Francisella tularensis in Swedish predators and scavengers.

Tularaemia is a zoonotic disease, in Europe caused by Francisella tularensis subsp. holarctica. Many lagomorphs and a variety of small rodents are wildlife species prone to develop clinical disease, while predators and scavengers are relatively resistant and may serve as sentinels. Blood samples from 656 Swedish wild predators and scavengers were serologically investigated using slide agglutination and microagglutination. In the slide agglutination test, 34 seropositive animals were detected, and they were found among all species investigated: brown bear (Ursus arctos), Eurasian lynx (Lynx lynx), raccoon dog (Nyctereutes procyonoides), red fox (Vulpes vulpes), wild boar (Sus scrofa), wolf (Canis lupus) and wolverine (Gulo gulo). Due to haemolysis the microagglutination test was more difficult to read at low titres, and only 12 animals were classified as seropositive. F. tularensis subsp. holarctica was detected by a polymerase chain reaction in lymphatic tissues of the head in one brown bear, one red fox and one wolf. The significance of this finding regarding possible latency of infection is not clear. In conclusion, the results of this study indicate that all predator and scavenger species included in this study may serve as sentinels for tularaemia in Sweden. Their role as reservoirs is unclear.

Serological testing of Schmallenberg virus in Swedish wild cervids from 2012 to 2016.

BACKGROUND: Schmallenberg virus (SBV) first emerged in Europe in 2011, and in Sweden in late 2012. The virus was still circulating in parts of Europe in 2015. In recent testing, the virus has not been detected in Swedish domestic animals, indicating that it is no longer circulating in Sweden. It is not known if the virus has circulated and is still circulating in Swedish wild cervid populations and whether wildlife can act as virus reservoirs. The aim of this study was to investigate whether SBV has circulated, and is still circulating among wild cervids in Sweden. RESULTS: Ninety-two sera from moose (Alces alces, n = 22), red deer (Cervus elaphus, n = 15), fallow deer (Dama dama, n = 44), and roe deer (Capreolus capreolus, n = 11) were collected and analyzed for antibodies against SBV. The sampling occurred in the southern and middle part of Sweden during three time periods: 1) before the vector season in 2012, 2) after the vector season in 2012, and 3) after the vector season in 2015. Animals from periods 1 and 2 were of varying ages, whereas animals collected in period 3 were born after the vector season 2013. Animals from period 1 (n = 15) and 3 (n = 47) were seronegative, but, 53% (16 of 30) of animals from period 2 were seropositive, determined by SBV competitive ELISA. Samples from period 2 were additionally analyzed for SBV-neutralizing antibodies. Such antibodies were detected in 16/16 SBV-N-antibody-positive, 3/12 negative and 2/2 doubtful sera. The two tests were in accordance at SBV-neutralizing antibody titers of 1:32 or higher. CONCLUSION: Our results show that SBV circulated among wild cervids during the vector season of 2012. Three years later, no SBV-antibodies were detected in animals born after the vector season 2013. The likely absence of SBV circulation in Sweden, in contrast to other parts of Europe, might be explained by the annual occurrence of a vector-free season due to climate conditions. Interpretations are limited by the small sample-size, but the results suggest that the SBV competitive ELISA has high specificity but might have slightly lower sensitivity compared to a seroneutralization assay, when using samples from wild cervids.

Dose related effects of LPS on endometrial epithelial cell populations from dioestrus cows.

Lipopolysaccharides (LPS) from Gram negative bacteria are involved in the pathogeny of uterine diseases in cows. This study aimed to investigate LPS effects on the growth of bovine endometrial epithelial cells (bEEC) and relationships between LPS response and tissue characteristics. Uteri from 35 females were characterized for parity and stage of oestrous cycle. Densities of glandular tissue (dGT), CD11b+ cells and Ki67+ cells were measured in the endometrial tissue. Cells from 13 dioestrus cows were exposed to 0, 2, 4, 8, 12, 16 or 24µg/mL LPS. Effects of parity and stage of the oestrous cycle on tissue characteristics and effects of LPS dosage, cow and tissue characteristics on changes in cell numbers were analyzed by ANOVA. The dGT was higher in metoestrus and dioestrus samples than in pro-oestrus ones whereas densities of CD11b+ and Ki67+ cells were higher at pro-oestrus (p<0.05-p<0.01). LPS influenced bEEC populations in a dose related manner. An increase in number of live cells was observed for dosages ranging from 2 to 12µg/mL LPS (p<0.0001 vs controls). No effect was found on numbers and frequencies of dead cells. With higher dosages, the numbers of live cells did not increase but the numbers of dead did increase. No relationships were observed between cow or tissue characteristics and growth patterns or frequencies of viable bEEC in controls nor in the response to LPS. To conclude this model is suitable for further studies on dysregulations induced by LPS in endometrial tissue.

Caries, Periodontal Disease, Supernumerary Teeth and Other Dental Disorders in Swedish Wild Boar (Sus scrofa).

Between January and December 2013, the dental and periodontal health of 99 Swedish wild boars (Sus scrofa) was investigated. Sampling occurred in conjunction with routine hunting at six large estates in the southern and middle parts of Sweden. All six of the estates use supplemental feeding. The weight of the animals, their sex and their dates of death were noted. Age was estimated using tooth eruption and tooth replacement patterns. The oral cavity was inspected and abnormalities were recorded on a dental chart modified for wild boars. The findings included supernumerary teeth, absence of teeth, mild class II malocclusion, severe tooth wear, periodontitis, calculus, caries, tooth fractures and the presence of enamel defects. Swedish wild boars suffer from different dental lesions and the impact of supplemental feeding on dental and periodontal health is still to be investigated.

Pregnancy rates after artificial insemination with cooled stallion spermatozoa either with or without single layer centrifugation.

A successful outcome after artificial insemination with cooled semen is dependent on many factors, the sperm quality of the ejaculate being one. Previous studies have shown that spermatozoa with good motility, normal morphology, and good chromatin integrity can be selected by means of colloid centrifugation, particularly single layer centrifugation (SLC) using species-specific colloids. The purpose of the present study was to conduct an insemination trial with spermatozoa from "normal" ejaculates, i.e., from stallions with no known fertility problem, to determine whether the improvements in sperm quality seen in SLC-selected sperm samples compared with uncentrifuged controls in laboratory tests are reflected in an increased pregnancy rate after artificial insemination. In a multicentre study, SLC-selected sperm samples and uncentrifuged controls from eight stallions were inseminated into approximately 10 mares per treatment per stallion. Ultrasound examination was carried out approximately 16 days after insemination to detect an embryonic vesicle. The pregnancy rates per cycle were 45% for controls and 69% for SLC-selected sperm samples, which is statistically significant (P < 0.0018). Thus, the improvement in sperm quality reported previously for SLC-selected sperm samples is associated with an increase in pregnancy rate, even for ejaculates from stallions with no known fertility problem.

Temporal and spatial variation in Anaplasma phagocytophilum infection in Swedish moose (Alces alces).

SUMMARY: The occurrence of Anaplasma phagocytophilum was investigated in spleen and serum samples from Swedish moose (Alces alces) in southern Sweden (island and mainland). Samples were analysed for presence of A. phagocytophilum DNA by real-time PCR (n = 263), and for Anaplasma antibodies with ELISA serology (n = 234). All serum samples had antibodies against A. phagocytophilum. The mean DNA-based prevalence was 26·3%, and significant (P < 0·01) temporal, and spatial variation was found. Island moose had significantly (P < 0·001) higher prevalence of A. phagocytophilum DNA than moose from the mainland areas. Two samples were sequenced to determine genetic variation in the 16S rRNA and groESL genes. Genetic sequence similarity with the human granulocytic anaplasmosis agent, equine granulocytic ehrlichiosis agent, and different wildlife-associated A. phagocytophilum variants were observed in the 16S rRNA and groESL genes. Our study shows that moose are exposed to A. phagocytophilum in Sweden, and represent a potential wildlife reservoir of the pathogen.

Short communication: Concentration of TGF-ß1, IL-10 and IL-6 in boar seminal plasma and TGF-ß1 level in different fractions of ejaculates.

Levels of the cytokines transforming growth factor (TGF)-ß1, interleukin (IL)-10 and IL-6 in the boar seminal plasma (SP) as well as TGF-ß1 level in different fractions of ejaculate were studied. These cytokines was chosen because of their expected effect on tissue immune response, i.e. suppressive (TGF-ß1 and IL-10) and pro-inflammatory (IL-6). Three whole ejaculates from five boars A-E, (n=15) were sampled weekly to evaluate the levels of seminal plasma TGF-ß1, IL-10 and IL-6 as well as their fluctuations over time. The effect of different storage temperatures, -20°C or -80°C, on the level of seminal plasma TGF ß1 was also tested (three boars, two fractions in one ejaculate). In addition, in 4 different fractions of ejaculates: the pre-sperm-rich (Pre-SRF), first 10 ml of sperm-rich (10SRF), the rest of the sperm-rich fraction (Rest-SRF) and the rest of the ejaculate (RE) fraction, were collected from three boars (A-C) on four different occasions for TGF-ß1 evaluation. In the whole ejaculates (n=15), a wide range in the concentration of the cytokines TGF-ß1 (20.4 - 766.5 pg/mL) and IL-10, (73.7 - 837.3 pg/mL), was found. For IL-6, the concentration was low (range 11.5 - 30.9 pg/ml) and only detected in four out of 15 collections (from two boars). The mean levels of TGF-ß1 and IL-10 between individual boars varied but were not statistical different. The level of TGF-ß1 in Pre-SRF, Rest-SRF and RE fractions was significantly lower in boar A than the other boars. A significantly higher concentration of TGF-ß1 was found in the 10SRF than in the other fractions. Different storage temperatures (-20°C or -80°C) did not affect the seminal plasma TGF-ß1 level after one year of storage. To conclude: Boar seminal plasma contained TGF- ß1 and IL-10 but with high individual variation. IL-6 was low or undetectable. The TGF- ß1 level was highest in the first 10 mL of the sperm-rich fraction of the ejaculate. Further studies are needed on the role of different levels of cytokine in boar semen on porcine female reproductive tissue, especially for TGF- ß1.

Restoration of seminal plasma to stallion spermatozoa selected by colloid centrifugation increases sperm progressive motility but is detrimental to chromatin integrity.

There is controversy about whether the presence of some seminal plasma (SP) in an equine insemination dose is necessary for promoting fertility. A new technique for improving stallion sperm quality, single layer centrifugation (SLC) using a species-specific colloid, Androcoll-E, selects a sperm subpopulation that is highly motile with normal morphology, intact membranes and good chromatin integrity from the rest of the ejaculate and removes SP. The present study was designed to investigate the effect of restoring homologous SP (5% and 10%) on the progressive motility, velocity, and chromatin integrity of SLC-selected stallion spermatozoa in 44 semen samples over time. Sperm progressive motility (P<0.01) and the proportion with class A velocity (>50 µm/sec) were increased in samples where SP was restored, whereas the proportion with class B velocity (10 to 50 µm/sec) was decreased compared with SLC samples. However, after 24 h cold storage of treated samples, progressive motility was not different for the SP-treated groups compared with SLC, whereas chromatin damage DNA fragmentation index (%DFI) was higher. In contrast, adding SP to untreated 24 h-stored SLC samples did not affect progressive motility although it did increase the proportion of spermatozoa with class A velocity. There was individual variation between stallions whether 5% or 10% SP produced a greater increase in progressive motility. In conclusion, 5% to 10% SP can be added back to SLC-selected samples if considered necessary to optimize fertility. However, it should be added immediately before insemination rather than before storage of the sperm dose, to benefit from the transient increase in sperm progressive motility and avoid increased chromatin damage.

Influence of seminal plasma, spermatozoa and semen extender on cytokine expression in the porcine endometrium after insemination.

The effects of semen components or extender alone on the expression of selected cytokines [interleukine (IL)-1ß, IL-6, granulocyte-macrophage colony stimulating factor (GM-CSF), IL-10 and transforming growth factor (TGF)-ß1] on the porcine endometrium were studied, as well as the presence of polymorphonuclear neutrophilic granulocytes (PMNs). In experiment (Exp) I, groups of gilts were sampled at 5-6h after insemination with fresh semen in extender (Beltsville thawing solution, BTS), spermatozoa in extender (Spz), seminal plasma (SP), or only BTS (control). In Exp II, gilts were sampled 35-40h after insemination with Spz, SP, BTS or only catheter inserted (as control). Immunohistochemical (IHC) labelling of IL-6, IL-10 and TGF-ß1 was evident, especially in surface and glandular epithelia of the porcine endometrium. There were no consistent differences in IHC-labelling of the cytokines in relation to different treatments. However, the scores for IL-6 and IL-10 in surface epithelium and sub-epithelial connective tissue compartments were higher at 35-40h than shortly (5-6h) after treatment. Cytoplasmic labelling in the sub-epithelial connective tissue was observed in scattered individual cells but not in PMNs. Shortly (5-6h) after insemination, there were no differences between animals inseminated with BTS (control) and the semen components for any of the cytokine mRNAs. Later however, at 35-40h, lower endometrial expression of TGF-ß1 mRNA was observed in the Spz and BTS groups compared with the control (catheter only). The same pattern was found for IL-10 (NS). The mRNA expression of IL-6 in the BTS inseminated group was higher compared to the control group. Insemination with SP resulted in significantly lower PMN cell infiltration in the sub-epithelial connective tissue compared with Spz or BTS groups shortly (5-6h) after insemination. Later (35-40h), a significant difference was found between SP (lower) and the control group (only catheter). To conclude, our results show that insemination and/or inseminated components modulated cytokine expression in the gilt endometrium. The semen extender BTS stimulated immune reactivity, as shown by down-regulation of the suppressive cytokine TGF-ß1. Insemination with solely SP clearly decreased PMN cell infiltration of the gilt endometrium. However, no clear relation between the cytokines studied and PMN cell presence was found.

Single layer centrifugation of stallion spermatozoa through Androcoll™-E does not adversely affect their capacitation-like status, as measured by CTC staining.

This study was designed to evaluate the effect of single layer centrifugation (SLC) and subsequent cold storage on stallion sperm capacitation-like status and acrosome reaction. Three stallions were included in the study, with three ejaculates per stallion. The samples were examined 4, 24 and 72 h after collection, extension and SLC, with storage at 6°C. Sperm capacitation-like status was investigated using the fluorescent dye chlortetracycline (CTC). There was no difference in capacitation-like status between colloid-selected and non-selected spermatozoa. Sperm motility decreased significantly during cold storage, whereas the proportion of apparently capacitated spermatozoa increased. There was no change in the proportion of acrosome-reacted spermatozoa. In conclusion, SLC through Androcoll™-E does not adversely affect the capacitation-like status of stallion spermatozoa, although it did increase with time during cold storage.

Stallion Sperm Viability, as Measured by the Nucleocounter SP-100, Is Affected by Extender and Enhanced by Single Layer Centrifugation.

On-stud assessment of stallion sperm quality can be problematic. A new instrument, the Nucleocounter SP-100, was validated for measuring stallion sperm concentration and viability. It was subsequently used to evaluate sperm viability in Kenney's extender and INRA96. There was a strong correlation between sperm concentrations measured by the Nucleocounter SP-100 and by the Bürker counting chamber (r = 0.84; P < .001). Similarly, there was a good correlation between sperm viability results from the Nucleocounter SP-100 and flow cytometric results (r = 0.73; P < .001). Sperm viability at 24 hours was significantly better for samples extended in INRA96 than in Kenney's extender (P < .001). Furthermore, sperm kinematics were better for stored samples in INRA96 than in Kenney's extender. Single Layer Centrifugation selected spermatozoa that maintained their viability better during storage for 24 hours than the uncentrifuged samples. In conclusion, the type of semen extender used and Single Layer Centrifugation were found to influence both the kinematics and viability of stallion spermatozoa. The Nucelocounter-SP100 was considered to be a useful instrument for rapidly measuring stallion sperm concentration and viability.

Cytokine expression in the gilt oviduct: effects of seminal plasma, spermatozoa and extender after insemination.

Effects of semen components [fresh semen in extender, spermatozoa in extender (Spz), seminal plasma (SP)], or extender alone (Beltsville thawing solution, BTS) on the expression of selected cytokines [interleukin (IL)-1beta, IL-6, IL-10 and transforming growth factor (TGF)-beta1)] as well as the presence of cells positive for CD8 or CD25 were studied in the pig oviduct. In addition, cytokines in SP and oviductal flushings were analyzed. In experiment (Exp) I, groups of gilts were sampled at 5-6h after insemination with SP, Spz, fresh semen in BTS or only BTS (control). In Exp II, gilts were sampled 35-40 h after insemination with SP, Spz, BTS or only catheter insertion (control). Most oviductal flushing samples were positive (> or =detectable limits) for IL-10 and TGF-beta1 but only few for IL-6. The IHC-labelling of IL-6, IL-10 and TGF-beta1 was evident, especially in the epithelial cells of the isthmus and infundibulum as well as in the cells of the regional (mesometrial) lymph node. Cilia of the epithelium were positive for IL-6 (strongest in the infundibulum) and TGF-beta1 (strongest in the isthmus) but negative for IL-10. There were no consistent differences in IHC-labelling of the cytokines in relation to different treatments, except at 35-40 h after insemination (Exp II), when IL-6 was slightly higher in epithelium of the SP group and IL-10 in the infundibular connective tissue was higher in the SP and Spz groups. In the isthmus and infundibulum, there were no differences between animals inseminated with BTS (control) and the semen components for any of the cytokine mRNAs at 5-6h after insemination (Exp I). However, later (35-40 h, Exp II), insemination with SP, Spz and BTS alone appeared to up-regulate TGF-beta1 mRNA expression compared with the control group (without any fluid infused). In all treatment groups, the mRNA level for TGF-beta1 was higher than for IL-1beta, IL-6 and IL-10. Higher mRNA levels of all cytokines were found in the isthmus compared with the infundibulum. Numbers of CD8-positive cells (both in epithelium and connective tissue) appeared higher in the infundibulum compared with the isthmus and were mostly higher shortly (Exp I) after treatment with SP, SPZ and BTS than later (Exp II) in both segments. CD25-positive cells were few and found solely in the sub-epithelial connective tissue. The results indicate that in the porcine oviduct, IL-6, IL-10 and TGF-beta1 are endogenous produced and that TGF-beta1 may have a more important role for immunomodulation than the other cytokines, especially in isthmus. Differences between isthmus and infundibulum in cytokine mRNA expression and in presence of CD8-positive cells indicate different patterns of immune reactivity in the upper and lower parts of the oviduct.

Single-layer centrifugation with Androcoll-E can be scaled up to allow large volumes of stallion ejaculate to be processed easily.

The objective of the current study was to optimize the volumes of Androcoll-E and sperm sample used in various sizes of centrifuge tube to scale up single-layer centrifugation (SLC) for routine use in the field. Although sperm suspensions of equivalent quality were produced using Androcoll-E in small and large tubes, the sperm yield was much lower in the latter (P<0.001). In contrast, in 200-mL tubes (XL), the yields were approximately 25% higher than those for the small tubes. An increased volume (4.5 mL) of extended ejaculate in small tubes (SLC-Inc) or 15 to 18 mL extended ejaculate on 15 mL of colloid of a reduced density, Androcoll-E-Large (SLC-Large), in 50-mL tubes were both found to give similar yields of motile spermatozoa as that of the SLC-Small method (SLC-Small, 49.7+/-18.6%; SLC-Inc, 53.3+/-17.1%; SLC-Large, 44.9+/-18.3%) and were found to be equivalent in quality (motility: 88.0+/-8.8%, 84.0+/-3.5%, 90.0+/-5.4%; normal morphology: 69.4+/-17.0%, 69.4+/-12.7%, 63.9+/-15.6%; viability: 78+/-16.7%, 83.8+/-12.5%, 80.05+/-14.6%; DNA fragmentation index: 14.7+/-10.9%, 12.8+/-8.1%, 11.6+/-7.6%, respectively). The processing of an "average" stallion Equus caballus ejaculate in approximately twenty-seven 10-mL tubes (SLC-Inc) or eight 50-mL tubes (SLC-Large) is feasible, the latter being considered more practical for on-stud use.

Comparison of density gradient and single layer centrifugation of stallion spermatozoa: yield, motility and survival.

REASONS FOR PERFORMING STUDY: A new, simpler, technique of colloidal centrifugation has recently been developed, designated single layer centrifugation (SLC). This technique requires evaluation by comparison with a density gradient for its ability to select the best quality spermatozoa and its practicality of use on studfarms. OBJECTIVE: To compare the effect of 2 methods of colloidal centrifugation, density gradient centrifugation and single layer centrifugation, on stallion sperm motility, yield and survival, using freshly collected extended stallion semen. METHODS: Aliquots of extended stallion semen from 10 stallions (38 ejaculates) were processed by the 2 methods of colloidal centrifugation. For both uncentrifuged and centrifuged samples, sperm yield was calculated and subjective sperm motility assessed over several days to provide an estimate of sperm survival. Some stored semen samples, held at 4 degrees C overnight, were also available for testing. RESULTS: For fresh, extended semen, a similar recovery yield of motile spermatozoa was seen for the 2 methods of preparation for single layers and density gradients, respectively. Sperm motility and survival rate were significantly improved by colloidal centrifugation compared to unprocessed ejaculate, without any significant difference between methods (SLC vs. gradient). However, the yield was reduced by 18-20% when cold-stored semen was used for centrifugation compared to fresh semen; and more variation between ejaculates was observed than for fresh ejaculates. Again, sperm motility and sperm survival were improved in the centrifuged sperm preparations compared to stored, unprocessed ejaculates. POTENTIAL RELEVANCE: The 2 colloid centrifugation techniques produce equivalent sperm preparations in terms of sperm quality. However, the SLC method would be more practical and convenient for use in the field.

Colloidal centrifugation with Androcoll-E prolongs stallion sperm motility, viability and chromatin integrity.

The objective was to investigate the changes in stallion sperm quality (sperm motility, viability, membrane integrity and chromatin integrity) occurring during cool storage, and to study the effect of sperm selection by single layer colloidal centrifugation on these parameters of sperm quality. Spermatozoa from 3 stallions (10 ejaculates, 3-4 per stallion) were selected by centrifugation through a single layer of colloid (SLC). The resulting sperm preparations and the control samples (extended but unselected semen samples) were stored at 5 degrees C for 48h. Assessments of sperm quality, such as sperm motility, viability (SYBR-14/PI staining), membrane stability (Annexin-V/PI staining) and chromatin integrity, were performed on aliquots of the selected sperm preparations and unselected samples on the day of collection (3h) and after 24 and 48h of storage. In the SLC-selected sperm samples, sperm motility, sperm viability, proportions of spermatozoa with normal morphology and with intact chromatin were significantly better than in unselected samples (motility: 77+/-4% vs. 64+/-8% at 3h; P<0.001; viability: 79.5+/-9% vs. 64.7+/-9%, P<0.001; normal morphology 89+/-6% vs. 69+/-9%; chromatin integrity DFI 11.3+/-5% vs. 22.1+/-10%). Membrane stability, however, was not different in the SLC-selected and unselected samples (74.6+/-8% vs. 69.3+/-8%). The deterioration seen in sperm quality in the unselected samples was prevented by SLC, so that sperm viability, membrane stability and chromatin integrity were unchanged in the selected samples by 48h compared to 3h (P<0.001), whereas the unselected samples were significantly worse by 48h (P<0.001). Furthermore, it should be possible to send an aliquot of a normal insemination dose (i.e. unselected spermatozoa) overnight to a reference laboratory for analysis of both plasma membrane and chromatin integrity. In conclusion, centrifugation of stallion spermatozoa through a single layer of colloid is a useful technique for selecting the best spermatozoa from an ejaculate and, moreover, sperm quality is maintained during storage.

Morphology and chromatin integrity of stallion spermatozoa prepared by density gradient and single layer centrifugation through silica colloids.

The objective was to investigate whether it is possible to improve the quality of stallion semen, with respect to sperm morphology and chromatin integrity, both of which have been linked to fertility, using either density gradient centrifugation (DGC) or a new method, hereby named single layer centrifugation (SLC). The two methods of colloidal centrifugation were evaluated using 38 ejaculates from 10 stallions. Sperm morphology, subjective motility and sperm chromatin integrity were compared in uncentrifuged samples and in centrifuged sperm preparations. The proportion of morphologically normal spermatozoa varied between stallions (p < 0.001) and was increased by both methods of colloidal centrifugation (median value before centrifugation 67.5%; after SLC 78%; after DGC 77%; p < 0.001). The incidence of certain abnormalities was reduced, e.g. proximal cytoplasmic droplets were reduced from 12.9% to 8.8% (p < 0.001), and mid-piece defects from 5.3% to 1.4% (p < 0.05). Similarly, sperm motility and chromatin integrity were significantly improved (p < 0.001), with no difference between the two centrifugation methods. Centrifugation through colloids can enrich the proportions of stallion spermatozoa with normal morphology and normal chromatin structure in sperm preparations. The new method, SLC, was as effective as DGC in selecting motile stallion spermatozoa with normal morphology and intact chromatin. SLC, being simpler to use than DGC, would be appropriate for routine use by stud personnel to improve stallion sperm quality in insemination doses.

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus.

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.

Sperm production and sperm morphology of Swedish Warmblood stallions.

The present study investigated daily sperm output and sperm morphology of fresh semen in eight Swedish Warmblood stallions aged 5-8 years. They were used for artificial insemination, and their fertility during the breeding season of semen collection exceeded 60% per cycle. One ejaculate of semen was collected daily for 10 consecutive days from each stallion. The gel-free volume was measured, and the sperm concentration was assessed with a Bürker chamber. The volume of gel-free fraction was multiplied by the sperm concentration to give the total number of spermatozoa (TSN). Sperm morphology was examined in ejaculates collected on days 2, 5 and 10. An aliquot from each ejaculate was fixed in 1 ml formol-saline immediately after collection and examined under a phase-contrast microscope (magnification 1000x) to assess morphological abnormalities. Furthermore smears were prepared and stained according to Williams (carbolfuchsin-eosin) for a more detailed examination of the sperm heads under a light microscope (magnification 1000x). Analysis of variance was applied to data. Total spermatozoa number decreased progressively during the first 8 days of collection, and daily sperm output (DSO) was calculated as mean TSN of collections on days 8-10, being 6.4 x 10(9) spermatozoa. The overall percentages of morphologically normal spermatozoa in ejaculates collected on days 2, 5 and 10 were above 70%, being significantly lower in ejaculate 2 (68.6%) compared with ejaculates 5 and 10 (72.9% respectively 75.3%).

Genetics of crossbred sow longevity.

The aim of this study was to estimate genetic parameters for longevity from Swedish crossbred sows to investigate the possibilities of selecting for this trait. Data were collected from 16 commercial piglet-producing herds, on crossbred (Landrace × Yorkshire) sows farrowing in the period 1 January 2001 to 31 December 2004. The data set with records on 10 373 sows was split into two sets according to the breed of the sire, i.e. Landrace sires (LS) or Yorkshire sires (YS). Removal hazard during productive life (PL) was analysed with survival analysis, using a sire model. Stayability from first to second litter (STAY12), stayability from first to third litter (STAY13), length of productive life (LPL) and lifetime production (LTP) were analysed with linear models, using an animal model. Females after the worst sire had 1.7 times higher (progeny of LS) and 2.4 times higher (progeny of YS) risk of removal than females after the best sire. Heritability for PL was estimated at 0.06 (LS) and 0.12 (YS). The heritabilities for the linear longevity traits ranged from 0.03 to 0.08. Genetic correlations between the four linear longevity traits were all high and positive (0.6 to 1.0), as were the phenotypic correlations (0.5 to 0.8). The correlations (Spearman rank) between the sire's estimated breeding values for all the five longevity traits were all significant (P < 0.001) and moderate to strong in both data sets. Estimated breeding value (EBV) correlations between the five longevity traits and traits included in the present Swedish breeding evaluation (Quality Genetics (QG)) were significant in a few cases. Significant and favourable EBV correlations were found between age at first farrowing and both STAY12 and STAY13 (-0.20 and -0.31), as well as between litter weight at 3 weeks and LPL and LTP (0.13 to 0.20). Significant and unfavourable EBV correlations were found between age at 100 kg and STAY12 (0.32), as well as between the exterior conformation score from testing station and PL (-0.20). The level of the estimated heritabilities for longevity indicates that genetic improvement of sow longevity would be possible. However, overall, there was no strong indirect selection for sow longevity with the current Swedish breeding evaluation (QG).

Factors affecting length of productive life in Swedish commercial sows.

The objective of this study was to investigate factors that might influence the length of productive life in Swedish crossbred (Landrace x Yorkshire) sows. The data set consisted of 20,310 sows farrowing between 2001 and 2004 in 21 commercial piglet-producing herds. Productive life (PL) was defined as the number of days between first farrowing and removal or termination of data collection. In addition to the overall risk analysis of PL, another 4 longevity traits were analyzed (competing risk analyses): reproductive disorder-determined length of PL (RPL), udder problem-determined length of PL (UPL), lameness-determined length of PL (LPL), and mortality-determined length of PL (MPL). Analyses were performed by using survival analysis, applying a Weibull model with 6 time-dependent and 1 time-independent variable (age at first farrowing). The factor with the largest contribution to the likelihood function for PL was days after farrowing, followed by parity, the herd x year combination, the total number of piglets born, days between weaning and next farrowing, farrowing month, and age at first farrowing. For all 4 competing risk traits, the factors contributing most to the likelihood function were days after farrowing, the herd x year combination, and parity, with a varied order between traits. The hazard for removal was greatest 30 to 40 d after farrowing (after weaning) for PL, UPL, and LPL (P < 0.001). However, for MPL the hazard was greatest just after farrowing (0 to 10 d), and for RPL the hazard peaked at 70 to 100 d after farrowing. The hazard for removal was, compared with parity 1, less in parities 2 to 7 and greater from parity 8 for PL (P < 0.001). The hazard was greatest in parity 1 (P < 0.01) for RPL, UPL, and LPL, whereas for MPL the hazard increased with greater parity number and was markedly greater from parity 9 (P < 0.001). Sows with litters of 9 piglets or less had a greater hazard for removal than sows with litters of 12 to 13 piglets (P < 0.001). Intervals between 120 and 122 d from weaning to the next farrowing showed the lowest hazard for removal (P < 0.001). The influence of farrowing month displayed no clear pattern for PL. Sows of 14 mo or older at their first farrowing had a 20% greater hazard for removal than younger sows (P < 0.001). The hazard for removal was greater for smaller litters in all parities but was more accentuated in greater parities. Overall, days after farrowing was the main risk factor for sow removal. Removal hazard was greatest shortly after weaning, and this peak increased with greater parity number.