A decreased expression of interferon stimulated genes in peri-implantation endometrium of embryo transfer recipient sows could contribute to embryo death.

Pig pregnancy succeeds thanks to a well-coordinated system ruling both maternal immune activation and embryonic antigen tolerance. In physiological pregnancies, the maternal immune system should tolerate the presence of hemi-allogeneic conceptuses from the pre-implantation phase to term, while maintaining maternal defence against pathogens. Allogeneic pregnancies, as after embryo transfer (ET), depict high embryo mortality during the attachment phase, calling for studies of the dynamic modifications in immune processes occurring at the maternal-foetal interface, for instance, of interferon (IFN)-stimulated genes (ISGs). These ISGs are generally activated by IFN secreted by the conceptus during the process of maternal recognition of pregnancy (MRP) and responsible for recruiting immune cells to the site of embryo attachment, thus facilitating cell-antigen presentation and angiogenesis. We performed RNA-Seq analysis in peri-implantation (days 18 and 24) endometrial samples retrieved from artificially inseminated sows (hemi-allogeneic embryos (HAL) group) or sows subjected to ET (allogeneic embryos (AL) group) to monitor alterations of gene expression that could be jeopardising early pregnancy. Our results showed that endometrial gene expression patterns related to immune responses differed between hemi- or allogeneic embryo presence, with allogeneic embryos apparently inducing conspicuous modifications of immune-related genes and pathways. A decreased expression (P < 0.05; FC < -2) of several interferon ISGs, such as CXCL8, CXCL10, IRF1, IRF9, STAT1, and B2M, among others was detected in the endometrium of sows carrying allogeneic embryos on day 24 of pregnancy. This severe downregulation of ISGs in allogeneic pregnancies could represent a failure of ET-embryos to signal IFN to the endometrium to warrant the development of adequate immunotolerance mechanisms to facilitate embryo development, thus contributing to elevated embryo death.

The role of semen and seminal plasma in inducing large-scale genomic changes in the female porcine peri-ovulatory tract.

Semen modifies the expression of genes related to immune function along the porcine female internal genital tract. Whether other pathways are induced by the deposition of spermatozoa and/or seminal plasma (SP), is yet undocumented. Here, to determine their relative impact on the uterine and tubal transcriptomes, microarray analyses were performed on the endocervix, endometrium and endosalpinx collected from pre-ovulatory sows 24 h after either mating or artificial insemination (AI) with specific ejaculate fractions containing spermatozoa or sperm-free SP. After enrichment analysis, we found an overrepresentation of genes and pathways associated with sperm transport and binding, oxidative stress and cell-to-cell recognition, such as PI3K-Akt, FoxO signaling, glycosaminoglycan biosynthesis and cAMP-related transcripts, among others. Although semen (either after mating or AI) seemed to have the highest impact along the entire genital tract, our results demonstrate that the SP itself also modifies the transcriptome. The detected modifications of the molecular profiles of the pre/peri-ovulatory endometrium and endosalpinx suggest an interplay for the survival, transport and binding of spermatozoa through, for instance the up-regulation of the Estrogen signaling pathway associated with attachment and release from the oviductal reservoir.

Stallion spermatozoa surviving freezing and thawing experience membrane depolarization and increased intracellular Na.

In order to gain insight of the modifications that freezing and thawing cause to the surviving population of spermatozoa, changes in the potential of the plasma membrane (Em) and intracellular Na+ content of stallion spermatozoa were investigated using flow cytometry. Moreover, caspase 3 activity was also investigated and the functionality of the Na+ -K+ ATPase pump was investigated before and after freezing and thawing. Cryopreservation caused a significant (p < 0.001) increase in the subpopulation of spermatozoa with depolarized sperm membranes, concomitantly with an increase (p < 0.05) in intracellular Na+ . These changes occurred in relation to activation of caspase 3 (p < 0.001). Cryopreservation reduced the activity of the Na-K+ pump and inhibition of the Na+ -K+ ATPase pump with ouabain-induced caspase 3 activation. It is concluded that inactivation of Na+ -K+ ATPase occurs during cryopreservation, an inhibition that could play a role explaining the accelerated senescence of the surviving population of spermatozoa.

Peroxidized mineral oil increases the oxidant status of culture media and inhibits in vitro porcine embryo development.

The use of oils with undetected alterations is a long-recognized problem for in vitro embryo production systems. Since peroxides in oils have been associated with reduced embryo production outcomes, our goals were (1) to evaluate the effects of a batch of mineral oil (MO) that was suspected to be altered on the in vitro production of pig embryos and (2) to determine oil peroxide values throughout culture and the transfer of oxidant agents from oil to culture media. Sunflower oil, which has a completely different chemical composition than MO but a higher oxidative status, and unaltered MO were used as controls. Oocyte maturation, fertilization and embryo development were affected differently depending on the oil overlay used. While the suspected MO was not able to sustain in vitro maturation and fertilization, the oocytes incubated in the presence of sunflower oil were matured and fertilized similarly to those of the unaltered MO group. Moreover, the cleavage rate of presumed zygotes cultured under the suspected MO was severely reduced compared with those cultured under the other oils, and none of the cleaved embryos developed to the blastocyst stage. Although the cleavage rates in the sunflower oil and unaltered MO groups were similar, embryos cultured under sunflower oil also failed to develop to the blastocyst stage. Our results revealed that the suspected MO and sunflower oil had similar levels of peroxides and that these levels were much higher than those of the unaltered MO. The total oxidant status was higher in media incubated under peroxidized oils than in fresh media or media incubated without an oil overlay or under unaltered MO, indicating that oxidant agents were transferred to the incubation media. However, unlike the sunflower oil group, the culture media incubated under the suspected MO had high levels of total oxidant status and low levels of hydrogen peroxide and reactive oxygen species, suggesting the presence of other unknown oxidant agents in that oil. These results indicate that a peroxidized MO overlay dramatically decreases embryo production outcomes. This decrease could be associated with the higher peroxide values of the oil but cannot be explained by the levels of hydrogen peroxide and reactive oxygen species transferred from the oil to the culture media. It is likely that different oxidant agent(s) and/or other toxic compounds present in the peroxidized MO are responsible for its damaging effects on oocytes and embryos.

Factors of importance when selecting sows as embryo donors.

The improvement in porcine embryo preservation and non-surgical embryo transfer (ET) procedures achieved in recent years represents essential progress for the practical use of ET in the pig industry. This study aimed to evaluate the effects of parity, weaning-to-estrus interval (WEI) and season on reproductive and embryonic parameters at day 6 after insemination of donor sows superovulated after weaning. The selection of donor sows was based on their reproductive history, body condition and parity. The effects of parity at weaning (2 to 3, 4 to 5 or 6 to 7 litters), season (fall, winter and spring), and WEI (estrus within 3 to 4 days), and their interactions on the number of corpus luteum, cysts in sows with cysts, number and quality of viable and transferable embryos, embryo developmental stage and recovery and fertilization rates were evaluated using linear mixed effects models. The analyses showed a lack of significant effects of parity, season, WEI or their interactions on any of the reproductive and embryonic parameters examined. In conclusion, these results demonstrate that fertilization rates and numbers of viable and transferable embryos collected at day 6 of the cycle from superovulated donor sows are not affected by their parity, regardless of the time of the year (from fall to spring) and WEI (3 or 4 days).

Immunolocalization of E-cadherin and ß-catenin in the cyclic and early pregnant canine endometrium.

Putative changes in E-cadherin and ß-catenin during implantation in dogs are of interest to study, as they are relevant proteins for epithelial integrity. E-cadherin and ß-catenin were immunolocalized in the canine endometrium during the estrous cycle and early pregnancy, using monoclonal antibodies. Both proteins were detected in all types of endometrial epithelia (surface epithelium [SE], superficial glandular, and deep glandular epithelia) at all stages of the estrous cycle and in early placental structures. E-cadherin depicted a gradient of intensity apparently being lowest in the SE to progressively increase toward the deepness of the endometrial glands, regardless of the stage of estrous cycle. The overall immunostaining was, however, weaker at diestrus. In pregnant samples, the trophoblast was conspicuously immunolabeled compared with the endometrial surface lining epithelium. In the latter, the cytoplasmic pattern predominated over the membrane-bound, as was also seen in the decidual cells of the placental labyrinth. In the early placenta, only trophoblast cells and lacunae retained membrane signals. ß-Catenin membrane labeling appeared relatively constant throughout the cycle, although a tendency toward a decrease in intensity was detected at the secretory stages. In addition, a dislocation of the immunoreaction from membrane to the cytoplasm was observed in both the SE and the glandular epithelia at particular stages of the cycle. In early pregnancy, a loss of the membranous pattern was observed in the SE and labyrinth, but neither on trophoblast nor in lacunae. The results show the existence of a softening of the adherens junctional complex in progestagen-dominated stages favoring embryo-maternal interactions and endometrial invasion during canine implantation.

The ubiquitous hyaluronan: Functionally implicated in the oviduct?

Hyaluronan (hyaluronic acid) is a simple, nonantigenic, nonsulfated glycosaminoglycan present everywhere in the extracellular compartments of the body. Noteworthy, it is highly conserved phylogenetically, from sauropsida to mammals; and plays a plethora of roles from embryonic/fetal development to adult physiological and pathological events, including tumor development. In reproduction, hyaluronan has proven related to initial events as sperm survival, buildup of the sperm reservoir in the oviduct, regulation of sperm capacitation, and prefertilization to later participate in embryo, fetal, and placental development. Synthesis, binding (via the CD44 membrane receptor), and degradation of hyaluronan occur in male and female genital organs, the oviduct being no exception. This review discusses our current knowledge on roles of this ubiquitous glycosaminoglycan on the survival of immunologically foreign spermatozoa in the pig oviduct, a relevant event for fertility. During preovulatory storage in the functional tubal sperm reservoir, spermatozoa are entrapped in a mucus-like tubal fluid. This fluid contains fluctuating levels of hyaluronan, which is synthesized by the lining epithelium by hyaluronan synthase 3. Both hyaluronan and its CD44 receptor are particularly evident in the deep mucosal furrows of the sperm reservoir, in which most spermatozoa are embedded in; kept alive, uncapacitated but also undetected by the immune system of the female. Hyaluronan is also present in the seminal plasma, and evidence points toward an involvement of hyaluronan and its receptor in the local (tubal and possibly uterine) production of antiinflammatory cytokines, such as interleukin-10, pertaining maternal immune tolerance of these foreign cells.

Retinoblastoma in the adolescent. Unusual clinical and histopathology findings.

CASE REPORT: A 17-year- old male with 2years history of an intraocular mass and progressive visual loss of the left eye. Spontaneous sclera rupture occurred during enucleation. Microscopic evaluation with H-E, PAS and immunohistochemistry (NSE, GAFP, SYN, CD99) revealed a small blue round cell malignant neoplasm with extensive necrosis and apoptosis. The optic nerve, ciliary body, choroid, anterior chamber, and sclera were infiltrated. SYN was positive and CD99 was negative in neoplastic cells, consistent with a poorly differentiated retinoblastoma. DISCUSSION: Retinoblastoma is the most frequent primary intraocular malignant tumour in childhood, but occasionally older patients can be affected. Immunohistochemistry is mandatory in poorly differentiated retinoblastomas.

Boar spermatozoa successfully predict mitochondrial modes of toxicity: implications for drug toxicity testing and the 3R principles.

Replacement of animal testing by in vitro methods (3-R principles) requires validation of suitable cell models, preferably obtained non-invasively, defying traditional use of explants. Ejaculated spermatozoa are highly dependent on mitochondrial production and consumption of ATP for their metabolism, including motility display, thus becoming a suitable model for capturing multiple modes of action of drugs and other chemicals acting via mitochondrial disturbance. In this study, a hypothesis was tested that the boar spermatozoon is a suitable cell type for toxicity assessment, providing a protocol for 3R-replacement of animals for research and drug-testing. Boar sperm kinetics was challenged with a wide variety of known frank mito-toxic chemicals with previously shown mitochondrial effects, using a semi-automated motility analyser allied with real-time fluorescent probing of mitochondrial potential (MitoTracker & JC-1). Output of this sperm assay (obtained after 30 min) was compared to cell viability (ATP-content, data obtained after 24-48 h) of a hepatome-cell line (HepG2). Results of compound effects significantly correlated (P<0.01) for all sperm variables and for most variables in (HepG2). Dose-dependent decreases of relative ATP content in HepG2 cells correlated to sperm speed (r=0.559) and proportions of motile (r=0.55) or progressively motile (r=0.53) spermatozoa. The significance of the study relies on the objectivity of computerized testing of sperm motility inhibition which is comparable albeit of faster output than somatic cell culture models. Sperm suspensions, easily and painlessly obtained from breeding boars, are confirmed as suitable biosensors for preclinical toxicology screening and ranking of lead compounds in the drug development processes.

Sex sorting increases the permeability of the membrane of stallion spermatozoa.

At present, the only repeatable means of selecting the sex of offspring is the Beltsville semen sorting technology using flow cytometry (FC). This technology has reached commercial status in the bovine industry and substantial advances have occurred recently in swine and ovine species. In the equine species, however, the technology is not as well developed. To better understand the changes induced in stallion spermatozoa during the sorting procedure, pooled sperm samples were sorted: sperm motility and kinematics were assessed using computer assisted sperm analysis, sperm membrane integrity was assessed using the YoPro-1 assay, while plasmalemmal stability and lipid architecture were assessed using Merocyanine 540/SYTOX green and Annexin-V, respectively. Lipid peroxidation was also investigated with the probe Bodipy(581/591)-C11. All assays were performed shortly after collection, after incubation and after sex sorting using FC. In order to characterize potential molecular mechanisms implicated in sperm damage, an apoptosis protein antibody dot plot array analysis was performed before and after sorting. While the percentage of total motile sperm remained unchanged, sex sorting reduced the percentages of progressive motile spermatozoa and of rapid spermatozoa as well as curvilinear velocity (VCL). Sperm membranes responded to sorting with an increase in the percentage of YoPro-1 positive cells, suggesting the sorted spermatozoa had a reduced energy status that was confirmed by measuring intracellular ATP content.

Localization of tumor necrosis factor in the canine testis, epididymis and spermatozoa.

Tumor necrosis factor (TNF), formerly known as Tumor necrosis factor alpha is now regarded as a natural component of the mammalian seminal plasma (SP). Although not completely clarified, its functions in the SP have been associated with paradoxal roles, such as sperm survival in the female genital tract, while at high levels negatively affect sperm survival and fertility potential. Recently, it has been discovered that canine inseminated spermatozoa display a strong immunoreaction for TNF when lining the female endometrium. As a continuation of this finding, the present work aimed at documenting TNF localization in the canine testes and epididymis and in freshly ejaculated spermatozoa (SPZ) through immunohisto- or cytochemistry. Immunoreaction for TNF was found in all samples used. In the dog testis, TNF immunoexpression was limited to the seminiferous tubules, where late round spermatids (SPD) showed weak intensity of immunostaining, while elongating and elongated SPD evidenced moderate and the residual bodies a strong intensity. In the epididymis, a gradual progressive increase of TNF immunolabelling was found throughout the epididymal regions, ranging from a weak intensity at the caput epididymis to a moderate intensity at the cauda. TNF immunolabelling was found in mature SPZ during the epididymal transit and also in freshly ejaculated SPZ, which showed a strong midpiece immunolabelling. Data presented here provide important information on expression of TNF in spermatozoa, which is acquired by the SPZ during their formation at the testis. It further provides the basis for subsequent studies on the physiological importance of cytokines in sperm function.

Tumour necrosis factor in the canine endometrium: an immunohistochemical study.

Tumour necrosis factor (TNF), a pleiotropic cytokine that regulates cell growth and differentiation as well as the synthesis of other cytokines, has been identified in the uterus of several species describing a cyclic pattern, eventually under ovarian steroid regulation. Information is yet limited on the presence of TNF protein in the canine endometrium during the oestrous cycle and early pregnancy. This study depicts the temporal immunolocalization of TNF in the bitch endometrium along the oestrous cycle and changes associated with the early steps of embryo invasion. TNF immunolabelling was found in both the stromal fibroblasts and epithelial components of the canine endometrium in all stages studied. Stromal immunostaining was more intense than that of the epithelia, in all the stages of the oestrous cycle. In addition, a tendency for a decrease in the surface epithelium intensity score was found in early dioestrus. A positive glandular content was only observed in anoestrus and proestrus stages. In early pregnancy (days 13-16), TNF immunolabelling was detected at the embryo-maternal surface, in the syncytium cords and the trophoblast, as well in the endometrial stroma and the basal endometrial glands, but not in the lacunar epithelium. The overall TNF immunoreactivity was higher in early pregnancy samples in comparison with those of the early dioestrus and dioestrus stages, suggesting it plays a role during implantation.

Follicular dynamics and oestrous detection in Thai postpartum swamp buffaloes (Bubalus bubalis).

This study characterized follicular activity and oestrous behaviour from 5 to 9 days post-calving up to the 4th ovulation postpartum (pp) in 16 multiparous (range 2-7 parities) Thai swamp buffalo cows (Bubalus bubalis), aged 4-12 years and weighing from 432 to 676 kg. Ovarian follicular activity was examined by transrectal ultrasonography (TUS) every morning. Oestrous detection was performed twice daily by direct personal observation of behaviour and for presence of clear cervical mucus discharge and indirectly by video camera recording during 21 h/day. A follicular wave-like pattern was present before the 1st ovulation leading to short oestrous cycles. Growth rates and maximum diameters of the ovulatory follicles did not differ between the 1st and 4th ovulations. However, growth rate for non-ovulatory dominant follicles (DF) before the 1st ovulation was lower than for the ovulatory follicle (p<0.05). In addition, the diameter of all ovulatory follicles (14.3 ± 0.46 mm, n=39) was significantly larger (p < 0.01) than those of the preceding last but one non-ovulatory DF (10.8 ± 0.20 mm, n = 5), but similar to the last preceding non-ovulatory DF diameter (12.92 ± 0.96 mm, n = 14). Short oestrous cycles were most common between the 1st and 2nd ovulations (93.75%, 15/16 cows, 10.2 ± 0.38 days) decreasing in prevalence thereafter (50%, 3/6 buffaloes, 12.0 ± 1.53 days). Oestrous signs were relatively vague around the 1st ovulation pp to become more easily detectable thereafter. This study suggests that properly fed swamp buffaloes could be mated successfully within 2 months pp, at their 2nd spontaneous ovulation, provided oestrous detection is at least performed daily at 06:00-08:00 hour.

Spermadhesin PSP-I/PSP-II heterodimer induces migration of polymorphonuclear neutrophils into the uterine cavity of the sow.

Seminal plasma (SP) is a complex fluid which exerts biological actions in the female reproductive tract. In pigs, SP elicits endometrial inflammation and consequent immune changes after mating. This study tested whether heparin-binding spermadhesins (HBPs) and the heterodimer of porcine sperm adhesions I and II (PSP-I/PSP-II) in SP recruit different lymphocyte subsets (CD2(+), CD4(+) and CD8(+) T cells) or polymorphonuclear leukocytes (PMNs) to the superficial endometrium or luminal epithelium and lumen, respectively, of oestrous sows. In Experiment 1, endometrial biopsies were taken between 2 and 120 min after infusion of uterine horns with HBPs, PSP-I/PSP-II or saline and evaluated by immunohistochemistry or histology. In Experiment 2, the uterus of oestrous sows was infused with PSP-I/PSP-II or saline to assess PMN numbers in the uterine lumen 3h later. PSP-I/PSP-II elicited CD2+ T cell recruitment from 10 min, and CD8(+) T cells from 60 min after infusion, while HBPs increased CD4(+) T cell recruitment by 120 min. PSP-I/PSP-II but not HBPs induced PMN migration to the surface epithelium by 10 min. PMN numbers were elevated 5-fold by 30 min and 7-fold from 60 min, with PMNs detectable in the lumen from 30 min after infusion. Six-fold more PMNs were collected from the uterine lumen of PSP-I/PSP-II-infused sows compared to controls at 3h after infusion. These data show that PSP-I/PSP-II heterodimer in seminal plasma has a predominant role in triggering the recruitment of uterine PMNs and T cells after mating, initiating a cascade of transient and long-lasting immunological events.

In vitro fertilizing capacity of frozen-thawed bull spermatozoa selected by single-layer (glycidoxypropyltrimethoxysilane) silane-coated silica colloidal centrifugation.

Barriers to the use of density gradient centrifugation for preparing animal spermatozoa for artificial insemination (AI) include the scarcity of animal-specific formulations and the daunting prospect of processing large volumes of ejaculate in small aliquots (1.5 ml extended semen). Recently, new colloid formulations have been tested in vitro in a modified procedure, centrifugation on a single layer of colloid. The present study investigated the fertilizing ability during in vitro fertilization (IVF) of frozen-thawed bovine spermatozoa following centrifugation through a single layer of glycerolpropylsilane (GS)-coated silica colloid with a species-specific formulation (patent applied for; treatment, T). Controls (C) included centrifugation through gradients of either the same colloid (C1) or Percoll (C2). Sperm recovery surpassed 50% for both C1-C2 and T (n.s.). Mean values of various parameters of computerized analysis of sperm motility did not differ between T and C1 (n.s.), and only the proportions of path straightness and linearity were lower in T vs C2 (p < 0.05). In T, the mean (+/-SD) percentages of fertilization rate, blastocyst development rate and the total number of blastomeres were 58.1 +/- 23.3%, 24.5 +/- 14.3% and 94.6 +/- 23.4%, respectively. The proportions did not differ significantly from controls (C1/C2). Therefore, centrifugation through a single layer of colloid offers an alternative method to density gradient centrifugation for selection of viable, potentially fertile frozen-thawed bull spermatozoa. This single-layer technique is gentle, versatile and convenient because it facilitates scaling-up the process of sperm preparation to allow larger numbers of spermatozoa (for instance, whole ejaculates) to be processed for AI.

Effects of continuous elevated cortisol concentrations during oestrus on concentrations and patterns of progesterone, oestradiol and LH in the sow.

This study investigated the effect of continuous elevated cortisol concentrations during standing oestrus on time of ovulation and patterns of progesterone, oestradiol and luteinising hormone (LH) in sows. The elevation of cortisol concentrations was achieved through repeated intravenous injections of synthetic adrenocorticotropic hormone (ACTH) every 2 h for approximately 48 h, from the onset of the second standing oestrus after weaning. Treatment was terminated when ovulation was detected (monitored by transrectal ultrasonography every 4h) or when the sow had received a maximum of 24 injections. The dose of ACTH (2.5 microg/kg) was chosen to mimic the cortisol concentrations seen during mixing of unfamiliar sows. The sows (n=14) were surgically fitted with jugular vein catheters and randomly divided into a control (C group where only NaCl solution were injected) or an ACTH group. Blood samples were collected every 2 h. In parallel with the blood sampling, saliva samples for cortisol analyses were taken from eight sows before onset of treatment and from four of the sows during treatment. There was no difference in time from onset of standing oestrus to ovulation between the two groups. The interval between the peaks of oestradiol and LH to ovulation was prolonged in the ACTH group compared to the C group (p<0.05), with a tendency towards an earlier decline of oestradiol in the ACTH group. Cortisol and progesterone concentrations were significantly elevated during treatment in the ACTH group (p<0.001), with cortisol peak concentrations occurring between 40 and 80 min after each ACTH injection. Cortisol concentrations in saliva and plasma were highly correlated (p<0.001). In conclusion, elevated cortisol concentrations from the onset of standing oestrus increase progesterone concentrations and prolong the interval between oestradiol and LH peaks to ovulation, the latter possible due to an early decline in oestradiol concentrations and a change of the LH peak outline. The effect these hormonal changes have on reproductive performance need to be further investigated. Saliva samples might be a useful and non-invasive method to assess cortisol concentrations in sows.

Short- and long-term effects of immunization against gonadotropin-releasing hormone, using Improvac, on sexual maturity, reproductive organs and sperm morphology in male pigs.

The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac Pfizer Ltd.), on sexual maturity, development of the reproductive organs, and the morphology of caudal epididymal spermatozoa in non-castrated male pigs. The pigs were slaughtered 4, 16 or 22 weeks after the second Improvac vaccination. A total of 80 crossbred non-castrated male pigs were included in this study comprising two experiments, a short-effect (Experiment 1) and a long-effect (Experiment 2). The first experiment included 56 pigs, 24 of them were maintained as controls and 32 were vaccinated twice, and slaughtered 4 weeks after the second vaccination. The second experiment included 24 pigs, 12 controls and 12 vaccinated twice, and slaughtered either 16 weeks (n=6) or 22 weeks (n=6) after the second vaccination. None of the immunized pigs was sexually mature at slaughter, i.e. 4, 16 or 22 weeks after second vaccination. Corresponding results of the control pigs showed that 50% had reached sexual maturity at the age corresponding to 4 weeks after the second vaccination, and 100% at slaughter 16, respectively, 22 weeks after vaccination. At 4, 16 and 22 weeks after second vaccination both testes weight and bulbourethral length were significantly reduced (p<0.001). The percentages of proximal droplets and abnormal heads were significantly lower in the control pigs than in the immunized pigs at slaughter 4 weeks after vaccination, whereas distal droplets were higher. For the other morphological parameters no significant differences were seen, but all mean values except for acrosome defects were numerically lower in the control pigs compared with the immunized pigs. For pigs slaughtered 16 or 22 weeks after vaccination, the vaccination effect was significant for percentages of proximal droplets, distal droplets, acrosome defects, acrosome abnormality and abnormal heads (p=0.017-0.001). The immunization clearly disrupted the number and morphology of the interstitial Leydig cells, lasting throughout the study period (4-22 weeks after vaccination). Spermatogenesis was also clearly affected in the immunized pigs, to various degrees, from mild disruption (spermatocyte loss, decrease of the normal number of layers of germ cells) to severe loss of germ cells including tubuli with Sertoli cells-only (complete disappearance of germ cells), also covering the entire study period. The results indicated that the effect of immunization persisted for at least 22 weeks after the second vaccination.

Post-thaw viability of bull AI-doses with low-sperm numbers.

Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 x 10(6) total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/PI), acrosome integrity (Carboxy-SNARF-1/PI/FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 degrees C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols.

Effects of exogenous ACTH during oestrus on early embryo development and oviductal transport in the sow.

This study was conducted to assess the effects of ACTH injections on the early development of embryos and their transportation to the uterus. Fifteen sows were monitored for ovulation using transrectal ultrasonography during the first two oestrous periods after weaning. The sows were randomly divided into a control group (C group, n = 8) and an ACTH-treated group (ACTH group, n = 7), and were all surgically fitted with intra-jugular catheters. From the onset of the second standing oestrus after weaning, the sows were injected (NaCl/synthetic ACTH) every 4 h. Blood samples were collected immediately before and 45 min after each injection. All sows were inseminated once 10-33 h before ovulation in their second oestrus after weaning. At 48 (n = 4) or 60 (n = 11) h after ovulation during their second oestrus, the sows were killed and the embryos retrieved from the oviduct and uterus. The embryos were counted and compared with the number of corpora lutea, cleavage rate was noted and, finally, the embryos were prepared for confocal laser scanning microscopy and transmission electron microscopy. There was no difference between the groups regarding cleavage rate, the cytoskeleton, or the number of active nucleoli. However, the ACTH group had significantly (p < 0.05) fewer ova/embryos retrieved (51%) than the C group (81%), and there was a tendency towards faster transportation to the uterus in the ACTH group, possibly because of high progesterone concentrations during treatment. To conclude, administration of ACTH every 4 h from onset of oestrus to 48 h caused significant loss of oocytes or embryos, and possibly faster transportation through the oviduct.

Effects of ACTH injections during estrus on concentrations and patterns of progesterone, estradiol, LH, and inhibin alpha and time of ovulation in the sow.

This study investigated whether injections of ACTH for 48 h, from the onset of the second standing estrus after weaning, had any impact on time of ovulation and patterns of progesterone, estradiol, luteinizing hormone (LH), and inhibin alpha. The studied sows (n=15) were fitted with jugular vein catheters and randomly divided into a control (C group) and an ACTH group. From the onset of standing estrus, the sows were injected (NaCl or synthetic ACTH, 5 microg/kg) every 4h; blood samples were collected immediately before and 45 min after each injection. Ovulation was monitored using ultrasonography. The ACTH-group sows stopped displaying signs of standing estrus sooner after ovulation in their second estrus, but no impact was found on time of ovulation. There were no significant differences in the intervals between LH peak, estradiol peak, and the onset of standing estrus between the C and ACTH groups. The cortisol and progesterone concentrations were significantly elevated (p<0.001) in samples taken 45 min after ACTH injection. There were minor differences in estradiol and LH concentrations between the groups. Overall inhibin alpha concentrations were significantly higher during the treatment period in the ACTH than in the C group, but there were no significant differences between samples taken either 45 min or 4h after injection. In conclusion, injections of synthetic ACTH during estrus in the sow apparently disturb the duration of signs of standing estrus and the hormonal pattern of progesterone, and possibly of inhibin alpha, estradiol and LH.