Boar semen cryopreservation: State of the art, and international trade vision.

Biosecurity is a major concern in the global pig production. The separation in time of semen collection, processing and insemination in the pig farm is a few days for chilled semen but it can be indefinite when using cryopreserved semen. Field fertility results of boar cryopreserved semen are close to chilled semen, which makes it a valuable resource for the establishment of semen genebanks, long-distance semen trade, and the implementation of other technologies such as the sex-sorted semen. But cryopreserved semen is far from being routine in pig farms. The most recent research efforts to facilitate its implementation include the use of additives before freezing, or in the thawing extender. Long-term preserved semen trade is a biosecurity challenge. To harmonize international trade of germplasm, the World Organization of Animal Health (WOAH) established a regulatory framework for all member countries. The present paper aims to review the latest advances of boar semen cryopreservation with special focus on the benefits of its inclusion as a routine tool in the pig industry. We also review recently reported field fertility results of cryopreserved semen, its international trade compared to chilled semen, and the regulatory framework involved. Boar cryopreserved semen is a valuable tool to control biosecurity risk, implement other technologies, and facilitate international trade. Research already demonstrated good field fertility results, but it still represents less than 0.1 % of the international trade. As boar cryopreserved semen gets closer to implementation, the correspondent authorities are reviewing the trade rules.

Defined Combinations of Cryomedia and Thawing Extenders Influence the Viable X-Y Boar Sperm Ratio in Vitro.

BACKGROUND: It is believed that plasma membrane X- and Y-chromosome bearing sperm are different; therefore the freezing and thawing process may affect X- and Y-sperm differently. OBJECTIVE: The objective of this study was to investigate the effect of cryomedia and thawing extenders on the survival of X and Y-sperm. MATERIALS AND METHODS: Three different cryomedia and thawing extenders were compared. Viable motile sperm were separated using a swim-up technique. Real-time PCR was used to identify the sperm type. RESULTS: Using CryoA for freezing and Beltsville-Thawing-Solution (BTS) as the thawing extender yielded significantly higher numbers of viable motile Y sperm (64 percent) than control (48 percent) (P < 0.01). Conversely, semen freezing with CryoC and thawing with Androstar Plus gave a significantly lower number of viable motile Y sperm (32 percent) than control (51 percent). CONCLUSION: Our results revealed that defined combinations of cryomedia and thawing extenders significantly altered the survival ratio of frozen-thawed X-Y sperm in vitro, which has potential implications for artificial insemination.

Low-density Lipoprotein Improves Motility and Plasma Membrane Integrity of Cryopreserved Canine Epididymal Spermatozoa.

Cryopreservation of caudal epididymal spermatozoa is an effective technique to conserve genetic potentials of superior dogs when it is not possible to collect ejaculated spermatozoa. Although hen egg yolk is commonly supplemented into the semen extender, active substances within the egg yolk which protect sperm against cryoinjury remain to be discovered. Among its compositions, low-density lipoprotein (LDL) has been reported to have a cryoprotective property for sperm cryopreservation. However, the effects of LDL on dog epididymal spermatozoa during cryopreservation have not yet been investigated. This study aimed to investigate the effects of LDL on epididymal spermatozoa quality following cryopreservation and thawing. After routine castration of 12 dogs, caudal epididymides from individuals were separated from the testes and cut into a few pieces in a Tris-buffer. Spermatozoa recovered from each sample were examined at once for sperm quality and divided into six groups of extender: no LDL, 20% egg yolk, 4%, 8%, 16%, and 24% LDL, before cryopreservation. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, morphology, plasma membrane integrity, and acrosome integrity were evaluated. The results revealed that 4% LDL and 20% egg yolk yielded significantly higher sperm motility (57.69% and 52.69%, respectively, p<0.05) than other LDLs. In addition, 4% LDL yielded the significantly highest plasma membrane integrity (70.54%, p<0.05). In conclusion, the supplementation of 4% LDL in Tris-glucose extender could be applied for cryopreservation of canine epididymal spermatozoa.

Breed of boar influences the optimal concentration of gamma-oryzanol needed for semen cryopreservation.

The aim of this study was to investigate the influence of boar breed on the optimal concentration of gamma-oryzanol on the qualities of cryopreserved boar semen. Semen was collected from 20 boars (10 Duroc, 5 Large white and 5 Landrace boars). The semen sample was divided into five groups (A-E) according to the concentration of gamma-oryzanol in extender II, that is 0, 0.08, 0.16, 0.24 and 0.32 mM, respectively. The semen was cryopreserved by nitrogen vapour and storage in nitrogen tank (-196°C). After storage for a week, samples were thawed at 50°C for 12 s and evaluated for progressive motility, sperm viability and acrosome integrity. The results demonstrated that gamma-oryzanol significantly improved progressive motility, viability and acrosome integrity of frozen-thawed boar semen. Considering the influence of breeds on the optimal concentration of gamma-oryzanol, for Duroc boar, gamma-oryzanol at 0.16 mM (group C) yielded the highest percentage of progressive motility, sperm viability and acrosome integrity. For Large white and Landrace boars, gamma-oryzanol at 0.24 mM (group D) showed a significantly higher percentage of progressive motility, viability (not significant in Landrace) and acrosome integrity than other concentrations. In conclusion, the optimal concentration of gamma-oryzanol needed for boar semen cryopreservation in lactose-egg yolk (LEY) freezing extender is not only depended on individual boar but also breed of boar, that is 0.16 mM for Duroc and 0.24 mM for Large white and Landrace.

L-carnitine Supplemented Extender Improves Cryopreserved-thawed Cat Epididymal Sperm Motility.

Cryopreservation of epididymal sperm is an effective technique to preserve genetic materials of domestic cats and wild felids when they unexpectedly die. However, this technique inevitably causes detrimental changes of cryopreserved-thawed spermatozoa, for example, by physical damage and excessive oxidative stress. L-carnitine is an antioxidant that has been used to improve sperm motility in humans and domestic animals. This study aimed to investigate the effects of L-carnitine on cat epididymal sperm quality following cryopreservation and thawing. After routine castration, cauda epididymides were collected from 60 cat testes. The epididymal spermatozoa from 3 cauda epididymides were pooled as 1 replicate. Spermatozoa samples (16 replicates) were examined for spermatozoa quality and then randomly divided into 4 groups: 0 mM L-carnitine (control), 12.5 mM, 25 mM and 50 mM L-carnitine. The sperm aliquots were then equilibrated and conventionally frozen. After thawing, sperm motility, plasma membrane integrity, DNA integrity and acrosome integrity were evaluated. The 25 mM L-carnitine significantly improved sperm motility compared with a control group (p<0.05), although this was not significantly different among other concentrations. In conclusion, supplementation of 25 mM L-carnitine in freezing extender improves cauda epididymal spermatozoa motility. The effects of L-carnitine on the levels of oxidative stress during freezing and thawing remains to be examined.

Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds.

During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

Study on the oestrous synchronization in gilts by using progestin altrenogest and hCG: its effect on the follicular development, ovulation time and subsequent reproductive performance.

The aim of this study was to further investigate the effect of using progestin altrenogest and hCG to synchronize the oestrous cycle and its effect on follicular development, ovulation time and subsequent reproductive performance. Thirty crossbred gilts were divided into three groups. Group A (control) received a 5 ml of normal saline for 18 consecutive days by individually top-dressing. Groups B and C gilts received 20 mg (5 ml) of progestin altrenogest for 18 consecutive days by individually top-dressing. On day 3 (72 h) after withdrawal of progestin altrenogest, Group C gilts received hCG (500 IU, im). The follicular development and ovulation time were examined by transabdominal ultrasonography. Subsequent reproductive performances, i.e. number of total born per litter (NTB), number of live born per litter (NBA), number of stillbirth per litter (NSB), average piglet birth weight (ABW), lactation length (LL) and weaning to oestrous interval (WOI), were recorded. None of the gilts in Group A showed oestrus within 10 days after withdrawal of normal saline. Groups B (eight of 10) and C gilts (four of 10) came into oestrus at 5.6 +/- 0.5 and 6.5 +/- 0.6 days after withdrawal of progestin altrenogest, respectively. The ovulation time of Groups B and C gilts took placed at 25.0 +/- 4.7 and 25.0 +/- 5.0 h after standing oestrus, respectively. The pre-ovulatory follicular size (diameter) of Groups B and C gilts was 8.0 +/- 2.0 and 11.0 +/- 3.0 mm, respectively. A tendency of larger litter size (NTB) in Group B gilts was found when compared with Group A gilts. To conclude, using progestin altrenogest alone can be used to synchronize the oestrous cycle in gilts without unenthusiastic effect on the follicular development, ovulation time and subsequent reproductive performances. However, treatment of gilts with hCG at day 3 (72 h) after withdrawal of altrenogest had unenthusiastic effect on oestrus synchronization.

The effect of dose and route of administration of R-cloprostenol on the parturient response of sows.

The aims of the present study were to further examine the efficacy of different doses and routes of R-cloprostenol administration on the parturition response in sows. Fifty crossbred multiparous sows (Landrace x Yorkshire) with an average parity number of 4.7 +/- 2.4 were allocated to induce farrowing by one of the following treatments: Group I (control, n = 10) injection with normal saline 2 ml administered intramuscularly (i.m.); Group II (n = 10) injection with 75 microg of R-cloprostenol administered i.m. (at 7 AM); Group III (n = 10) injection with 75 microg of R-cloprostenol (at 7 AM) together with 10 IU of oxytocin (24 h after injection of R-cloprostenol) administered i.m.; Group IV (n = 10) injection with 37.5 microg of R-cloprostenol (at 7 AM) administered into perivulva region; Group V (n = 10) injection with 37.5 microg of R-cloprostenol (at 7 AM) administered into perivulva region together with 10 IU of oxytocin (24 h after injection of R-cloprostenol) administered i.m. The following parameters: pre-farrowing maternal behaviour, restless behaviour, R-cloprostenol or oxytocin injection to farrowing interval, expulsion intervals, duration of farrowing, total number of piglets born, litter birthweight, umbilical cord morphology and the degree of meconium staining were record. There were no significant differences among groups for the pre-farrowing maternal behaviours. In all the sows, the restless behaviour was not observed. There were no significant effect of oxytocin administration (10 IU, i.m.) on the percentage of umbilical cord morphology and the degree of meconium staining in different groups. There were no significant effect of route and dose of administration on the number of total piglet born, piglet born alive, stillbirth, mummy and litter birthweight. No significant effects of the different groups were found on the R-cloprostenol and oxytocin injection to farrowing interval, expulsion interval and farrowing duration. In conclusion, the present results demonstrated that a half dose (37.5 microg) of R-cloprostenol administered into the perivulva region was effective for inducing farrowing as the full recommended dose (75 microg) administered into the neck region (i.m.) and with no restless behaviour.

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 sow endosalpinx at different stages of the oestrous cycle and at anoestrus: studies on morphological changes and infiltration by cells of the immune system.

The aim of this study was to investigate the morphological changes of the sow endosalpinx and the distribution of leukocytes throughout the oestrous cycle and at anoestrus. Nineteen crossbred sows (Swedish Landrace x Swedish Yorkshire) at late dioestrus (three), prooestrus (three), oestrus (three), early dioestrus (three), dioestrus (three) and anoestrus (four) were used. Oviductal samples from three different parts (isthmus, ampulla and infundibulum), taken immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue or stored in a freezer at -70 degrees C until analysed by immunohistochemistry (prooestrus and anoestrus) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. During all stages, a lower degree of morphological changes (pseudostratification, mitosis and secretory granules) was found in the isthmus compared with ampulla and infundibulum. In ampulla and infundibulum, pseudostratification, mitotic activity and secretory granules of the epithelium were high at prooestrus/oestrus. Cytoplasmic protrusions of epithelial cells with some extruded nuclei were prominent in ampulla and infundibulum at all stages except for oestrus and early dioestrus. Lymphocytes as well as CD2- and CD3-positive cells were the predominant immune cells in the epithelial layer. The numbers of lymphocytes and CD3-positive cells did not differ among segments and stages. Numbers of CD2-positive cells did not differ between prooestrus and anoestrus while the numbers were significantly higher in the infundibulum than in ampulla and isthmus. Neutrophils were only occasionally found and mainly in the infundibulum. In the subepithelial connective tissue layer, the two most commonly observed immune cell types were lymphocytes and plasma cells. The numbers of lymphocytes as well as CD2- and CD3-positive cells was lower in isthmus than in the other segments (p < or = 0.001). Higher numbers of plasma cells (p < or = 0.001) were found in infundibulum than in ampulla and isthmus. The numbers of lymphocytes and plasma cells were not significantly different between stages of the oestrous cycle. However, the number of neutrophils differed and were highest at prooestrus in ampulla and infundibulum. The numbers of CD2-, CD3- and CD79-positive cells did not differ between prooestrus and anoestrus whereas for CD14- and SWC3-positive cells, the numbers were higher at prooestrus (p < or = 0.05) than at anoestrus. In the oviduct, the morphology differed in ampulla and infundibulum with oestrous cycle stages, which indicates an effect by ovarian steroid hormones. The immune cell infiltration was less influenced by cyclic changes. However, the immune cell infiltration (in the connective tissue) in the upper part, especially infundibulum, differed significantly from the one in the lower part, isthmus, indicating different immune functions within various parts of the oviduct.

Immunohistochemical studies on oestrogen receptor alpha (ERalpha) 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.

Immune cell infiltration of normal and impaired sow endometrium.

The paper reviews the physiological infiltration of immune cells, leukocytes, in the sow endometrium during different stages of the normal oestrous cycle, after mating and during early pregnancy. The mechanisms for development of endometritis in relation to oestrous cycle stages are also described.

Influence of CRH and ACTH administration on endocrine profile and ovulation in sows.

Grouping of sows is a stressful event until the ranking is established. The purpose of this study was to simulate stress by repeated administration of porcine corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH)/tetracosactide and to study its influence on endocrine profile and ovulation. Four multiparous sows were used and blood was collected every 2 h from the onset of pro-oestrus until 12 h after ovulation. The first oestrus after weaning was used to check ovulation and acclimate the sows to their environment. The second oestrus after weaning was used as control. At their third oestrus CRH (0.6 microg/kg) and at their fourth oestrus ACTH (5 microg/kg) were given every 4 h from onset of oestrus until ovulation. The total 'area under the curve' of cortisol was twofold larger in two of four sows during the CRH treatment period, and two- to fourfold larger (p < or = 0.05) during the ACTH treatment period, compared with the corresponding control period. In three sows, there was no clear effect of either CRH or ACTH on the levels of oestradiol 17beta, luteinizing hormone (LH) or on the timing of ovulation. One sow was different in all hormonal patterns and also in the timing of ovulation. In all four sows, ACTH treatment lowered the baseline level of prostaglandin F(2 alpha)-metabolite. Therefore, we conclude that stage of the oestrous cycle seems to be of importance when investigating the influence of exogenous administration of CRH/ACTH on hormonal pattern and ovulation time in the sow.

Influence of post-ovulatory insemination on sperm distribution, pregnancy and the infiltration by cells of the immune system, and the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium.

This study investigates the distribution of leucocytes, CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following post-ovulatory insemination in relation to clinical findings and pregnancy outcome. Crossbred multiparous sows were inseminated once either at 15-20 h after ovulation [experiment 1, slaughtered at 20-25 h (5-6 h after artificial insemination (AI), group 1-A, n = 4), at 70 h after ovulation (group 1-B, n = 4), on day 11 (group 1-C, n = 4, first day of standing oestrus = day 1) or on day 19 (group 1-D, n = 4)] or 30 h after ovulation [experiment 2, slaughtered at 5-6 h after AI (group 2-A, n = 4) or on day 19 (group 2-D, n = 3)]. The uterine horns were flushed to control for the presence of spermatozoa and neutrophils and/or for recovery of oocytes and/or embryos. Mesometrial uterine samples were plastic embedded and stained. Cryofixed uterine samples were analysed by immunohistochemistry using mAbs to lymphocyte subpopulations and MHC class II molecules. Light microscopy was used to examine surface (SE) and glandular epithelia (GE), and connective tissue layers, both subepithelially (SL) and glandular (GL). In experiment 1, group 1-A, only one sow had spermatozoa in the utero-tubal junction (UTJ). Marked/moderated numbers of neutrophils and spermatozoa were observed in the flushings of two sows. In group 1-B, altogether 23 of 48 oocytes were cleaved. Day 11 (1-C), embryos with small diameter were observed. Day 19 (1-D), no embryos were found but small pieces of foetal membrane were observed in one of the sows. In group 1-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. For T lymphocyte subpopulations, in the SE, most CD2+ cells were found in group 1-A. For both SE and GE in all groups, the number of CD8+ cells was significantly larger than that of CD4+ cells. In experiment 2, group 2-A, no sow had spermatozoa in the UTJ or in the uterine flushings. At day 19, no sow was pregnant. In group 2-A, large numbers of neutrophils were found within the SE and SL but with high individual variation. At day 19, high E2 levels showed a hormonal prooestrous stage but the endometrial neutrophil infiltration normally expected at pro-oestrus was absent. In conclusion, post-ovulatory insemination (about 18 h after ovulation) resulted in impaired spermatozoa transport within the uterus and embryonic degeneration. In sows post-ovulatory inseminated at a later stage (30 h after ovulation), no sow was pregnant. In both experiments, disturbed immune cell patterns were observed in some individuals.

Influence of pre-ovulatory insemination and early pregnancy on the distribution of CD2, CD4, CD8 and MHC class II expressing cells in the sow endometrium.

The aim was to investigate the distribution of CD2(+), CD4(+) and CD8(+) lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium following pre-ovulatory insemination and during early pregnancy. Crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were inseminated once at 15-20 h before ovulation. The sows were slaughtered at 5-6h (group I, n=4) after AI or at 20-25 h (group II, n=4) and 70 h (group III, n=4) after ovulation, day 11 (group IV, day 1=first day of standing oestrus, n=3) and day 19 (group V, n=3). Uterine horns were flushed to control for the presence of spermatozoa and neutrophils (groups I-IV) and/or for recovery of oocytes and/or embryos (groups II-IV, control of pregnancy). Cryofixed mesometrial uterine samples were analysed by immunohistochemistry with an avidin-biotin-peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface (SE) and glandular (GE) epithelia as well as connective tissue layers in subepithelial (SL) and glandular (GL) areas were examined by light microscopy. Taking all groups and different tissue layers together, the most commonly observed positive cells were CD2(+) cells (P

Immunohistochemical studies on oestrogen receptor alpha (ER alpha) and the proliferative marker Ki-67 in the sow uterus at different stages of the oestrous cycle.

In order to better understand physiological changes during the different stages of the oestrous cycle, immunohistochemistry was used in the present study to investigate the distribution of oestrogen receptor alpha (ER alpha) as well as the proliferative marker Ki-67, in the sow uterus during the oestrous cycle. Uterine samples were collected from multiparous sows with normal reproductive performance at selected stages of the oestrous cycle: at late dioestrus (d 17), prooestrus (d 19), oestrous (d 1), early dioestrus (d 4) and dioestrus (d 11-12), respectively. The tissue samples were fixed in 10% formaldehyde, embedded in paraffin and subjected to immunohistochemistry using monoclonal antibodies against ER alpha (C-311) and Ki-67 (MM-1). In general, the immunostaining of both ER alpha and Ki-67 was confined to nuclei of the target cells. Variations were seen, not only at the different stages of the oestrous cycle, but also in the different tissue compartments of the uterus. In the epithelia, the strongest ER alpha staining and highest amount of positive Ki-67 cells were found at early dioestrus. In the myometrium, the highest levels of staining of both ER alpha and Ki-67 positive cells were found at pro-oestrus and oestrus. For the proliferative marker, Ki-67, no positive cells were found at dioestrus and late dioestrus in the epithelium and myometrium. In the connective tissue stroma (subepithelial layer), the highest number of ER alpha positive cells were found at oestrus, which was significantly different compared with other stages (p< or = 0.05), whereas the levels of Ki-67 positive cells were relatively low and did not differ between the stages examined. Significant correlations between the number of ER alpha positive cells in the stroma and Ki-67 positive cells in the epithelia were observed. This suggests indirect regulatory mechanisms on epithelial proliferation via ER alpha in the stroma. In conclusion, these findings in the sow uterus show that the presence of ER alpha as well as Ki-67 protein varies not only between different stages of the oestrous cycle but also between different tissue compartments of the uterus. These findings indicate various regulatory mechanisms and stress the importance of localising ER alpha and proliferating cells in different uterine tissues.

Influence of pre-ovulatory insemination and early pregnancy on the infiltration by cells of the immune system in the sow endometrium.

The aim of this study was to investigate the distribution of leukocytes in the sow endometrium following insemination and during early pregnancy. Cross-bred multiparous sows (Swedish Landrace x Swedish Yorkshire) were artificially inseminated (AI) once at 20-15 h before ovulation. Blood samples were collected from the jugular vein 1 h before slaughter for analyses of oestradiol-17beta and progesterone levels. The sows were slaughtered at 5-6 h (group I, n = 4) after AI or at different times after ovulation: 20-25 h (group II, n = 4), 70 h (group III, n = 4), day 11 (group IV, n = 3; first day of standing oestrus = day 1) and day 19 (group V, n = 3). Uterine horns were flushed to control for the presence of spermatozoa and neutrophils (groups I-IV) and/or for recovery of oocytes and/or embryos (groups II-IV, control of pregnancy). Mesometrial uterine samples were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelia as well as subepithelial and glandular connective tissue layers were examined by light microscopy. A marked number of neutrophils and spermatozoa were observed in the flushings from the uterine horns of sows slaughtered at 5-6 h after insemination. All animals slaughtered after ovulation were pregnant but no morphological effect of pregnancy was observed until day 11. In the surface epithelium, the largest numbers of intraepithelial lymphocytes were found in groups II and III, the smallest number was found in group V. The largest number of lymphocytes within the glandular epithelium was found in group III. The largest number of macrophages within the surface and glandular epithelia were found in group I. Neutrophils were found within the surface epithelium only in groups I and II. In the subepithelial connective tissue layer, a high infiltration of neutrophils was found in groups I and II while the largest number of eosinophils was found in group IV. The largest number of lymphocytes was observed in group V. In conclusion, this study showed a variation in the infiltration and distribution of neutrophils, lymphocytes, macrophages, eosinophils and plasma cells in the endometrium following insemination and during different stages of early pregnancy. Particularly, the pattern of lymphocyte presence on day 19 of pregnancy, indicate that the lymphocyte function may play a role during embryonic attachment in the pig.

Corrigendum to "The sow endometrium at different stages of the oestrus cycle: studies on morphological changes and infiltration by cells of the immune system." [Anim. Reprod. Sci. 65 (2001) 95-114].

The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4+/-0.7 (mean+/-S.D.) were used. Blood samples were collected from the jugular vein 1 h before slaughter for analyses of oestradiol-17beta and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy (LM). The significantly highest surface and the glandular epithelium were observed at oestrus and dioestrus, respectively. The largest number of capillaries (underneath the surface epithelium) was found at oestrus. In the surface epithelium, the largest number of intraepithelial lymphocytes (IELs, round nucleus) was found at early dioestrus. The largest number of lymphocytes and macrophages within the glandular epithelium were found at early dioestrus and oestrus, respectively. In the subepithelial connective tissue layer, the most common type of leukocytes during all stages was the lymphocyte. The largest numbers of lymphocytes and neutrophils were found at oestrus while the largest number of eosinophils was found at dioestrus. The dominating cells of the immune system in the connective tissue of the glandular layer were lymphocytes and macrophages. The significantly largest numbers of lymphocytes and plasma cells were found at early dioestrus and dioestrus, respectively. The number of lymphocytes in the connective tissue of the glandular layer and the number of plasma cells in the subepithelial layer were positively correlated with the plasma level of progesterone (P < or = 0.05). The numbers of capillaries and neutrophils in the subepithelial layer underneath the surface epithelium as well as the number of macrophages in both surface and glandular epithelium were positively correlated with the plasma level of oestradiol-17beta (P < or = 0.05). In conclusion, the present study showed a variation om the infiltration and distrobution of lymphocytes, neutrophils, eosinophils, macrophages, mast cells, and plasma cells in the sow endometrium during different stages of the oestrous cycle. Also morphological parameters (e.g. height of surface and glandular epithelium, capillaries density and degree of oedema) varied throughout the oestrous cycle.

Corrigendum to "The sow endometrium at different stages of the oestrous cycle: studies on the distribution of CD2, CD4, CD8 and MHC class II expressing" cells. [Anim. Reprod. Sci. 68 (2001) 99-109].

The aim of this study was to investigate the distribution of CD2(+), CD4(+), CD8(+) lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire), with an average parity number of 3.4 +/- 0.7 (mean +/- S.D.) were used. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus (day 17, n = 3), prooestrus (day 19, n = 3), oestrus (day 1, n = 3), early dioestrus (day 4, n = 3) and dioestrus (days 11-12, n = 3), were stored in a freezer at -70 degrees C until analysed by immunohistochemistry with an avidin-biotin peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface and glandular epithelium as well as connective tissue layers in subepithelial and glandular areas were examined by light microscopy. For the T lymphocyte subpopulations, all oestrous cycle stages and different tissue layers taken together, the most commonly observed cell type was CD2(+) cells. The largest number of CD2(+) cells within the surface and glandular epithelium were observed at oestrus and early dioestrus. In the surface epithelium, a larger number of CD8(+) cells compared with CD4(+) cells were observed and no CD4(+) cells were found within the glandular epithelium at any stage of the oestrous cycle. In the subepithelial and glandular connective tissue layers, during the oestrus cycle stages, a larger number of CD4(+) cells compared with CD8(+) cells were found. Endothelial cells in the connective tissue generally expressed MHC class II. However, no obvious differences between oestrous cycle stages were observed. For other cells than endothelial cells, the result was as follows. In the surface epithelium, a large number of MHC class II expressing cells was observed at oestrus compared with the other stages. No MHC class II expressing cells were found at late dioestrus and dioestrus. MHC class II expressing cells were also found in the glandular epithelium, and in the subepithelial and glandular connective tissue layers during all oestrous cycle stages but with no significant differences between stages. In conclusion, the present study showed a variation in the distribution of T lymphocyte subpopulations (CD2(+), CD4(+) and CD8(+)) and MHC class II expressing cells in the sow endometrium during different stages of the oestrous cycle. Also a variation between different tissue layers was found. It is suggested that helper and cytotoxic function of the immune system have primary locations in different tissue layers of the endometrium.

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

The aim of present study was to investigate the influence of pre-compared with post-ovulatory insemination, on the distribution of spermatozoa in the oviduct, the accessory sperm counts on the zona pellucida and early embryonic development. Thirty-six crossbred multiparous sows (Swedish Landrace x Swedish Yorkshire) were artificially inseminated once either at 20-15 h before (group AIB) or at 15-20 h after (group AIA) ovulation by using a pooled semen of two boars. Thereafter, they were randomly allocated to one of five groups: slaughter at 5-6h after AI (group I-AIB), at 20-25 h after ovulation (groups II-AIB and II-AIA), at 70 h after ovulation (groups III-AIB and III-AIA), on day 11 (groups IV-AIB and IV-AIA, first day of standing oestrus=day 1) and on day 19 (groups V-AIB and V-AIA). The plasma levels of oestradiol-17beta and progesterone differed significantly (P