Effects of increased ambient temperature and supplemental altrenogest prior to pregnancy establishment in gilts

Heat stress (HS) mitigation strategies are critically needed to combat the substantial economic effects on animal agriculture. The manifestations of seasonal infertility include delayed puberty onset, reduced conception rates, decreased litter size, and increased wean to estrus interval. To assess the effects of HS during early gestation and evaluate a benefit of supplemental altrenogest (ALT) as a mitigation strategy, thirty crossbred post-pubertal gilts (157 ± 11 kg) were subjected to estrous synchronization via 14 d oral administration of ALT. Artificial insemination during estrus was performed and gilts were then placed into one of four treatment groups; heat stress (HS; 35 ± 1 οC for 12h/31.60 ± 1 οC for 12h) with (HSALT, n = 7) or without (HSCON, n = 7) 15 mg/d ALT supplementation or thermal neutral (TN; 20 ± 1 οC) conditions with (TNALT, n = 8) or without (TNCON, n = 8) 15 mg/d ALT supplementation until 12 d post-estrus (dpe). Administrating ALT occurred at 0600 h from 3-12 dpe and rectal temperatures (TR) and respiration rates (RR) were recorded. Blood was collected via jugular venipuncture on 0, 4, 8 and 12 dpe. Gilts were euthanized humanely at 12 dpe followed by collection of ovarian tissue, and uterine flushing for conceptus collection. In HS compared to TN gilts, RR and TR were increased (P < 0.01) but unaffected by ALT supplementation. Feed intake (FI) was reduced (P < 0.01) by HS but unaltered by ALT treatment. Corpora lutea (CL) weight was reduced (P < 0.01) in HSCON gilts when compared to TNCON and HSALT gilts despite progesterone (P4) concentrations in serum and luteal tissue not being affected by treatment (P ≥ 0.10). CL diameter was reduced (P ≤ 0.05) in HSALT gilts compared to other treatments. Interleukin-1β (IL1B) uterine flush concentration was not affected (P > 0.20) by environment or ALT supplementation, although moderate (P = 0.06) interaction between environment and ALT existed, as IL1B concentration in TNALT was increased (P = 0.03) compared to TNCON gilts. While environment did not affect conceptus development (P = 0.90), ALT supplementation advanced conceptus elongation (P < 0.01). Collectively, these data demonstrate that HS may affect luteal development prior to pregnancy establishment, and ALT increases conceptus elongation by12 dpe.

Formation of fibrin at sights of conceptus adhesion in the ewe

In ruminants, various molecules are involved in regulating conceptus attachment and adhesion; however, molecules that maintain the conceptus adhesion have not been well characterized. We hypothesized that conceptus must produce a molecule(s), yet uncharacterized or overlooked, which maintain conceptus adhesion to the uterine epithelium. In this study, we aimed to identify new candidate(s) in conceptus secretory proteins responsible for maintaining conceptus adhesion in sheep. We performed RNA-sequence analysis with ovine conceptuses, followed by endometria obtained from pregnant animals on day 15 (P15: pre-attachment), 17 (P17: right after attachment), and 21 (P21: post-attachment; adhesion) and iTRAQ analysis of uterine flushing on P15 and P17. To identify the proteins secreted from conceptuses, we cross-referenced the transcriptome and proteome data. These analyses identified 16 and 26 proteins as conceptus secretory proteins on P15 and P17, respectively. Gene ontology analysis revealed that the conceptus secretory proteins were enriched in those categorized to fibrinolysis and coagulation. RT-qPCR analysis verified that the expression levels of transcripts in conceptuses encoding coagulation factors, fibrinogen subunits, and fibrinolysis factors were significantly higher on P21 than on P15 or P17, which were supported by those through in situ hybridization, western blotting and immunohistochemistry. Histology analysis confirmed that fibrin protein was present at the conceptus adhesion region on P21. These results suggest that in addition to the numerous adhesion molecules so far characterized, fibrin is a new candidate molecule for maintaining conceptus adhesion for pregnancy continuation in ruminants.

Evaluation of fertility and subfertility in adult alpacas and tuis using ultrasonography, endometrial cytology and bacterial isolation

The objective of this study was to compare the uterine health between fertile, sub-fertile alpacas and tuis using transrectal ultrasonography, endometrial cytology and bacterial isolation. A total 10 tuis (young mature females without breeding with average age of 1.5 years) and 20 adult alpacas of the Suri breed were used. In turn, the adult females were divided into two groups of 10 animals each according to their reproductive history: fertile group (parturition every year) and sub-fertile group (1 to 2 years without pregnancy). In all females, the thickness of the cervix and uterine horns was determined by transrectal ultrasonography. On the other hand, endometrial cytology and bacterial isolation were performed from samples obtained by uterine flushing. A Kruskal-Wallis and a Chi-square tests were used to compare ultrasonography and cytology groups. A greater thickness of the cervix and both uterine horns (p˂0,05) was observed in the fertile alpacas with respect to the sub-fertile and tuis. The percentage of PMN in tuis and sub-fertile alpacas was < 2%, while in fertile alpacas the percentage of PMN were: 6 animals with < 2% PMN, 2 animals with 2-5% PMN and two other alpacas with > 5% PMN. The bacteria isolated were: Bacillus lechiniformis and Escherichia coli in the three groups studied, Staphylococcus saprophyticus and Bacillus cereus in tuis and fertile alpacas, Staphylococcus aureus in tuis and sub-fertile, Bacillus spp. and Micrococcus spp. in fertile and sub-fertile alpacas, Bacillus lactic acid, Staphylococcus epidermidis and Citrobacter spp. in fertile alpacas, Enterococcus spp., Bacillus subtilis and Klebsiella spp. in sub-fertile and Enterobacter spp. in tuis. The low percentage of PMN in endometrial cytology in sub-fertile alpacas would indicate the absence of endometritis at the time of the study. However, the lower thickness of the cervix and uterine horns observed in sub-fertile alpacas suggest that it would be necessary to perform uterine biopsies in order to evaluate if there is any association between the thickness of the uterine wall and the presence of degenerative and/or inflammatory changes observed on histopathological examination.

1. A new uterine flushing curette 2. New aluminium uterine probe 3. Improved Syringe for Transfusion of Saline fluid into the Cellular Tissue

In theory, none of these instruments is anything new or original: the Donalde instrument, manufactured by Arnold and Son in Manchester, resembles the Fritsch-Bosemann catheter, whose beak is elongated in the form of a sharp spoon; its diameter is equal to No. 14 of Duncan's expander (approximately No. 8-9 Hegar).

30 Uterine flushing as a non-invasive method to access inflammatory cytokines in sows reproductive tract after parturition

Inflammatory mediators, such as cytokines, can reduce feed intake and drastically modify the partitioning of nutrients. Thus, it may increase the animal’s maintenance requirements, reduce milk production and piglet growth, impact reproductive performance, and reduce sow lifetime productivity. The objective of this experiment was to evaluate a non-invasive method, not tested before in swine, to assess cytokine profiles from post-partum uterine lavage. The uteri of second and third parity sows (n=14) were flushed with sterile saline solution (0.9%) on days 2, 4, and 14 post-parturition. An artificial insemination (AI) catheter, in its plastic covering, to avoid vaginal contents, was inserted through the vagina into the cervix. A flexible inner catheter was passed through the outer AI catheter and into the uterine horn. Sterile saline (30-40 mL) was flushed through the inner catheter into one uterine horn. After inner catheter removal, a tube was attached to the end of the outer AI catheter to allow collection of the natural back-flow of uterine lavage fluid (avg 24.7% recovery). Uterine fluid collected was immediately centrifuged and the supernatant stored at -20°C. Samples were freeze-dried, re-suspended in sterile saline (2 mL), and stored at -80°C. Cytokine profiles of the uterine fluid were evaluated using a multiplex ELISA panel including interleukin (IL)-1β, IL-6, IL-8, IL-4, IL-10, tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). Cytokine concentrations were calculated relative to protein content (pg/mg of protein). IFN-γ and TNF-α were lower than the limit of detection in most samples (5/38 and 1/38, respectively). IL-4 and IL-10 concentrations did not differ among days of collection (P &gt;0.14). IL-8 was greater on day 4 than on days 2 or 14 (P&lt; 0.05). IL-1β and IL-6 were greater on days 2 and 4 than on day 14 (P&lt; 0.05). Uterine flushing is a novel non-invasive method to access uterine inflammatory cytokines in sows.

Location relative to the corpus luteum affects bovine endometrial response to a conceptus

In cattle, embryo transfer into the uterine horn contralateral to the corpus luteum results in a higher incidence of pregnancy loss compared to transfer into the ipsilateral horn. We have previously reported temporal changes in the endometrial transcriptome during the estrous cycle which differ between uterine horns. The objective of this study was to compare the transcriptomic response of endometrium from the ipsilateral and contralateral horns to an elongating conceptus. Cross-bred beef heifers (n = 16) were synchronized and either used to generate day 14 conceptuses following the transfer of in vitro-produced blastocysts or to obtain day 14 endometrial explants. Conceptuses were recovered on day 14 by post-mortem uterine flushing, placed individually on top of explants collected from the ipsilateral (IPSI-D14) or the contralateral (CONTRA-D14) uterine horn of cyclic heifers, and co-cultured for 6 h. The response to a conceptus was markedly different between uterine horns, with 61 and 239 differentially expressed genes (DEGs; false discovery rate <0.05) in the ipsilateral and contralateral horns, respectively, compared to their controls. Direct comparison between IPSI-D1 and CONTRA-D14 revealed 32 DEGs, including CXCL11, CXCL10, IFIT2, RSAD2 and SAMD9. Gene Ontology analysis of these 32 genes revealed ten enriched biological processes, mainly related to immune response and response to an external stimulus. These data indicate that the endometrial response to the presence of a conceptus varies between uterine horns in the same uterus and may contribute to the higher incidence of pregnancy loss following embryo transfer to the contralateral horn.