality attributes. In conclusion, boar fertility after AI with freshly diluted semen can be predicted based on the evaluation of sperm morphology and chromatin integrity.     

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.     

Boar sperm production: Use in artificial insemination — A review

The production of spermatozoa increases with boar age from puberty to adulthood. Increasing collection frequency to more than once or twice a week increases the quantity of spermatozoa recovered per boar. Possible improvement of quality by collecting more often remains open to discussion. Sperm production is mainly a result of testis size, which varies with individuals and families. Selection on female ovulation rate leads to more rapid testis growth of males in the families selected. There is a correlation between pregnancy rate obtained after insemination with a given board, small size (direct boar effect) and embryonic survival rate at 25 days. Keeping boars at a high temperature lowers the quantity of spermatozoa recovered, pregnancy rate and embryonic survival in the sows inseminated. Fresh sperm may be stored for 2 days without decreasing its fertilizing ability (dose of 3 × 10⁹ spz. per AI). It may be efficiently used on day 3 of storage (double dose). The preparation and use of deep-frozen sperm is simple. Efficacious use is highly correlated with a good knowledge of sow oestrus. The number of sows which can be inseminated with one deep-frozen ejaculate is limited to 8–10 due to the number of motile spermatozoa after thawing.     

Fertility in Single‐ovulating and Superovulated Dairy Heifers after Insemination with Low Dose Sex‐sorted Sperm

The present study was performed to test fertility in single‐ovulating and superovulated dairy heifers after insemination with low dose sex‐sorted sperm under field conditions. Some parameters, including the dosage, deposition site and timing, were assessed with the pregnancy rates after artificial insemination (AI). Moreover, the use of oestrus synchronization in combination with sorted sperm was evaluated. Besides that, we also improved the embryo production efficiency in superovulated dairy heifers by optimizing the timing of inseminations and repartitioning the sexed sperm dosage among multiple inseminations. The conception rate (52.8%) in heifers after low dose (2 × 10⁶) insemination with sorted sperm deep into the uterine horn did not differ (p > 0.05) from that (59.6%) of conventional AI (1 × 10⁷ non‐sorted sperm) and that of deep insemination with low dose non‐sorted sperm (57.7%). There was also no difference (p > 0.05) between conception rates after single (51.7%) and double (53.8%) deep insemination with sorted semen. Heifers inseminated with sorted sperm at synchronous oestrus had a lower pregnancy rate (48.1%) than heifers at spontaneous oestrus (53.6%), but this did not reach statistical difference (p > 0.05). The average number of transferable embryos collected in vivo from heifers inseminated with sorted sperm (4.81 ± 2.04) did not differ (p > 0.05) from that obtained from heifers after insemination with non‐sorted sperm (5.36 ± 2.74). Thus, we concluded that the pregnancy rate after deep intra‐uterine insemination with low dose sorted sperm was similar to that of non‐sorted sperm, which was either also deposited at a low dose deep intra‐uterine or into the uterine body. Sychronization of oestrus can be beneficial in combination with sorted sperm to optimize the organization and management of dairy herds. The results from superovulated heifers demonstrated that our insemination regime can be used to obtain a comparable embryo production efficiency with sorted sperm than with non‐sorted sperm.     

Sperm Distribution and Its Relation to Ovulation in Artificially Inseminated Heifers

Contents: Blood samples were taken in ten heifers every two hours during prooestrus and oestrus for determination of the LH peak, which was used to calculote the time of expected ovulation. Artificial insemination with frozen/thawed semen was performed at different times from the beginning of oestrus and the animals were slaughtered two, twelve or 24 hours after insemination. The genital tract was flushed and the spermatozoa recovered from the flushing solution by microscopic investigation. Heifers slaughtered two hours after insemination and more than 19 hours before ovulation had more spermatozoa in the uterotubal junction (UTJ) than in the isthmus and ampulla. This sperm distribution differed from that in the other animals. In animals which were slaughtered closer to ovulation and in those who had already ovulated at slaughter, approximately the same number of spermatozoa were recovered from the UTJ and isthmus and only a few spermatozoa were recovered from the ampulla. Motile spermatozoa were found in both the isthmus, UTJ and uterus at two and twelve hours after insemination and approximately ten hours before ovulation. Their number decreased with time but some motile spermatozoa were also recovered 24 hours after insemination and after ovulation. The distribution of spermatozoa in the oviducts ipsi-and contralateral to the corpus haemorrhagicum-bearing ovary was compared in heifers that had ovulated. There were no differences in the number of oviductal segments containing spermatozoa, nor were there any differences within animal in the number of spermatozoa in the oviducts. It was concluded that ovulation does not seem to cause great changes in the distribution of spermatozoa in the oviducts. Inhalt: Spermienverteilung und deren Beziehung zur Ovulation bei kunstlich besamten Jungrindern Von 10 Färsen wurden wahrend des Diöstrus und Östrus Blutproben im Abstand von 2 Std. entnommen, um den LH-Gipfel festzustellen. Dieser wurde jür die Bestimmung des erwarteten Ovulationstermins herangezogen. Inseminationen mit Gefriersperma wurden von Brunstbeginn an in verschiedenen Intervallen vorgenommen. Die Tiere wurden 2, 12 oder 24 Std. nach der Besamung getötet, die Geschlechtsorgane gespült und die Spermien in den Spülflüssigkeiten mikroskopisch nachgewiesen. Färsen, die 2 Std. nach der Besamung und mehr als 19 Std. vor der Ovulation getötet wurden, hat-ten mehr Spermien im Ostium uterinum des Eileiters (OU) als im Isthmus und Ampulle. Diese Verteilung war von jener der anderen Tiere verschieden. Tiere, die kürzere Zeit vor der Ovulation getötet wurden, oder bei der Schlachtung schon ovuliert hatten, zeigten etwa dieselbe Anzahl von Spermien in OU und Isthmus, in der Ampulle wurden nur wenig Spermien gefunden. Bei Färsen, die ovuliert hatten, wurde die Sper-mienverteilung in den ipsi- und contralateralen Eileitern zum Corpus haemowhagicum des Ovars verglichen. Die Spermienxahlen in den Eileitersegmenten waren nicht verschieden und auch im rechten und linken Eileiter gleich. Es wurde gefolgert, daβ die Ovulation keinen groβen Einfluβ auf die Spermienverteilung in den Eileitern bewirkt.     

Incidence of Unilateral Fertilizations after Low Dose Deep Intrauterine Insemination in Spontaneously Ovulating Sows under Field Conditions

A new procedure for non-surgical deep intrauterine insemination (DUI) in unrestrained sows hormonally induced to ovulate, has been reported. In comparison with standard artificial insemination (AI), with this procedure, the sperm numbers inseminated can be reduced 20-fold without reducing the reproductive performance of these hormonally treated sows. The present study evaluated, using two experiments, the reproductive performance applying 20-fold different sperm numbers per AI dose using DUI or standard AI in spontaneously ovulating sows, under field conditions. In experiment 1, AI was applied to crossbred sows at 12, 24 and 36 h after onset of spontaneous oestrus using one of the following two regimes: (i) DUI (treatment) with 0.15 x 10(9) fresh boar spermatozoa in 5 ml of Beltsville thawing solution (BTS) extender (n = 95), and (ii) standard cervical AI (control) with 2.85 x 10(9) fresh spermatozoa in 95 ml of BTS extender (n = 95). The farrowing rates of the two groups of sows were statistically similar (NS). However, a decrease (p < 0.002) in litter size and the total number of pigs born alive was observed in sows inseminated with the DUI procedure. In experiment 2, 42 post-weaned oestrus sows were inseminated following the same design described for experiment 1 during spontaneous oestrus. On day 6 after onset of oestrus, the proximal segment of the uterine horns of the sows were flushed under surgery to retrieve eventual embryos and evaluate the success of fertilization per cornua (e.g. occurrence of effective uni- vs bilateral sperm transport rendering uni- or bilateral, complete or partial fertilization). Retrieved embryos were assessed for cleavage and number of accessory spermatozoa. Although identical overall pregnancy rates were achieved in both insemination groups, the percentage of sows with partial bilateral fertilization and unilateral fertilization was markedly higher (p < 0.05) in the DUI group (35%) compared with the control (standard AI) group (5%), with a consequent lower (p < 0.001) percentage of viable early embryos after DUI. The number of accessory spermatozoa in the zona pellucida of the embryos was highly variable, but higher (p < 0.001) in control animals than in DUI-AI. No accessory spermatozoa were found in oocytes retrieved from sows depicting unilateral fertilization. In conclusion, DUI in spontaneously ovulating sows with 0.15 x 10(9) spermatozoa renders similar farrowing rates but a lower litter size compared with use of standard AI with a 20-fold higher sperm dose. The lower litter size ought to be related to a decreased distribution of spermatozoa after DUI leading to a higher incidence of partial bilateral and unilateral fertilization.     

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.     

Is the Function of the Porcine Sperm Reservoir Restricted to the Ovulatory Period?

The uterotubal junction (UTJ) and caudal isthmus are recognized as a functional pre-ovulatory sperm reservoir (SR). Spermatozoa are released from the SR in a complex and concerted action. However, whether this functionality is restricted only to the ovulatory period is still open to debate. Our study was aimed to analyze the presence of spermatozoa within the UTJ (SR), isthmus (ISTH) and ampulla (AMP) after laparoscopic intrauterine insemination (LIUI) either in the peri- (PERI) or post-ovulatory (POST) period or at mid cycle (MID). Each uterine horn of estrus synchronized gilts (n=12) was inseminated with 20 ml sperm (29.5×10⁶ cells/ml). Oviducts were recovered 7 h after LIUI and separated into the UTJ, ISTH and AMP, and sections were flushed with 10 ml PBS+EDTA solution. After centrifugation, the sperm pellet was evaluated by Čeřovský staining. The median sperm numbers in the PERI, POST and MID groups were 578, 171 and 789 in the UTJ; 545, 233 and 713 in the ISTH; and 496, 280 and 926 in the AMP, respectively, and there were differences between the POST and MID groups (P<0.05) but not between the oviductal sections of each group (P>0.05). Compared with the MID group, the percent of intact sperm cells was higher (P<0.01) in the PERI and POST groups (32.8 vs. 66.4 and 76.8%). Also, the percentages of aberrations in the acrosome and tail were higher (P<0.05) in the MID group. Based on this, it can be assumed that the sperm reservoir is active during different phases of the estrus cycle. However, the mid-cycle oviduct environment considerably impairs sperm cell quality.     

A new device to inseminate cows at the base of the uterine horns

A new device (Chapingo device) to deposit semen at the base of the uterine horns of cattle was developed at Universidad Autonoma Chapingo, Mexico. Nine Holstein heifers were inseminated by transvaginal laparoscopy, using a laparoscope for cattle and the Chapingo device. A dose of sexed semen (2.1 × 10⁶ spermatozoa) was deposited at the base of the uterine horn ipsilateral to the ovary where the preovulatory follicle was identified. Insemination was achieved in all the heifers, taking on average 13.7 ± 3.1 min per animal. In all cases, it was possible to see both ovaries, the base of the uterine horns and the oviducts. After the procedure, none of the heifers showed any type of complications such as haemorrhage, adhesions or trauma. On days 21 and 22 after insemination, four of the nine heifers (44.4%) returned into oestrus; on day 30 after insemination, one heifer was found to be pregnant by ultrasound. The results show the feasibility of generating pregnancies by transvaginal laparoscopy in heifers inseminated with sexed semen.     

Establishment of a Pregnancy Following Intravaginal Insemination with Epididymal Semen from a Dog Castrated due to Benign Prostatic Hyperplasia

Benign prostatic hyperplasia was diagnosed in an American Staffordshire Terrier of high breeding value presenting concurrent haematuria. Castration as a treatment was synchronized with the oestrus cycle of a bitch selected for insemination. After castration the cauda epididymis was flushed with Gent semen extender and collected spermatozoa were filtered and analysed by Hamilton Thorn computer assisted sperm analysis. A total of 7 ml semen containing 742 x 10(6) spermatozoa with 76.5% mean motility was used for insemination. Intravaginal insemination of the bitch was performed with an insemination catheter for dogs (Kruuse, Marslev, Denmark) on the day when plasma progesterone levels reached 9.9 ng/ml. Normal pregnancy without complications resulted in eight live-born puppies 63 days after insemination. This is the first report of a normal pregnancy and birth of puppies from a bitch inseminated with epididymal semen obtained from a dog affected by benign prostate hyperplasia.     

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.