Ülle Jaakma

Fixed time deep intracornual insemination of heifers at synchronized estrus

The aim of the study was to determine the efficiency of single fixed time deep intracornual insemination using 2 x 10(6) spermatozoa compared with single standard dose deep intracornual insemination and single and dual standard dose (40 x 10(6)) uterine body (conventional) insemination in heifers at synchronized estrus. Estrus was synchronized in 275 virgin heifers by administration of two doses of PGF(2)alpha 14 days apart. Deep intracornual inseminations with low (ICI-LD1, n=102) and standard (ICI-SD1, n=56) dose of semen and the single standard dose conventional inseminations (AI-SD1, n=66) were performed 80-82 h after the second PGF(2)alpha treatment. Ultrasonography was used to identify the first dominant (presumed ovulatory) follicle, and semen was deposited either close to the utero-tubal junction (n=69 in ICI-LD1 and n=23 in ICI-SD1) or in the middle part of the uterine horn (n=28 in ICI-LD1 and n=28 in ICI-SD1) ipsilateral to the ovary bearing the first dominant follicle. The dual standard dose conventional inseminations were performed 72 and 96 h after the second PGF(2)alpha treatment (AI-SD2, n=51). The pregnancy rate in the ICI-LD1 group (68.0%) did not differ significantly (P>0.05) from the ICI-SD1 group (56.9%) or the AI-SD2 group (65.9%) and was significantly higher (P<0.05) than in the AI-SD1 group (54.2%). The site of intacornual deposition of semen, near the utero-tubal junction or in the middle of the horn, had no effect on the pregnancy rate. The pregnancy rate in all the groups was not affected by the intensity of expression of estrous signs.

Effects of sperm treatments on the in vitro development of bovine oocytes in semidefined and defined media

Bovine in vitro matured oocytes were inseminated with frozen-thawed spermatozoa prepared by A) swim-up through Fert-TALP supplemented with hyaluronic acid (HYA, 1 mg/ml), heparin (5.0 microg/ml) and bovine serum albumin (BSA, 6 mg/ml) or B) washing by centrifugation in modified Brackett-Oliphant medium (mBO) supplemented with 10 mM caffeine-sodium benzoate. For Method A, in vitro fertilization (IVF) was performed in Fert-TALP supplemented with 6 mg/ml BSA, 5.0 microg/ml heparin, 20 microM D-penicillamine, 10 microM hypotaurine and 1 microM epinephrine. For Method B it was performed in mBO medium supplemented with 10 mg globulin-free BSA/ml and 10 microg heparin/ml. Presumptive zygotes were cultured in 1 of 3 culture media: 1) BSAITS - TCM 199 supplemented with 10 mg/ml BSA and ITS (5 microg/ml insulin, 5 microg/ml transferrin, and 5 ng/ml sodium selenite); 2) BECM - bovine embryo culture medium; and 3) BECM supplemented with ITS. Altogether, a significantly higher proportion of oocytes developed to the blastocyst stage after insemination with spermatozoa prepared by Method A than by Method B (17.9 vs 7.1%, respectively; P < 0.001). For Method A, the cleavage rate and the proportion of zygotes with >2 cells 48 h after insemination did not differ significantly between any of the 3 culture media assayed, but blastocyst formation was significantly stimulated in BSAITS and BECMITS compared with that in BECM (20.7 and 22.1% vs 10.7%, respectively; P < 0.05). For Method B, the cleavage rate and the proportion of zygotes with >2 cells were significantly lower in BSAITS than in BECM and BECMITS (56.4 and 28.7% vs 71.6 and 42.1%; and 70.2 and 51.1%, respectively; P < 0.05). However, no significant differences were recorded in blastocyst development rates between any of the culture media assayed (6.4 to 7.4%; P > 0.05).

Bovine sperm plasma membrane proteomics through biotinylation and subcellular enrichment

A significant proportion of mammalian fertilization is mediated through the proteomic composition of the sperm surface. These protein constituents can present as biomarkers to control and regulate breeding of agricultural animals. Previous studies have addressed the bovine sperm cell apical plasma membrane (PM) proteome with nitrogen cavitation enrichment. Alternative workflows would enable to expand the compositional data more globally around the entire sperms surface. We used a cell surface biotin‐labeling in combination with differential centrifugation to enrich sperm surface proteins. Using nano‐LC MS/MS, 338 proteins were confidently identified in the PM‐enriched proteome. Functional categories of sperm–egg interaction, protein turnover, metabolism as well as molecular transport, spermatogenesis, and signal transduction were represented by proteins with high quantitative signal in our study. A highly significant degree of enrichment was found for transmembrane and PM‐targeted proteins. Among them, we also report proteins previously not described on bovine sperm (CPQ, CD58, CKLF, CPVL, GLB1L3, and LPCAT2B) of which CPQ and CPVL cell surface localization was further validated. A descriptive overview of the bovine sperm PM integral and peripheral proteins is provided to complement future studies on animal reproduction and its relation to sperm cell surface. All MS data have been deposited in the ProteomeXchange with identifier PXD001096 (http://proteomecentral.proteomexchange.org/dataset/PXD001096).

Morphological Quality of Oocytes and Blood Plasma Metabolites in Repeat Breeding and Early Lactation Dairy Cows

The objectives of the study were to evaluate the morphological quality of oocytes in repeat breeder and early lactation cows and to determine the possible associations between the quality of oocytes and a range of blood metabolites. Oocyte quality and a range of metabolites were compared between 29 repeat breeder and 13 early lactation cows. The yield of oocytes from the repeat breeders was lower than that from the early lactation cows (4.4 ± 0.2 vs 5.4 ± 0.6, p < 0.05). Percentages of abnormal oocytes for the repeat breeders and the early lactation cows were 52.5% and 37.9%, respectively (p < 0.001). An excess of abnormal oocytes to normal was found in 55.2% of the studied repeat breeders (65.8% vs 34.2%, p < 0.05). Total protein, glucose and aspartate aminotransferase did not differ (p > 0.05) between the repeat breeders with an excess of abnormal oocytes (81 ± 1.0 g/l, 3.5 ± 1.0 mmol/l and 68.5 ± 3.7 U/l), those with the prevalence of normal oocytes (84 ± 1.0 g/l, 3.6 ± 0.1 mmol/l and 73.2 ± 3.5 U/l) and the early lactation cows (83 ± 2.0 g/l, 3.7 ± 0.1 mmol/l and 74.5 ± 3.6 U/I). The repeat breeders with an excess of abnormal oocytes had higher (p < 0.05) urea (5.2 ± 0.2 mmol/l) level than in those with the prevalence of normal oocytes (4.8 ± 0.2 mmol/l) and the early lactation cows (4.7 ± 0.2 mmol/l). A trend for higher total cholesterol and lactate dehydrogenase activity was found in the repeat breeders with an excess of abnormal oocytes. In conclusion, it is suggested that possible causes of repeat breeding in dairy cows may include impaired oocytes. An excess of abnormal oocytes in the repeat breeder cows was associated with elevated blood plasma levels of urea.

Myosin heavy chain pattern in the Akhal-Teke horses

This study investigates the myosin heavy chain (MyHC) isoform composition in the gluteus medius muscle of the Akhal-Teke horses using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Fifteen horses aged between 1.5 and 23.5 years were used in this study and divided into three age groups: 1.5 to 4 (n = 6), 9 to 13 (n = 5) and 18.5 to 23.5 years (n = 4). The average content of the MyHC I isoform was 11.72 ± 1.07% (variation between individuals: 7.09% to 20.14%). The relative content of the MyHC IIa and IIx isoforms was subsequently 38.20 ± 1.46% (30.73% to 48.78%) and 50.07 ± 1.10% (43.8% to 56.78%) from the total MyHC. The MyHC pattern in the skeletal muscles of the Akhal-Teke horses shows that the muscles of these horses have a high capacity both for endurance and speed.

Sequencing and annotated analysis of an Estonian human genome

In present study we describe the sequencing and annotated analysis of the individual genome of Estonian. Using SOLID technology we generated 2,449,441,916 of 50-bp reads. The Bioscope version 1.3 was used for mapping and pairing of reads to the NCBI human genome reference (build 36, hg18). Bioscope enables also the annotation of the results of variant (tertiary) analysis. The average mapping of reads was 75.5% with total coverage of 107.72 Gb. resulting in mean fold coverage of 34.6. We found 3,482,975 SNPs out of which 352,492 were novel. 21,222 SNPs were in coding region: 10,649 were synonymous SNPs, 10,360 were nonsynonymous missense SNPs, 155 were nonsynonymous nonsense SNPs and 58 were nonsynonymous frameshifts. We identified 219 CNVs with total base pair coverage of 37,326,300 bp and 87,451 large insertion/deletion polymorphisms covering 10,152,256 bp of the genome. In addition, we found 285,864 small size insertion/deletion polymorphisms out of which 133,969 were novel. Finally, we identified 53 inversions, 19 overlapped genes and 2 overlapped exons. Interestingly, we found the region in chromosome 6 to be enriched with the coding SNPs and CNVs. This study confirms previous findings, that our genomes are more complex and variable as thought before. Therefore, sequencing of the personal genomes followed by annotation would improve the analysis of heritability of phenotypes and our understandings on the functions of genome.

Globin mRNA reduction for whole-blood transcriptome sequencing

The transcriptome analysis of whole-blood RNA by sequencing holds promise for the identification and tracking of biomarkers; however, the high globin mRNA (gmRNA) content of erythrocytes hampers whole-blood and buffy coat analyses. We introduce a novel gmRNA locking assay (GlobinLock, GL) as a robust and simple gmRNA reduction tool to preserve RNA quality, save time and cost. GL consists of a pair of gmRNA-specific oligonucleotides in RNA initial denaturation buffer that is effective immediately after RNA denaturation and adds only ten minutes of incubation to the whole cDNA synthesis procedure when compared to non-blood RNA analysis. We show that GL is fully effective not only for human samples but also for mouse and rat, and so far incompletely studied cow, dog and zebrafish.

Effect of insemination-related factors on pregnancy rate using sexed semen in Holstein heifers

The objectives were to determine the effects of insemination with sexed semen at spontaneous and induced estrus, fixed-time insemination at synchronized estrus, the deposition site, estrous intensity, housing, age, body weight, and bull on the pregnancy rate in Holstein heifers, and to compare the quality traits of sexed sperm with those of unsexed semen. The study was conducted on 3206 heifers, housed in three free-stall barns and in four tie-stall facilities. After synchronization by two prostaglandin F2α (PGF2α) treatments 14 days apart, 281 heifers were inseminated conventionally and 118 intracornually with sexed semen, and 532 and 148 heifers, respectively, with unsexed semen 80–82 h after the second treatment. At spontaneous estrus, 1129 heifers were inseminated with sexed and 529 with unsexed semen, and at estrus induced by a single PGF2α treatment 185 heifers were inseminated with sexed and 284 with unsexed semen. Heifers were inseminated conventionally with sexed semen 12 h after detection of estrus, and with unsexed semen according to the a.m.– p.m rule. Sexed and unsexed semen doses from five bulls were evaluated for motility, morphology, membrane integrity, and chromatin stability. Overall pregnancy rate with sexed semen (41.7%) was 80.8% of that with unsexed semen (51.6%) and was lower than with unsexed semen irrespective of the type and intensity of estrus, and deposition site. Insemination at spontaneous estrus resulted in a higher pregnancy rate (53.4%) than at induced (41.9%) or synchronized (44.7%) estrus. Pregnancy rates did not differ after intracornual (44.9%) or conventional insemination (48.4%). Strong estrus resulted in higher pregnancy rate (by 14.4% points) compared to weak estrus. The type of housing, age, and weight of heifers had no effect irrespective of the type of semen. The total, progressive and linear motility, and membrane integrity were lower and proportions of immotile sperm greater, for sexed than for unsexed semen.