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Eduardo S Ribeiro, José E P Santos and William W Thatcher

Elongation of the preimplantation conceptus is a prerequisite for successful pregnancy in ruminants and depends on histotroph secretion by the endometrium. Lipids are an essential component of the histotroph, and recent studies indicate that lipids have important roles in the elongation phase of conceptus development. The onset of elongation is marked by dynamic changes in the transcriptome of trophectoderm cells, which are associated with lipid metabolism. During elongation, the trophectoderm increases transcript expression of genes related to uptake, metabolism and de novo biosynthesis of fatty acids and prostaglandins. Expression of the gene PPARG increases substantially, and activation of the transcription factor PPARG by binding of lipid ligands appears to be crucial for the coordination of cell biology during elongation. Lipids accumulated in the epithelial cells of the endometrium during diestrus are likely the most important source of fatty acids for utilization by the conceptus and become available in the uterine lumen through exporting of exosomes, microvesicles, carrier proteins and lipoproteins. Targeting of uterine lipid metabolism and PPARG activity during preimplantation conceptus development through nutraceutical diets may be a good strategy to improve pregnancy survival and reproductive efficiency in ruminants.

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Ronaldo L A Cerri, Heloísa M Rutigliano, Ricardo C Chebel and José E P Santos

Length of dominance of the ovulatory follicle and exposure to oestradiol (OE2) during proestrus can affect fertility. Lactating cows had their oestrous cycle pre-synchronized and were subjected to one of the four synchronization treatments. Cows in the oestrus detection (OD) treatment received GnRH on day 6 of the oestrous cycle, PGF 7 days later, and were inseminated at detected oestrus. The remaining cows were subjected to the Ovsynch (OVS) protocol (day 0 GnRH, day 7 PGF, day 9 GnRH, and timed artificial insemination (AI) 12 h later) starting on day 3 (OVS3) or day 6 (OVS6 and OVS6E) of the oestrous cycle. Cows in the OVS6E treatment received an injection of 0.5 mg oestradiol cypionate 36 h before AI. Ovaries were examined by ultrasonography and blood was sampled for progesterone and OE2 concentrations. Uteri were flushed 6 days after AI and recovered embryos–oocytes evaluated. Diameter of the ovulatory follicle at AI differed (P<0.01) among treatments, and it was the largest for OVS3 cows, which also had extended (P<0.01) length of follicular dominance. During proestrus, OD and OVS6E cows had increased (P<0.01) OE2 concentrations. Fertilization was not altered by treatments, and maximum fertilization was achieved when the number of accessory spermatozoa was >7. Proportions of viable embryos in relation to embryos and embryos–oocytes recovered were smaller for OVS3 cows (P<0.01) than the other treatments, and embryos from OVS3 cows also had fewer (P<0.01) blastomeres and tended (P=0.09) to have a lower proportion of live blastomeres. Extending the period of follicle dominance did not alter fertilization but reduced (P<0.001) embryo quality. Embryo quality was compromised even when the dominance of the ovulatory follicle was extended by only 1.5 days.

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Rachel L Piersanti, Anthony D Horlock, Jeremy Block, José E P Santos, I Martin Sheldon and John J Bromfield

Metritis is associated with reduced fertility in dairy cows, but the mechanisms are unclear because the disease resolves several weeks before insemination. One hypothesis is that metritis causes persistent changes in granulosa cells during follicle development, which might be evident in the transcriptome of granulosa cells from dominant follicles weeks after parturition. To test this hypothesis, we collected the follicular fluid and granulosa cells from dominant follicles 63 days post partum from cows previously diagnosed with metritis, at least 6 weeks after resolution of the disease and from cows not diagnosed with metritis (control cows). Bacterial lipopolysaccharide was detected in follicular fluid, and concentrations were associated with follicular fluid IL-8 and glucose concentrations. Transcriptome analysis using RNAseq revealed 177 differentially expressed genes in granulosa cells collected from cows that had metritis compared with control cows. The most upregulated genes were ITLN1, NCF2, CLRN3, FSIP2 and ANKRD17, and the most downregulated genes were ACSM1, NR4A2, GHITM, CBARP and NR1I3. Pathway analysis indicated that the differentially expressed genes were involved with immune function, cell–cell communication, cell cycle and cellular metabolism. Predicted upstream regulators of the differentially expressed genes included NFκB, IL-21 and lipopolysaccharide, which are associated with infection and immunity. Our data provide evidence for a persistent effect of metritis on the transcriptome of granulosa cells in ovarian follicles after the resolution of disease.

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Letícia D P Sinedino, Paula M Honda, Letícia R L Souza, Adam L Lock, Maurice P Boland, Charles R Staples, William W Thatcher and José E P Santos

The objectives were to determine the effects of supplementing docosahexaenoic acid (DHA)-rich algae on reproduction of dairy cows. Holstein cows were assigned randomly to either a control (n = 373) or the same diet supplemented daily with 100 g/cow of an algae product containing 10% DHA (algae, n = 366) from 27 to 147 days postpartum. Measurements included yields of milk and milk components, fatty acids (FA) profiles in milk fat and plasma phospholipids, resumption of ovulation by 57 days postpartum, pregnancy per artificial insemination (AI) and expression of interferon-stimulated genes in leukocytes. Feeding algae increased resumption of estrous cyclicity (77.6 vs 65.9%) and pregnancy at first AI (47.6 vs 32.8%) in primiparous cows. Algae increased pregnancy per AI in all AI in both primiparous and multiparous cows (41.6 vs 30.7%), which reduced days to pregnancy by 22 days (102 vs 124 days) compared with control cows. Pregnant cows fed algae had greater expression of RTP4 in blood leukocytes compared with those in pregnant control cows. Feeding algae increased the incorporation of DHA, eicosapentaenoic acid, conjugated linoleic acid isomers cis-9 trans-11, trans-10 cis-12 and total n-3 FA in phospholipids in plasma and milk fat. Yields of milk and true protein increased by 1.1 kg/day and 30 g/day respectively, whereas fat yield decreased 40 g/day in algae compared with that in control. Supplementing DHA-rich algae altered the FA composition of lipid fractions and improved reproduction in dairy cows. The benefits on reproduction might be mediated by enhanced embryo development based on changes in interferon-stimulated gene expression.

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Fernando A Rivera, Luís G D Mendonça, Gláucio Lopes Jr, José E P Santos, Rolando V Perez, Marcel Amstalden, Abelardo Correa-Calderón and Ricardo C Chebel

Fertility of lactating dairy cows is associated with reduced progesterone (P4) concentration compared with nonlactating animals. The objective of the current study was to determine whether P4 during growth of the first follicular wave (FFW) affects embryo quality. Lactating Holstein cows at 33±3 days post partum were allocated to one of three treatments. Cows in the FFW and FFW with P4 (FFWP) treatments started the superstimulation protocol on day 1 of the estrous cycle and second follicular wave (SFW) cows started the superstimulation protocol on estrous cycle day 7. Cows were superstimulated with 400 mg of NIH-FSH-P1 (FSH) given twice daily for 5 days, two prostaglandin F (PGF) injections given with the ninth and tenth injections of FSH, GNRH given 48 h after the first PGF injection, and timed insemination 12 and 24 h after the GNRH injection. Cows in the FFWP treatment received two intravaginal P4 inserts during the superstimulation. Embryos were recovered 6.5 days after artificial insemination and excellent/good and fair embryos were frozen and transferred. Blood was sampled daily from estrous cycle day 0 until insemination from donor cows. During the superstimulation protocol, P4 was (P<0.01) greatest for SFW cows followed by FFWP and FFW cows respectively. The percentage of embryos–oocytes from SFW and FFWP cows classified as excellent/good and fair embryos was (P=0.02) greater than those of FFW cows. Pregnancy per embryo transfer was not (P≥0.73) affected by embryo donor treatment. Reduced embryo quality of cows induced to ovulate the follicles from the first follicular wave is a consequence of reduced P4 during follicle growth.

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Anthony D Horlock, Rachel L Piersanti, Rosabel Ramirez-Hernandez, Fahong Yu, Zhengxin Ma, KwangCheol C Jeong, Martin J D Clift, Jeremy Block, José E P Santos, John J Bromfield and I Martin Sheldon

Infection of the postpartum uterus with pathogenic bacteria is associated with infertility months later in dairy cattle. However, it is unclear whether these bacterial infections lead to long-term changes in the reproductive tract that might help explain this infertility. Here we tested the hypothesis that infusion of pathogenic bacteria into the uterus leads to changes in the transcriptome of the reproductive tract 3 months later. We used virgin Holstein heifers to avoid potential confounding effects of periparturient problems, lactation, and negative energy balance. Animals were infused intrauterine with endometrial pathogenic bacteria Escherichia coli and Trueperella pyogenes (n = 4) and compared with control animals (n = 6). Three months after infusion, caruncular and intercaruncular endometrium, isthmus and ampulla of the oviduct, and granulosa cells from ovarian follicles >8 mm diameter were profiled by RNA sequencing. Bacterial infusion altered the transcriptome of all the tissues when compared with control. Most differentially expressed genes were tissue specific, with 109 differentially expressed genes unique to caruncular endometrium, 57 in intercaruncular endometrium, 65 in isthmus, 298 in ampulla, and 83 in granulosa cells. Surprisingly, despite infusing bacteria into the uterus, granulosa cells had more predicted upstream regulators of differentially expressed genes than all the other tissues combined. In conclusion, there were changes in the transcriptome of the endometrium, oviduct and even granulosa cells, 3 months after intrauterine infusion of pathogenic bacteria. These findings imply that long-term changes throughout the reproductive tract could contribute to infertility after bacterial infections of the uterus.